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Studies into the mechanism of action of murine interleukin-3 Sorensen, Poul Henrik Bredahl 1990

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STUDIES INTO THE MECHANISM OF ACTION OF MURINE INTERLEUKIN-3 by POUL HENRIK BREDAHL SORENSEN B.Sc, The University of British Columbia. 1980 M.D.. The University of British Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1990 © Poul H.B. Sorensen. 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ii A B S T R A C T The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hemopoietic cell line, B6SUtA. These cells were first used to identify the mIL-3 surface receptor as a monomeric 67 kDa protein with a pi of approximately 6.2. Further studies suggested the presence of an additional mIL-3 binding protein with an apparent molecular mass of 140 kDa. Then, in an attempt to gain some insights into the mechanism of action of mIL-3, molecules other than mIL-3 were tested to determine their effects on cell proliferation. Murine granulocyte -macrophage colony-stimulating factor (mGM-CSF) was found to be as potent as mIL-3 in stimulating B6SUtA cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-0-tetradecanoyl-phorbol-13-acetate (TPA), a known activator of protein kinase C, both stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA cells exposed for brief periods to mIL-3, mGM-CSF or TPA were analyzed for changes in phosphorylation patterns using metabolic 32p-labeling. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68 kDa cytosolic protein, while all three agents stimulated the serine-specific phosphorylation of a 67 kDa membrane protein. Furthermore, using antibodies to phosphotyrosine, it appeared that mIL-3 stimulated tyrosine phosphorylation of 67 kDa and 140 kDa membrane proteins, as well as of 40, 55 and 90 kDa cytosolic proteins. The 90 kDa protein was also tyrosine phosphorylated in response to mGM-CSF, suggesting that this phosphorylation results from a common step in mIL-3 and mGM-CSF-stimulated signaling pathways. These phosphotyrosine containing proteins were not detected in TPA-treated cells. Moreover, evidence from a variety of studies is presented that the 140 kDa but not the 67 kDa mIL-3 receptor becomes phosphorylated on tyrosine residues when B6SUtA cells bind mIL-3. iii T A B L E O F CONTENTS Page ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii ACKNOWLEDGEMENTS ix Page Chapter I INTRODUCTION 1 A. General Concepts of Hemopoiesis 1 B. Control of Hemopoiesis 7 1. Role of the Stroma in Hemopoiesis 7 2. Role of Regulatory Molecules in Hemopoiesis 9 2.1. Types of Factors Active in the Hemopoietic System 9 2.1.1. Class I Factors 12 Interleukin-3 12 GM-CSF 15 2.1.2. Class II Factors 16 G-GSF 16 M-CSF 17 Erythropoietin 18 Interleukin-5 18 2.1.3. Class III Factors 19 Interleukin-1 19 Interleukin-4 19 Interleukin-6 20 TC-1 Synergistic Activity 20 Mesenchymal Cell Activators 20 2.1.4. Class IV Factors 21 2.2. In Vivo Actions of the CSFs 24 2.3. Colony-stimulating Factors and Myeloid Leukemia 27 C. Mechanism of Action of Polypeptide Growth Factors 3 1 1. Cellular Responses to Growth Factors 32 1.1. Ion Fluxes 33 1.2. GTP-binding Proteins and Receptor-effector coupling 3 1 1.3. Elevation of cAMP Levels 36 1.4. Phosphoinositide Hydrolysis and the Activation of Protein Kinase C 37 1.5. Tyrosine-specific Protein Phosphorylation 43 1.6. Changes in Gene Expression 47 2. Signal Transduction Pathways Involved in Mitogenesis 47 3. Mechanism of Action of the CSFs 52 D. Thesis Objectives 56 E. References 58 iv Page Chap t e r II M A T E R I A L S A N D M E T H O D S 79 A . Mate r ia l s 79 B. Ce l l s 80 C . C e l l Prol iferat ion A s s a y s 80 D . Iodinat ion of mIL-3 81 E . B i n d i n g S tud ies w i th 1 2 5 I - m I L - 3 81 F . Radio labe l ing of the mIL-3 Receptor U s i n g S A S D 82 G . One- and Two-d imens iona l G e l Elec t rophores is 84 H . Labe l ing of Ce l l s w i th [ 3 2 P ] Or thophosphate 85 I. S t i m u l a t i o n wi th Fac tors and Subce l lu l a r F rac t iona t ion 85 J. Phosphoamino A c i d A n a l y s i s 87 K . Aff ini ty Pur i f ica t ion of Ant iphospho ty ros ine An t ibod ie s 88 L . Wes te rn Blot A n a l y s i s of Cytosol ic a n d Membrane Proteins wi th Ant iphospho ty ros ine Ant ibod ies 89 M . Immunoprec ip i t a t ion of Phosphopro te ins w i th Ant iphospho ty ros ine Ant ibod ies 8 9 N . Ant iphospho ty ros ine An t ibody Immunoprec ip i t a t ion after C h e m i c a l C r o s s l i n k i n g of 1 2 5 I - m I L - 3 to Intact Cel l s 90 O. F luoresce ina t ion of mIL-3 90 P. B io t iny la t ion of mIL-3 91 Q. Affini ty Precipi ta t ion of 3 2 P - L a b e l e d Proteins U s i n g Fluoresce ina ted and Bio t iny la ted mIL-3 91 R. References 93 Chap t e r III I D E N T I F I C A T I O N O F T H E M U R I N E I N T E R L E U K I N - 3 R E C E P T O R U S I N G A C L E A V A B L E . P H O T O R E A C T I V E C R O S S L I N K I N G A G E N T 94 A . In t roduct ion 94 B . Resul t s 95 1. B i n d i n g of 1 2 5 I - m I L - 3 to mIL-3-Dependent C e l l L ines 95 2. Sca t cha rd A n a l y s i s of the B i n d i n g of 1 2 5 I - m I L - 3 to . B 6 S U I A and F D C - P 1 Cel l s 9 8 3. Identification of the mIL-3 Receptor U s i n g S A S D 9 8 4. Receptor Identification U s i n g Glu ta ra ldehyde and D S S as C r o s s l i n k i n g Agents 104 C . D i s c u s s i o n 105 D . References 1 12 Chap te r IV I N T E R L E U K I N - 3 , G M - C S F A N D A C T I V A T O R S O F P R O T E I N K I N A S E C I N D U C E D I S T I N C T P H O S P H O R Y L A T I O N E V E N T S IN M U R I N E M U L T I P O T E N T I A L H E M O P O I E T I C C E L L S 1 14 A . In t roduct ion 114 B . Resul t s 115 1. Effects of V a r i o u s Agents on the Prol iferat ion of m l L -3-dependent B 6 S U t A ! Cel l s 115 2. Phosphory la t ion of Cytosol ic Proteins in Intact Factor-S t imula t ed Ce l l s 120 3. T ime Course a n d Concen t ra t ion Dependence of cp68 Phosphory la t ion 123 4. Phosphoamino A c i d A n a l y s i s of 3 2 P - l a b e l e d cp68 127 V Page 5. Phosphorylation of Membrane Proteins in Intact Factor-stimulated Cells 127 6. Time Course and Concentration Dependence of mp67 Phosphorylation 131 7. Phosphoamino Acid Analysis of ^^P-labeled mp67 136 8. Treatment of Gels with Alkali after SDS-PAGE to Enrich for Phosphotyrosine 136 C. Discussion 137 D. References 144 Chapter V INTERLEUKIN-3 STIMULATES TYROSINE-SPECIFIC PROTEIN PHOSPHORYLATION IN FACTOR-DEPENDENT B6SUtAj CELLS 146 A. Introduction 146 B. Results '147 1. Western Blot Analysis of Cytosolic and Membrane Phosphoproteins using Antiphosphotyrosine Antibodies 147 2. Immunoprecipitation of Phosphotyrosine-containing Proteins using Antiphosphotyrosine Antibodies 148 3. Time Course Study of mIL-3-induced Tyrosine Phosphorylation 153 4. Concentration Dependence of mIL-3-induced Tyrosine Phosphorylation 153 5. Phosphoamino Acid Analysis of Immunoprecipitated Phosphoproteins 157 C. Discussion 157 D. References 161 Chapter VI INTERLEUKIN-3 STIMULATES THE TYROSINE PHOSPHORYLATION OF THE 140 kDa INTERLEUKIN-3 RECEPTOR 166 A. Introduction 166 B. Results 167 1. Crosslinking of 1 2 5I-mIL-3 to B6SUtA 1 Cells and Immunoprecipitation with Antiphosphotyrosine Antibodies 167 2. Effect of SDS Denaturation of Cell Lysates on Antiphosphotyrosine Immunoprecipitation 170 3. Affinity Precipitation of Phosphorylated mIL-3 Binding Proteins 173 C. Discussion 175 D. References 180 Chapter VII SUMMARY AND CONCLUSIONS 182 A. Identification of the mIL-3 Receptor 182 B. Murine mIL-3, mGM-CSF and TPA Stimulate Distinct Phosphorylation Events in BeSUtAj Cells 183 C. Tyrosine Phosphorylation is an Early Event in the Stimulation of BeSUtAj Cells by mIL-3 185 D. Murine IL-3 Stimulates Tyrosine Phosphorylation of the 140 kDa mIL-3 Receptor 186 E. General Comments 186 F. References 190 vi LIST OF TABLES Page TABLE 1. Major Murine Polypeptide Factors Active in the Hemopoietic System 11 TABLE 2. Binding of 1 2 5I-mIL-3 to Murine Hemopoietic Cell Lines 96 TABLE 3. Ability of Various Agents to Substitute for mIL-3 in Stimulating [3H]Thymidine Incorporation into BeSUtA! Cells 116 TABLE 4. Apparent Molecular Masses of Receptors for Murine CSFs 184 TABLE 5. Murine IL-3-induced Protein Phosphorylation in B6SUtA 1 Cells 188 vii LIST OF FIGURES Page Figure 1. Schematic Representation of the Murine Hemopoietic System 6 Figure 2. Schematic Representation of Postulated Early Signals in the Mitogenic Response 39 Figure 3. Signal Transduction Pathways in Mitogen-stlmulated Cell Proliferation 54 Figure 4. Time Course of Binding of mIL-3 to B6SUtA Cells 97 Figure 5. Scatchard Analysis of the Binding of 125I-mIL-3 to B6SUtA and FDC-P1 Cells 100 Figure 6. Scheme for the Cross-linking of mIL-3 to its B6SUtA Cell Surface Receptor 102 Figure 7. Incorporation of 1 2 5 I from 125I-SASD-mIL-3 into B6SUtA and P815 Cells 103 Figure 8. Two-dimensional O'Farrell Gel Electrophoresis of Plasma Membranes from B6SUtA Cells Labeled with 125I-SASD-mIL-3 106 Figure 9. Cross-linking of 125I-mIL-3 to the mIL-3 Receptor on B6SUtA Cells Using Glutaraldehyde and Disuccinimidyl Suberate 109 Figure 10. Effects of mIL-3, GM-CSF and TPA on the Proliferation of B6SUtA^ Cells 118 Figure 11. Effects of TPA on Growth and Viability of BeSUtAj Cells Treated with mIL-3 or GM-CSF 121 Figure 12. Time Course Study of TPA-induced Inhibition of Proliferation 122 Figure 13. Effects of mIL-3, GM-CSF and TPA on the Phosphorylation of Cytosolic Proteins in B6SUtA^ Cells 124 Figure 14. Time Course of cp68 Phosphorylation 126 Figure 15. Concentration Dependence of cp68 Phosphorylation 128 Figure 16. Phosphoamino Acid Analysis of cp68 129 Figure 17. Analysis of Membrane Phosphoproteins by One-Dimensional SDS-PAGE 130 Figure 18. Analysis of Membrane Phosphoproteins by Two-Dimensional Gel Electrophoresis 132 Figure 19. Time Course of mp67 Phosphorylation 134 ig re 20. Concentration De endence of mp67 Phosphorylation 138 VU1 Figure 21. Phosphoamino Acid Analysis of mp67 Figure 22. Alkali Treatment of SDS-PAGE Gels to Enrich for Phosphotyrosine Figure 23. Western Blot Analysis of Phosphoproteins from B6SUtA 1 Cells Using Antiphosphotyrosine Antibodies Figure 24. Immunoprecipitation of BGSUtA]^ phosphoproteins with Antiphosphotyrosine Antibodies Figure 25. Two-Dimensional Gel Analysis of mIL-3-induced Tyrosine Phosphorylation in B6SUtA^ Cells Figure 26. Time Course of mIL-3-lnduced Tyrosine Phosphorylation Figure 27. Dose Dependence of mIL-3-induced Tyrosine Phosphorylation Figure 28. Phosphoamino Acid Analysis of the mIL-3-induced Phosphotyrosine-Containing Proteins Figure 29. Antiphosphotyrosine Precipitation of ^ 2^I-mIL-3 Crosslinked to B6SU1A.! Cells Figure 30. Antiphosphotyrosine Precipitation of 1 2 ^I-mIL-3 in the Presence of Various Phosphoamino Acids Figure 31. Antiphosphotyrosine Precipitation of SDS Treated DSS-crosslinked 1 2 5 I -mIL-3 -B6SUtA 1 Cell Protein Complexes Figure 32. Detection of Phosphorylated mIL-3 Binding Proteins Using Fluoresceinated and Biotinylated mIL-3 Figure 33. Phosphoamino Acid Analysis of p 140 Page 139 140 149 151 152 154 156 158 168 171 172 174 176 ix A C K N O W L E D G M E N T S When one sets out to negotiate the unchartered waters of a new scientific challenge, navigational dilemmas are perhaps not to be unexpected. I therefore want to thank Dr. Gerald Krystal for somehow maintaining direction of what must have seemed at times a rudderless ship. His extensive knowledge and relentless curiosity were key to the completion of this thesis. I would like to thank the other senior staff scientists of the Terry Fox Laboratory for sharing their expertise and for pointing out the importance of collaboration in science, as well as the Terry Fox secretarial staff, especially Judy Wong, for help in preparing the thesis. Thanks must of course go to members of the S 2 society for their continuous support. I also want to express my appreciation to Drs. Derek Applegarth and Don Brooks for helping to point my interests towards research early in my education, and to the members of my supervisory committee for their guidance and interest. Above all, I want to thank my family for the quiet inspiration they so deftly provide, without which this work could not have been achieved. As we live, we are transmitters of life. And when we fail to transmit life, life fails to flow through us... And if, as we work, we can transmit life into our work, life, still more life, rushes into us to compensate, to be ready and we ripple with life through the days. D. H. Lawrence 1 C H A P T E R I INTRODUCTION A. G E N E R A L CONCEPTS OF HEMOPOIESIS Hemopoiesis is the process whereby mature red and white cells are maintained in the circulating blood. Because most of these cells have relatively short lifespans, they need to be constantly replaced throughout life. In addition, the system must be capable of adapting to conditions of hemopoietic stress, such as blood loss or infection, by rapid fluctuations in the levels of appropriate mature cells. These demands are met by the generation of multiple blood cell types from pluripotent hemopoietic stem cells through accurately controlled and tightly coupled cell division and differentiation. The hemopoietic stem cell compartment of the adult resides mainly in the bone marrow, where it gives rise to progenitors of both the myeloid and lymphoid cell lineages (1-4). These committed progenitors then undergo an orderly sequence of developmental steps leading to the formation of terminally differentiated hemopoietic cells inc luding members of the erythrocyt ic , granulocyt ic , monocyt ic and megakaryocytic l ineages, and, in the lymphopoietic system, B cells and T cells. The bone marrow therefore consists of a mixture of representatives of various cell lineages at different stages of differentiation. The vast majority of these cells are in the later stages of maturation and are thus recognizable by well-established morphological criteria, while only a very small percentage exist as more primitive progenitor cells (5). The hemopoietic system has been structurally described as a hierarchy consisting of three major cellular compartments. The most primitive of these, and that from which blood cell formation is initiated, is the pluripotent stem cell compartment. Although these cells are not morphologically identifiable and are present in very low numbers, they have two 2 properties which distinguish them from other cells and which are essential for the maintenance of hemopoiesis: they have unrestricted differentiation potential, i.e., they have genetic potential to undergo development into all of the various blood cell lineages (6), and they have extensive self-renewal capacity, i.e., they can divide to form daughter cells which are exact copies of themselves (7). Early evidence for the existence of pluripotent stem cells came from the demonstration that lethally irradiated mice could be repopulated with functional hemopoietic tissue, in the form of macroscopic nodules on the surfaces of their spleens, after the intravenous injection of bone marrow cells from normal histocompatible donor mice (8). Subsequently, cytological studies (6,9) and cytogenetic examination using radiation-induced chromosomal markers (3,10) revealed that cells within each splenic nodule were derived from single pluripotent cells, and that these stem cells could give rise to progeny that repopulated both the myeloid and lymphoid organs of the host. The presence of splenic nodules of hemopoietic tissue in lethally irradiated recipient mice formed the basis for the first quantitative colony assay for pluripotent stem cells - the so-called spleen colony assay - and defined the cell initiating these spleen colonies as the CFU-S (colony forming unit - spleen) (8). There is now much evidence to suggest that cells responsible for the generation of self-maintaining, non-transient spleen colonies are similar if not identical to cells capable of long-term hemopoietic repopulation in mice (11). Furthermore, recent studies using retrovirally marked murine marrow cells have confirmed that single cells are capable of repopulating the entire myeloid and lymphoid systems of primary and secondary recipients (12). Evidence for a pluripotent hemopoietic stem cell in the human system has come from studies of various hematological disorders. First, the Philadelphia chromosome (Ph1) has been found in myeloid cells and B cells, but not marrow fibroblasts, of most patients with chronic myelogenous leukemia (CML) (13,14). Second, women with CML who are heterozygous at the locus on the X chromosome encoding the enzyme glucose-6-phosphate dehydrogenase (G6PD) show only a single enzyme isotype in their hemopoietic cells (15,16). strongly suggesting that these cells are descendents of a cell with considerable 3. differentiation potential. T h i r d , one patient with sideroblastic anemia was demonstrated to e x p r e s s a s i n g l e G 6 P D isotyp e i n m y e l o i d c e l l s as w e l l as B a n d T c e l l s (15). F o u r t h , a n a l y s i s of r e s t r i c t i o n fragment length p olymorphisms w i t h i n the X - l i n k e d h ypoxanthine phosp h o r i b o s y l transferase gene of a patient after allogeneic bone marrow t r a n s p l a n t a t i o n f rom a heterozygous female donor revealed long-term m o n o c l o n a l hemopoiesis of donor origin (17). D u r i n g n o r m a l hemopoiesis the m a j o r i t y of stem c e l l s are not u n d e r g o i n g m i t o t i c d i v i s i o n , b u t are r a p i d l y m o b i l i z e d i n t o a c y c l i n g s t a t e b y p e r t u r b a t i o n s i n more differentiated compartments (18). What regulates the decision for a stem cel l to self-renew or to become committed to a p a r t i c u l a r lineage is not known, b u t two theories have been p r o p o s e d . The d e t e r m i n i s t i c t h e o r y p o s t u l a t e s t h a t the b e h a v i o r of a s t e m c e l l i s a consequence of specific inductive microenvironmental signals from the s u r r o u n d i n g bone marrow s t r o m a (19,20). The s t o c h a s t i c theory, on the other h and, proposes that loss of self-renewal and commitment to differentiation is a random event with a probability that is i n t r i n s i c to the stem c e l l (21,22). A c c o r d i n g to t h i s s e cond model, the role of e x t e r n a l i n f l u e n c e s are e i t h e r to m o d i f y the p r o b a b i l i t y of c o m m i t m e n t or to a l l o w f u r t h e r development of an already committed ce l l . It is l i k e l y t h a t elements of b o t h models w i l l prove to be c o r r e c t a n d one c a n h y p o t h e s i z e tha t s t e m c e l l development i s b a s i c a l l y a stochastic process that can be modulated by the hemopoietic microenvironment. Whatever the m echanism, the d e c i s i o n of stem c e l l s to d i f f e r e n t i a t e r e s u l t s i n the f o r m a t i o n of c o m m i t t e d p r o g e n i t o r c e l l s , w h i c h c o m p r i s e the s e c o n d m a j o r c e l l u l a r c o m p a r t m e n t of the h e m o p o i e t i c system. P r o g e n i t o r s are a t r a n s i e n t p o p u l a t i o n of proliferating cells that are committed to only one or two lineages. As with pluripotent stem cells, they are present at very low frequencies i n n ormal hemopoietic t i s s u e s and have no distinctive morphological features. Therefore, progenitors c a n be identified only indirectly by the differentiated progeny they give rise to when these c e l l s are grown i n vit r o . S u c h hemopoietic colony assays involve suspending cells to be tested i n semi-solid media (agar, methylcellulose, or a plasma clot) containing appropriate nutrients, serum, and a source of 4 hemopoietic growth factors (see below). The lineage specificity of a progenitor can then be determined from the visual appearance of the resulting colony. Progenitors are in fact defined by the types of colonies they produce. In terms of the size and composition of the colony and its time of maturation. These parameters have been shown to be reproducible indicators of the proliferative and differentiation potential of different types of progenitors (23,24). Cells that give rise to colonies of granulocytes and macrophages were, for historical reasons, initially termed CFU-C (colony forming unit - in culture). They have more recently been named CFU-granulocyte/macrophage (CFU-GM). Similarly, megakaryocytic progenitors have been named CFU-megakaryocyte (CFU-Mk or CFU-Meg). At least three different types of erythroid colonies appearing sequentially in vitro have been documented in both the human and murine systems (25,26). The primitive burst-forming unit-erythroid (BFU-E) gives rise to large multiclustered (>8) colonies (hence the term burst-forming), consisting of greater than 1000 hemoglobin-synthesizing erythroblasts. The more mature BFU-E produces colonies of only 3-8 clusters of approximately 30 erythroblasts each, while the cell type giving rise to single or paired clusters oi erythroblasts is designated as the colony forming unit - erythroid (CFU-E). These properties reflect the decreasing migratory abilities of differentiating progenitors in semi-solid medium. The progenitors described thus far give rise to colonies restricted to one, or at most, two myeloid lineages. Lymphoid bipotent (B and T cell) or unipotent (B or T cells) progenitors have recently been described using retroviral marker analysis in marrow repopulation studies (27,28). Also recently documented is the clonal growth of human hemopoietic progenitors with multilineage differentiation potential (29-32). Colonies developing from these cells have been shown to contain granulocytes, erythrocytes, macrophages and megakaryocytes, and the cell of origin has therefore been designated the CFU-GEMM. Mixed human colonies have also been shown to contain mast cells (33). eosinophils (33), basophils (34), and cells with lymphoid markers (35-39). Similar findings with the murine system (40) have led investigators to query the relationship between CFU-5 GEMM and the previously mentioned CFU-S, with the current view being that they represent at least overlapping populations (41,42). Very recent evidence for a colony forming cell more primitive than the CFU-GEMM has emerged from the identification of murine colonies composed primarily of undifferentiated blast cells (43). The progenitors of these colonies give rise to a high incidence of secondary stem cell colonies as well as GEMM colonies after replating. These progenitors, designated S-cells (stem cells), show a delayed initiation of proliferation, possibly due to the relative resistance they show to being stimulated into a cycling state. A human equivalent of the murine S-cell was originally observed in umbilical cord blood (44), and has now been demonstrated in adult bone marrow (45). Studies are currently in progress to determine the relationship between S-cells and cells capable of long-term hemopoietic repopulation in vivo. Progenitors give rise to members of the third major hemopoietic compartment. This compartment is quantitatively the largest and consists of cells that are morphologically recognizable. The compartment includes two classes of cells. The first class is comprised of precursor cells (eg. proerythroblasts and erythroblasts) which have a very limited proliferative potential and undergo a small number of divisions leading to terminal -maturation. The second class consists of fully differentiated, non-dividing blood cells which are released into the circulation to perform their respective functions. The above model of hemopoiesis, which is summarized in Figure 1, is therefore based on a hierarchical structuring of the hemopoietic system. Aside from stem cells, which can self-renew, proliferation leads to a progressively more mature phenotype with a concomitant loss of proliferative potential (46,47). Although disturbances in hemopoiesis do occur and may result in a range of diseases from anemia to leukemia, the system normally functions with extraordinary constancy. This is attributed at least in part to a complex interplay of cellular and molecular control mechanisms. ULTIMATE STEM CELL • • Lymphopoiesis ERYTHROID PROGENITORS Primitive BFU E •Mi large erythroid colony or buist tit Mature BFU E CFU I small erythroid colony or burst erylhroid cluster sell-renewal @ MYELOID STEM CELL (CFU S. CFU GEMM) MEGAKARYOCYTE PROGENITORS I © • cfu Meg large megakaryocyte colony small megakaryocyte colony GRANULOCYTE PROGENITORS 1 © •; CFU C large granulocyte colony small granulocytic colony RED CELIS PI ATEIETS GRANULOCYTES & MACROPHAGES ifture 1. S c h e m a t i c r e p r e s e n t a t i o n of (he h i e r a r c h i c a l s t r u c t u r e of the m u r i n e h e m o p o i e t i c s y s t e m a s determined by clonogeiiic assays l o r p l i n i p o t r n l and committed progenitor cells. 7 B. CONTROL OF HEMOPOIESIS The hemopoietic system requires an exquisite level of control to m a i n t a i n both overall cel l numbers and the balance between various mature cell types. While some of the control m echanisms appear to involve intimate contact between hemopoietic cells and cells of the m icroenvironment located i n the sites of blood c e l l formation, there is now overwhelming e v i d e n c e to s u g g e s t t h a t a g r o u p of d i f f u s i b l e r e g u l a t o r y g l y c o p r o t e i n s , the c o l o n y s t i m u l a t i n g factors (CSFs), plays an essential role i n the normal proliferation of hemopoietic c e l l s . A l t h o u g h the m a i n focus of t h i s t h e s i s i s on the C S F s , i n p a r t i c u l a r the m u r i n e m y e l o i d f a c t o r , I n t e r l e u k i n - 3 , a d i s c u s s i o n of the c o n t r o l of h e m o p o i e s i s w o u l d be i n c o m p l e t e w i t h o u t a c o n s i d e r a t i o n of t h e r e g u l a t o r y r o l e of t h e h e m o p o i e t i c microenvironment. 1. Role of the Stroma i n Hemopoiesis A r e g u l a t o r y r o l e for the m i c r o e n v i r o n m e n t was o r i g i n a l l y s u g g e s t e d by the morphological observation that developing hemopoietic cells exist i n close association with a complex stromal c e l l network (48). Included i n this network are mesenchymally derived fibr o b l a s t s , endothelial cells, adipocytes, osteoblasts, and osteoclasts (49). Fixed marrow macrophages have also been considered as part of the stroma, although they are derived from hemopoietic stem cells i n contrast to the above cells. Stromal cells participate i n the f o r m a t i o n of and are i n contact with an intricate three-dimensional meshwork of fibrous and n o n - f i b r o u s p r o t e i n s k n o w n as the e x t r a c e l l u l a r m a t r i x (ECM). The p r o t e i n s of the marrow E C M i n c l u d e collagen types I, III, and V, proteoglycans, l a m i n i n , fibronectin, and the recently described marrow-specific adhesion molecule, hemonectin (50). It has been postulated that the collagens, proteoglycans, and l a m i n i n form nests for hemopoietic cells (51,52), and that fibronectin and hemonectin mediate the adhesion of hemopoietic cells to stromal cells or to the E C M (50,53). Other lin e s of evidence also suggest a role for the marrow stroma i n the c o n t r o l of h e m o p o i e s i s . It h a s b e e n s h o w n t h a t C F U - S d e v e l o p i n g i n the s p l e e n are m a i n l y 8 erythropoietic i n nature, while those developing i n the marrow are m a i n l y granulopoietic, suggesting th a t the d i f f e r e n t i a t i o n of CFU-S i s i n f l u e n c e d by its m i c r o e n v i r o n m e n t (19). Also, mice carrying the so-called Steel m utation ( S l / S l ^ ) can have their macrocytic anemia c u r e d by b e i n g i n f u s e d w i t h n o r m a l donor spleen cel l s , b u t not by the t r a n s p l a n t a t i o n of n o r m a l marrow stem cells (54). However, hemopoietic stem c e l l s f rom S l / S l ^ mice c a n c u r e a n e m i c m i c e w h o s e s t e m c e l l s are d e f e c t i v e (54). T h e r e f o r e , the h e m o p o i e t i c abnormality i n S l / S l ^ mice appears to reflect a stromal cel l defect rather t h a n a stem cell defect. These findings are supported by studies u s i n g the long-term bone marrow cultu r e s y stem, a l i q u i d c u l t u r e s y s t e m w h i c h i s c a p a b l e of m a i n t a i n i n g the s e l f - r e n e w a l a n d d i f f e r e n t i a t i o n p o t e n t i a l of hemopoietic stem c e l l s , i n c l u d i n g CFU-S (55). The a b i l i t y of these c u l t u r e s to s u p p o r t hemopoiesis i n v i t r o has been a t t r i b u t e d to the fo r m a t i o n , i n cu l t u r e flasks, of a complex adherent layer that putatively serves as the i n vitro equivalent of the m a r r o w s t r o m a (56-58). U s i n g m u r i n e long-term bone m a r r o w c u l t u r e s , it was demonstrated that hemopoietic c e l l s from S l / S l ^ mice c o u l d be s u p p o r t e d by adherent l a y e r s f r o m n o r m a l mice, w h i l e a d h e r e n t l a y e r s f r o m S l / S l ^ m i c e were i n c a p a b l e of supporting hemopoiesis (56). T h e s e a n d o t h e r d a t a (59,60) s u p p o r t the c o n c e p t t h a t c l o s e r a n g e c e l l u l a r interactions, the nature of which remain largely unknown, are involved i n the regulation of hemopoiesis. A n obvious candidate for the m e c h a n i s m by w h i c h s t r o m a l c e l l s influence hemopoiesis is the l o c a l p r o d u c t i o n of the aforementioned colony s t i m u l a t i n g factors by these cells. In fact, it has been known for some time that, i n a d d i t i o n to lymphocytes (see below), a v a r i e t y of c e l l types i n c l u d i n g e n d o t h e l i a l c e l l s (61,62) a n d f i b r o b l a s t s (63) are ca p a b l e of p r o d u c i n g CSFs. More recently, it h a s been d e m o n s t r a t e d tha t f i b r o b l a s t s , i n c l u d i n g t h o s e d e r i v e d from the marrow, a n d e n d o t h e l i a l c e l l s c a n be s t i m u l a t e d to produce C S F s by the monocyte-derived factor, Interleukin-1 (64-69). These data w i l l be d i s c u s s e d further i n a later section, but serve at t h i s point to promote the n o t i o n that the m i c r o e n v i r o n m e n t may i n f l u e n c e h e m o p o i e t i c c e l l d evelopment by the e l a b o r a t i o n of h e m o p o i e t i c growth factors. W h e t h e r other forms of c e l l - c e l l c o m m u n i c a t i o n between 9 stromal and colony-forming cells are also involved, remains to be determined. Intriguingly, it has recently been shown that glycosaminoglycans produced by marrow stromal cells can bind hemopoietic growth factor "molecules (70), further underscoring the putative significance of the CSFs in the regulation of hemopoiesis. 2. Role of Regulatory Molecules in Hemopoiesis Analysis of colonies grown in vitro led to the recognition that hemopoietic cells are unable to survive or to proliferate unless they are specifically stimulated (70). It was then established that hemopoietic progenitor cells can develop in soft agar in the absence of stromal cells, provided that the cultures are supplemented with conditioned medium from a variety of normal and leukemic cell lines, or from various tissues including populations of activated T lymphocytes (71). This, in turn, led to the discovery of a series of regulatory glycoprotein molecules that promote the proliferation and differentiation of specific subsets of hemopoietic cells (72). Because the factors were originally identified by their ability to stimulate progenitor cells to form colonies in vitro, they have been, as mentioned, called colony stimulating factors. In fact, analysis of the types of colonies formed in response to various CSFs have permitted the characterization of many of the biological activities of the CSFs. This has been greatly facilitated by the purification to homogeneity of many of these factors, as well as the cloning of their genes and production of large quantities of pure factors by recombinant DNA technology. However, this has also led to the appreciation that other molecules may be involved in the regulation of hemopoiesis. 2.1. Types of factors active in the hemopoietic system. Based on recent data regarding biological activities of highly purified native or recombinant molecules, there appear to be at least four classes of regulatory molecules active in the hemopoietic system (73,74). The CSFs make up the first two classes, which consist of molecules that can stimulate colony formation in vitro. Class I factors act on pluripotent stem cells and immature progenitors, and as such are relatively lineage non-10 specific. They are survival factors required for cell self-renewal and proliferation and contribute to the differentiation of these cells. Class II factors are restricted in their actions to more mature progenitor cells, and are required for the development of specific lineages. They also influence the survival and functional activity of mature cells. Recently, evidence has emerged for the existence of a third class of hemopoietic growth factors influencing the development of progenitors. These factors have no colony- stimulating activity when used alone, but appear to synergize with and thereby enhance the activities of Class I and II factors. Finally, Class IV factors consist of molecules whose major hemopoietic actions are inhibitory. They are therefore considered as negative regulators of hemopoiesis. Members of each of the four classes wil l now be discussed individual ly . Pr ior to this , however, several important points should be noted. First, as wi l l become apparent below, certain hemopoietic growth factors display properties of more than one class. The significance of this property in vivo remains to be determined. Second, since the various factors are produced by multiple cell types (see below), the assignment of specific biological activities to factors may be complicated by the presence of factor-producing accessory cells in target cell preparations under study. Therefore, such assignments may be influenced by the purity of target cell populations. Third, the purification of primitive hemopoietic progenitor cells has still not been achieved (75). Therefore, it remains to be determined if the overlap of in vitro activities observed when comparing certain CSFs reflects st imulation of identical target cells, or whether the factors are acting on different target cells. Lastly, a number of terms relating to the nomenclature of growth factors should be defined. Cytokines are cellular polypeptide products that are secreted to act as transmitters of regulatory signals among cells. Monokines are cytokines released by monocytes, while lymphokines are cytokines released by antigen-activated T lymphocytes. Finally, interleukins are lymphokines which, in addition to having other functions, are thought to play a role in lymphocyte development The following discussion is limited to factors active in the myeloid system, with the main focus on the Class I factors interleukin-3 and granulocyte-macrophage colony-stimulating factor. The major murine hemopoietic growth factors are presented in Table 1. 11 TABLE 1 - Major Murine Polypeptide Factors Active in the Hemopoietic System FACTOR ALTERNATIVE NAMES APPARENT MOLECULAR MASS (kDa) MAJOR TARGET CELLS 1. CLASS I (multi-lineage factors) Interleukin-3 (IL-3) Multi-CSF, BPA, HCGF, PSF, MCGF, CSF-2a 23-30 CFU-S, CFU-GEMM, C F U - G M / G / M , CFU-EO CFU-Meg, CFU-E. BFU-E, Mast cells Granulocyte-macrophage CSF (GM-CSF) MGI-IGM 23 CFU-GM/G/M, CFU-GEMM, *CFU-Meg, CFU-EO, Granulocytes, Eosinophils II. CLASS II (lineage- specific factors) Granulocyte CSF (G-CSF) MGI-IG 25 CFU-G, CFU-GM, WEHI-3B cell line Macrophage CSF (M-CSF) Erythropoietin (Ep) Interleukln-5 (mIL-5) BCGF-II, EDF CSF-1 45-60 70 (homodimer) 34-36 Pre-B cells, CFU-EO CFU-M, CFU-G, Macrophages BFU-E, CFU-E III. CLASS HI (synergistic factors) Interleukln-1 (IL-1) Hemopoietln-1 22 Activated T and B cells, Neutrophils, Fibroblasts, Endothelial cells, 9CFU-GEMM Interleukin-4 (IL-4) BSF-1 15-20 Activated B and T cells. Mast cells, CFU-GEMM, CFU C, Interleukln-6 (IL-6) IFN-P2, HGF, BSF-2 26 Pre-B cells, CFU-blast, 9CFU-GEMM, Hybridoma, TC-1 Synergistic Activity Platelet-derived Growth Factor (PDGF) "60 60 (heterodimer) CFU-M Fibroblasts, Endothelial cells Tumour Necrosis Factor-a (TNF-ct) 17 Fibroblasts, Endothelial cells IV. CLASS IV (inhibitory factors) Lactoferrin 14 CFU-GEMM, CFU-GM, CFU-G, CFU-M, CFU-E Inhibitory and Stimulatory Marrow Factors Interferon--/ (IFN-Y) Transforming Growth Factor-P(TGF-P) MAF 50-100 (Inhibitory) 30-50 (stimulatory) 38-80 25 (homodimer) CFU-S Macrophages, 7CFU-S, ?CFU-GEMM, Other cell typ Primitive myeloid progenitor cells High concentrations required. By gel filtration. Abbreviations: CSF, colony-stimulating factor; BPA, burst-promoting activity; HCGF, hemopoietic cell growth factor; PSF, persisting-cell-stimulating factor; MCGF, mast cell growth factor; EO, eosinophil; MGI. macrophage-granulocyte inducer; BCGF-II, B cell growth factor II; EDF, eosinophil differentiation factor; BSF. B cell stimulating factor; INF, interferon; HGF, hybridoma growth factor; MAF, macrophage-activating factor 12 2.1.1. Class I Factors Interleukin-3. Murine interleukin-3 (mIL-3) was initially identified as a T-cell derived factor that stimulated the enzyme 20a-hydroxysteroid dehydrogenase in cultures of splenic lymphocytes from athymic mice (76). It was subsequently purified to homogeneity (77) and shown to possess other activities including burst-promoting activity (BPA) (78) as well as the activities of hematopoietic cell growth factor (HCGF) (79). persisting-cell stimulating factor (PSF) (80). and multi-CSF (81). Current evidence supports the concept that all these previously reported activities reside in the same macromolecule, and that this factor plays an important role in regulating the growth and differentiation of early hemopoietic progenitors. As yet, only antigen- or mitogen-activated T lymphocytes have been documented as a normal cellular source for mIL-3 (82). However, this factor is also produced constitutively by the murine myelomonocytic leukemia cell line, WEHI-3B (77,82), and by a T cell hybridoma (83). The apparent molecular mass of purified mIL-3 has a range of 23-30 kDa. probably due to a varying carbohydrate content, the carbohydrate moiety does not appear to be necessary for biological activity in vitro. As an indication of its potency, the specific activity of mIL-3 is 0.05-0.2 ng /ml for half-maximal proliferation of target cells, corresponding to approximately 10" 1 2 M (72). Clones of cDNA for mIL-3 have recently been isolated from a WEHI-3B-derived cDNA library (84), and from a T cell-derived cDNA library (85). On the basis of the cDNA sequence, the mature polypeptide consists of 140 amino acids and has a molecular mass of 15 kDa. There are four potential glycosylation sites, and four cysteines which are likely linked by mandatory disulphide bridges (86). The mIL-3 gene exists in single-copy fonn and is composed of four introns and five exons. Linkage studies with the protooncogene C-erb B. known to be on chromosome 11, have localized the mIL-3 gene to chromosome 1 1 (87). Interestingly, chromosome 11 also contains the locus for the hemoglobin a complex and, as noted below, the locus for murine granulocyte-macrophage colony-stimulating factor (GM-CSF). In fact, the spectacular increase in transcription of both mIL-3 and GM-13 CSF genes when various T-cell clones are stimulated with antigens or activators such as concanavalin A, suggests some form of coordinate expression of the two genes (88). Evidence also exists for the presence of a longer variant of the mIL-3 gene in some cells, raising the possibility of a larger, membrane-bound form of mIL-3 (89). Recombinant mIL-3 has been produced using both a monkey COS cell (90,91) and E. coli expression system (92). The biological properties of both recombinant forms in vitro are similar to those of native mIL-3 (90-92). Chemical synthesis of this molecule has also recently been achieved (93). The testing of pure mIL-3 in various in vitro bioassays has demonstrated that this molecule has a wide range of biological properties. These properties include not only the stimulation of granulocyte and/or macrophage colony formation, but also the stimulation of BFU-E, CFU-E, eosinophil and megakaryocyte progenitors (72). This factor also induces the proliferation of mast cells (94) and various mIL-3 dependent hemopoietic cell lines (90). This spectrum of activities is consistent with a role for mIL-3 in the regulation of a hemopoietic stem cell which is capable of becoming committed to a variety of lineages. In fact, it was found several years ago that mIL-3 can increase the number of erythroid bursts when added to bone marrow colony assays containing the erythroid CSF erythropoietin (95), and hence mIL-3 was said to have burst-promoting activity (BPA). Similar ly , it was shown that mIL-3 is capable of increasing the responsiveness of certain cells to macrophage colony-stimulating factor (M-CSF), thereby synergizing with M-CSF in the formation of macrophage colonies (96). From these studies it was proposed that mIL-3 was acting by expanding the population of early progenitors that would subsequently differentiate to become committed to particular lineages. More recently, it has been shown that mIL-3 can support the growth in vitro of cells which can give rise to CFU-S (972). Furthermore, mIL-3 is the only hemopoietic growth factor known to induce the surface expression of the Thy-1 antigen (87). It is thought that mIL-3 induces the differentiation of mIL-3-responsive, Thy-1 negative stem cells in the bone marrow, which then transiently express Thy-1 and become committed to the various hemopoietic 14 l i n e a g e s (87) . T h e p h y s i o l o g i c a l s i g n i f i c a n c e o f T h y - 1 e x p r e s s i o n o n t h e s e c e l l s r e m a i n s to b e d e t e r m i n e d . T h e T h y - 1 a n t i g e n i s a l s o e x p r e s s e d i n t h e T l y m p h o c y t e l i n e a g e (54) , a n d a r o l e fo r m I L - 3 i n e a r l y T c e l l d i f f e r e n t i a t i o n h a s b e e n h y p o t h e s i z e d . T h i s i s c o n s i s t e n t w i t h t h e d e m o n s t r a t i o n t h a t a p o p u l a t i o n o f c e l l s a p p e a r i n g l a t e i n f e t a l d e v e l o p m e n t w h i c h e x p r e s s 2 0 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e a c t i v i t y a n d T h y - 1 a l s o e x p r e s s T c e l l r e c e p t o r s (87). I n a d d i t i o n , t h e r e a r e l a r g e p o s t - n a t a l i n c r e a s e s i n 2 0 a - h y d r o x y s t e r o i d d e h y d r o g e n a s e a c t i v i t y i n b o t h t h e t h y m u s a n d s p l e e n (s i tes o f T c e l l d e v e l o p m e n t ) , a n d t h e r e i s a r a p i d i n c r e a s e i n t h e f r e q u e n c y o f p e r i p h e r a l T c e l l s w h i c h c a n b e i n d u c e d t o s e c r e t e m I L - 3 a n d o t h e r l y m p h o k i n e s . W h e t h e r t h e s e e f f ec t s a r e a c o n s e q u e n c e o f t h e s t i m u l a t i o n o f a c o m m o n p r o g e n i t o r f o r l y m p h o i d a n d m y e l o i d c e l l s r e m a i n s to b e d e t e r m i n e d . A l s o n o t k n o w n i s w h e t h e r m I L - 3 r e g u l a t e s t h e d i f f e r e n t i a t i o n o f p r o g e n i t o r s o f t h e B c e l l l i n e a g e . R e c e n t l y , h o w e v e r , i t w a s r e p o r t e d t h a t a s e r i e s o f c e l l l i n e s w i t h t h e p h e n o t y p i c p r o p e r t i e s o f e a r l y B c e l l s a s w e l l a s i m m u n o g l o b u l i n g e n e r e a r r a n g e m e n t s r e q u i r e d m I L - 3 f o r g r o w t h i n v i t r o ( 9 8 , 9 9 ) . T h e e x i s t e n c e o f h u m a n i n t e r l e u k i n - 3 (h IL-3) w a s p r e d i c t e d f r o m s t u d i e s w i t h m I L - 3 . b u t i t w a s n o t u n t i l t h e r e c e n t i s o l a t i o n o f t h e h u m a n g e n e , u s i n g a g i b b o n T c e l l - d e r i v e d c D N A i n t e r m e d i a t e , t h a t h I L - 3 w a s i d e n t i f i e d a n d o b t a i n e d i n a p u r i f i e d f o r m ( 1 0 0 ) . T h i s m o l e c u l e i s a g l y c o p r o t e i n w i t h a n a p p a r e n t m o l e c u l a r m a s s o f 2 0 - 2 6 k D a , a g a i n d e p e n d i n g o n t h e e x t e n t o f g l y c o s y l a t i o n . T h e h u m a n g e n e h a s f i v e e x o n s o f s i m i l a r s i z e a n d a r r a n g e m e n t a s i n t h e m u r i n e g e n e , a n d t h e c o d i n g r e g i o n h a s a p p r o x i m a t e l y 45% h o m o l o g y w i t h t h e m I L - 3 g e n e . T h i s s e q u e n c e d i v e r g e n c e i s s u b s t a n t i a l l y g r e a t e r t h a n tha t o b s e r v e d w i t h o t h e r C S F g e n e s , a n d i t h a s b e e n s u g g e s t e d t h a t e a r l y a c t i n g C S F s m a y be m o r e s p e c i e s s p e c i f i c t h a n C S F s a c t i n g o n l i n e a g e - r e s t r i c t e d c e l l s (101) . T h e h I L - 3 g e n e h a s b e e n l o c a l i z e d t o c h r o m o s o m e 5 . P r e l i m i n a r y r e s u l t s i n d i c a t e t h a t h I L - 3 h a s a l a r g e s p e c t r u m o f a c t i v i t i e s , w i t h t a r g e t c e l l s r a n g i n g f r o m p l u r i p o t e n t s t e m c e l l s t o t h e v a r i o u s m a t u r e c o m m i t t e d p r o g e n i t o r s (100) . 15 Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF). A m u r i n e f a c t o r c a p a b l e o f s t i m u l a t i n g g r a n u l o c y t e a n d / o r m a c r o p h a g e c o l o n y f o r m a t i o n w a s f i r s t p u r i f i e d t o h o m o g e n e i t y f r o m l u n g c o n d i t i o n e d m e d i u m ( 1 0 2 ) . T h i s f a c t o r , c a l l e d G M - C S F , i s a g l y c o p r o t e i n w i t h a n a p p a r e n t m o l e c u l a r m a s s o f 2 3 k D a , a l t h o u g h t h e e x a c t s i z e v a r i e s w i t h t h e e x t e n t o f g l y c o s y l a t i o n . G M - C S F s t i m u l a t e s 5 0 % c o l o n y f o r m a t i o n a t a c o n c e n t r a t i o n o f a p p r o x i m a t e l y 1 0 " 1 2 M . T h i s f a c t o r i s f o u n d i n m a n y t i s s u e s a n d i s s y n t h e s i z e d b y a v a r i e t y o f c e l l t y p e s , i n c l u d i n g T c e l l s a n d m e s e n c h y m a l c e l l s (103) . O l i g o n u c l e o t i d e p r o b e s w e r e c o n s t r u c t e d b a s e d o n N - t e r m i n a l s e q u e n c e d a t a f r o m p u r i f i e d G M - C S F ( 1 0 4 ) , a n d t h e s e p r o b e s w e r e u s e d to i s o l a t e c D N A s f o r G M - C S F f r o m a l u n g c e l l - d e r i v e d l i b r a r y ( 1 0 5 ) a n d a T c e l l - d e r i v e d l i b r a r y ( 1 0 6 ) . T h e m a t u r e G M - C S F p o l y p e p t i d e h a s 1 2 4 a m i n o a c i d s a n d h a s a n a p p a r e n t m o l e c u l a r m a s s o f 14 k D a . T h e r e a r e f o u r c y s t e i n e r e s i d u e s w h i c h a r e l i k e l y l i n k e d i n m a n d a t o r y d i s u l p h i d e b r i d g e s , a s t r e a t m e n t w i t h p - m e r c a p t o e t h a n o l d e s t r o y s t h e b i o l o g i c a l a c t i v i t y o f G M - C S F . T h e g e n e for G M - C S F e x i s t s i n s i n g l e - c o p y f o r m , a n d , a s m e n t i o n e d , h a s b e e n l o c a l i z e d to c h r o m o s o m e 11 i n t h e m o u s e (105) . T h e g e n e c o n t a i n s f o u r e x o n s , b u t , a s i n t h e c a s e o f m I L - 3 , t h e r e i s e v i d e n c e f o r t h e e x p r e s s i o n o f a l o n g e r v a r i a n t w h i c h m a y e n c o d e a l a r g e r , c e l l s u r f a c e -b o u n d p r o d u c t (107) . E x p r e s s i o n o f t h e m u r i n e G M - C S F g e n e h a s b e e n a c h i e v e d u s i n g b o t h m o n k e y C O S c e l l s ( 106 ) a n d a b a c t e r i a l e x p r e s s i o n s y s t e m ( 1 0 8 ) . B o t h n a t i v e a n d r e c o m b i n a n t m u r i n e G M - C S F a r e p o t e n t s t i m u l a t o r s o f g r a n u l o c y t e a n d m a c r o p h a g e c o l o n y f o r m a t i o n i n v i t r o , a n d , a t h i g h e r c o n c e n t r a t i o n s , c a n s t i m u l a t e e o s i n o p h i l a n d m e g a k a r y o c y t e c o l o n y f o r m a t i o n ( 8 6 , 1 0 8 ) . T h e s e a c t i o n s a p p e a r to i n v o l v e d i r e c t s t i m u l a t i o n o f p r o g e n i t o r c e l l s . G M - C S F a l s o e n h a n c e s t h e f o r m a t i o n o f m i x e d c o l o n i e s a n d c o l o n i e s d e r i v e d f r o m p r i m i t i v e B F U - E , b u t o n l y w h e n e r y t h r o p o i e t i n i s a d d e d to t h e c u l t u r e ( 1 0 3 ) . T h e r e f o r e , t h i s m a y c o n s t i t u t e a n i n d i r e c t a c t i o n o n C F U - G E M M a n d B F U - E , o r o n e t h a t r e q u i r e s t h e p r e s e n c e o f o t h e r f a c t o r s (73) . I n c o m p a r i s o n w i t h m I L - 3 , t h e n , m u r i n e G M - C S F i s a m o r e p o t e n t s t i m u l a t o r o f g r a n u l o c y t e - m a c r o p h a g e c o l o n i e s , a n d a l e s s p o t e n t s t i m u l a t o r o f m i x e d c o l o n i e s a n d e r y t h r o i d b u r s t s . 16 M u r i n e GM-CSF has also been shown to induce a number of f u n c t i o n a l changes i n m a t u r e c e l l s (72,86). It i n h i b i t s n e u t r o p h i l m i g r a t i o n a n d i s a p o t e n t a c t i v a t o r of n e u t r o p h i l s a n d eosinophils. In addition, it increases n e u t r o p h i l phagocytic activity and i n c r e a s e s t h e e x p r e s s i o n of a f a m i l y of c e l l a d h e s i o n m o l e c u l e s o n the s u r f a c e s of neutrophils.. T h e h u m a n f o r m of G M - C S F was f i r s t p u r i f i e d to h o m o g e n e i t y f r o m m e d i u m conditioned by the h u m a n T-lymphotropic v i r u s II (HTLV-II) infected T-lymphoblast cell line Mo (109). The gene for h u m a n GM-CSF was cloned by c o n s t r u c t i n g a Mo ce l l l i n e c DNA library, transfecting monkey COS cells, and screening for transient expression of GM-CSF (110). The gene is present i n single-copy form, and encodes a mature protein of 127 amino acids. The apparent molecular mass is 14-35 k b a , due to variable glycosylation. There is 6 0 % sequence identity to murine GM-CSF at the amino acid level and 7 0 % homology at the nuc l e o t i d e level, a l t h o u g h there is no cross-species r e a c t i v i t y (111). The gene has been l o c a l i z e d to c h r o m o s o m e 5 (112), a n d i s deleted i n the 5q~ syndrome. The b i o l o g i c a l p r o p e r t i e s of h u m a n GM-CSF are very s i m i l a r to those d e s c r i b e d for m u r i n e GM-CSF (109,111,113,114). There is no evidence to date of a role for either m u rine or h u m a n GM-C S F i n the regulation of lymphoid progenitors. 2.1.2. Class II Factors G r a n u l o c y t e C o l o n y - S t i m u l a t i n g F a c t o r ( G - C S F ) . Both murine and h u m a n forms of G-CSF have been identified and purified to homogeneity. M u r i n e G-CSF, a glycoprotein of apparent molecular mass 25 kDa, is synthesized i n vitro by a wide range of t i s s u e s and has been p u r i f i e d from mouse l u n g c o n d i t i o n e d m e d i u m (115). H u m a n G-CSF was first p u r i f i e d to a p p a r e n t h o m o g e n e i t y f r o m m e d i u m c o n d i t i o n e d b y the h u m a n b l a d d e r c a r c i n o m a c e l l l i n e 5 6 3 7 (116), a n d h a s a n a p p a r e n t m o l e c u l a r m a s s of 19.6 k D a Treatment of the molecule with the enzyme O-glycanase reduced the molecular mass from 19.6 k D a to 18.8 kDa, s u g g e s t i n g t h a t the m o l e c u l e is O-glycosylated (117). I n t e r n a l di s u l p h i d e bridges appear to be required for biological activity of both forms of G-CSF, and 17 t h e y a r e a c t i v e a t p i c o m o l a r c o n c e n t r a t i o n s . T h e g e n e f o r h u m a n G - C S F ( 1 1 8 ) , b u t n o t m u r i n e G - C S F , h a s b e e n c l o n e d a n d a p p e a r s to b e p r e s e n t i n s i n g l e - c o p y f o r m . P u r i f i e d m u r i n e a n d h u m a n G - C S F s h o w a l m o s t c o m p l e t e c r o s s - r e a c t i v i t y , i n t e r m s o f r e c e p t o r - b i n d i n g a n d b i o l o g i c a l a c t i v i t y , w h e n n o r m a l a n d l e u k e m i c m u r i n e a n d h u m a n c e l l s a r e t e s t e d ( 1 1 9 ) . A t l o w to m e d i u m c o n c e n t r a t i o n s , b o t h f o r m s s t i m u l a t e e x c l u s i v e l y t h e f o r m a t i o n o f g r a n u l o c y t e c o l o n i e s . A t h i g h e r c o n c e n t r a t i o n s , G - C S F a p p e a r s t o s t i m u l a t e a f e w d i v i s i o n s o f G M p r o g e n i t o r s ( 7 3 , 8 6 ) . I n t h e p r e s e n c e o f e r y t h r o p o i e t i n , h u m a n G - C S F m a y i n d i r e c t l y i n d u c e t h e f o r m a t i o n o f c o l o n i e s d e r i v e d f r o m B F U - E a s w e l l a s m i x e d c o l o n i e s ( 1 1 6 ) . M u r i n e G - C S F h a s b e e n s h o w n to h a v e a p o w e r f u l c a p a c i t y to i n d u c e t e r m i n a l d i f f e r e n t i a t i o n i n W E H I - 3 B m y e l o m o n o c y t i c l e u k e m i c c e l l s ( 1 1 5 ) , a n d a l s o s t i m u l a t e s a n t i b o d y d e p e n d e n t c e l l - m e d i a t e d c y t o t o x i c i t y o f b o t h m u r i n e a n d h u m a n n e u t r o p h i l s (120) . M a c r o p h a g e C o l o n y - S t i m u l a t i n g F a c t o r ( M - C S F ) . M o u s e M - C S F ( a l s o c a l l e d C S F -1) c a n b e s y n t h e s i z e d b y a v a r i e t y o f c e l l t y p e s , i n c l u d i n g f i b r o b l a s t s , e m b r y o n i c c e l l s , a n d u t e r i n e c e l l s (86). It w a s p u r i f i e d to h o m o g e n e i t y f r o m L c e l l c o n d i t i o n e d m e d i u m (121) , a n d i s a d i m e r o f a p p a r e n t m o l e c u l a r m a s s 7 0 k D a . T h e m o l e c u l e c o n s i s t s o f i d e n t i c a l 3 5 k D a s u b u n i t s w i t h c a r b o h y d r a t e a c c o u n t i n g fo r a l l b u t 14 k D a . T h e s u b u n i t s h a v e n o b i o l o g i c a l a c t i v i t y a n d d o n o t b i n d a n t i - M - C S F s e r u m . S i m i l a r l y , h u m a n M - C S F i s a g l y c o p r o t e i n o l t w o i d e n t i c a l 3 5 - 3 8 k D a s u b u n i t s , a n d h a s b e e n p u r i f i e d f r o m u r i n e ( 1 2 2 ) . It c a n a l s o be s y n t h e s i z e d b y a v a r i e t y o f t i s s u e s . T h e g e n e for h u m a n M - C S F h a s b e e n c l o n e d (123) , a n d h a s b e e n l o c a l i z e d t o t h e l o n g a r m o f c h r o m o s o m e 5 . T h e r e i s e v i d e n c e f o r d i f f e r e n t i a l p r o c e s s i n g o f p r i m a r y t r a n s c r i p t s , a n d f o r t h e e x i s t e n c e o f m e m b r a n e - b o u n d , a s w e l l a s s e c r e t e d f o r m s o f M - C S F (123) . B o t h m u r i n e a n d h u m a n M - C S F s t i m u l a t e p r e d o m i n a n t l y m a c r o p h a g e p r o g e n i t o r s ( 7 2 , 124 ) . T h e h u m a n f o r m i s a c t i v e o n m o u s e c e l l s , b u t m u r i n e M - C S F d o e s n o t a p p e a r to b e a c t i v e i n t h e h u m a n s y s t e m . It h a s a l s o b e e n d o c u m e n t e d t h a t m u r i n e M - C S F h a s the a b i l i t y to s t i m u l a t e a s m a l l p e r c e n t a g e o f g r a n u l o c y t e p r o g e n i t o r s (72), a l t h o u g h t h i s a c t i o n m a y b e i n d i r e c t ( 7 3 ) . W h e n m u r i n e M - C S F i s c o m b i n e d w i t h t h e h u m a n c y t o k i n e 18 h e m o p o i e t i r i - 1 , w h i c h i s i t s e l f n o t a C S F ( a n d i s n o w k n o w n to b e i d e n t i c a l to i n t e r l e u k i n - 1 ( 1 2 4 ) ) . i t s t i m u l a t e s t h e f o r m a t i o n o f v e r y l a r g e m a c r o p h a g e c o l o n i e s ( 1 2 6 ) . T h e r e f o r e , h u m a n M - C S F m a y r e q u i r e t h e p r e s e n c e o f o t h e r f a c t o r s f o r o p t i m a l s t i m u l a t i o n o f h u m a n t a r g e t c e l l s . It h a s a l s o b e e n s h o w n t h a t n a t i v e m u r i n e M - C S F i n c r e a s e s p l a s m i n o g e n -d e p e n d e n t f i b r i n o l y t i c a c t i v i t y o f m a t u r e m o u s e p e r i t o n e a l m a c r o p h a g e s (127) . E r y t h r o p o i e t i n (Ep). B o t h m u r i n e a n d h u m a n E p h a v e b e e n i d e n t i f i e d , b u t b e c a u s e h u m a n E p c a n b e r e a d i l y p u r i f i e d f r o m p a t i e n t s w i t h a p l a s t i c a n e m i a ( 1 2 8 , 1 2 9 ) a n d i s a c t i v e o n a w i d e v a r i e t y o f s p e c i e s , i n c l u d i n g m i c e , t h e m a j o r i t y o f s t u d i e s h a v e b e e n p e r f o r m e d w i t h h u m a n E p . T h i s f a c t o r i s a t r u e c i r c u l a t i n g h o r m o n e t h a t i s m o s t l i k e l y p r o d u c e d b y t u b u l a r c e l l s w i t h i n t h e k i d n e y ( 1 3 0 , 1 3 1 ) . It h a s a n a p p a r e n t m o l e c u l a r m a s s o f 3 4 - 3 9 k D a ( c o m p a r e d to 3 4 - 3 6 k D a f o r t h e m u r i n e f ac to r ) , w i t h t h e p o l y p e p t i d e b a c k b o n e c o n t r i b u t i n g 1 8 . 4 k D a , a n d t h e r e s t b y b o t h N - l i n k e d a n d O - l i n k e d s u g a r r e s i d u e s . T h e r e a r e f o u r c y s t e i n e r e s i d u e s , a t l e a s t t w o o f w h i c h a r e i n v o l v e d i n d i s u l p h i d e b o n d s a s b i o l o g i c a l a c t i v i t y i s l o s t o n r e d u c t i o n . T h e g e n e f o r h u m a n E p h a s b e e n c l o n e d a n d e x p r e s s e d i n m o n k e y C O S c e l l s (132) . O n l y a s i n g l e g e n e h a s b e e n d e t e c t e d , a n d h a s b e e n l o c a l i z e d t o c h r o m o s o m e 7 q l l - q 2 2 ( 1 3 3 ) . N a t i v e a n d r e c o m b i n a n t E p s t i m u l a t e t h e p r o l i f e r a t i o n a n d t e r m i n a l d i f f e r e n t i a t i o n o f C F U - E a n d a s u b s e t o f r e l a t i v e l y m a t u r e B F U - E ( 1 3 4 - 1 3 5 ) . It a p p e a r s t h a t a s e c o n d s u b s e t o f B F U - E , p r e s u m a b l y l e s s m a t u r e , c a n s u r v i v e i n t h e a b s e n c e o f E p i f i n t e r l e u k i n - 3 o r G M - C S F i s p r e s e n t ( 1 3 6 ) . T h i s p o p u l a t i o n t h e n p r o l i f e r a t e s a n d t e r m i n a l l y d i f f e r e n t i a t e s i f E p i s a d d e d to t h e c u l t u r e s . I n t e r l e u k i n - 5 (IL-5). M u r i n e I L - 5 w a s i n i t i a l l y d e s c r i b e d a s a g r o w t h a n d d i f f e r e n t i a t i o n f a c t o r fo r m o u s e B c e l l s (137) , a n d w a s g i v e n t h e n a m e B c e l l g r o w t h f a c t o r II ( B C G F - I I ) . It h a s a n a p p a r e n t m o l e c u l a r m a s s o f 4 5 - 6 0 k D a . a n d i t s g e n e h a s b e e n c l o n e d ( 1 3 8 ) . I n t e r l e u k i n - 5 i s i n c l u d e d i n t h e p r e s e n t d i s c u s s i o n b e c a u s e , i n a d d i t i o n to s t i m u l a t i n g B c e l l s , i t i n d u c e s t h e d i f f e r e n t i a t i o n o f b o n e m a r r o w p r o g e n i t o r c e l l s i n t o e o s i n o p h i l s (138) . T h e f a c t o r h a s t h e r e f o r e a l s o b e e n c a l l e d e o s i n o p h i l d i f f e r e n t i a t i o n f a c t o r ( E D F ) . T h e g e n e f o r h u m a n I L - 5 h a s r e c e n t l y b e e n c l o n e d b y t w o g r o u p s ( 1 3 9 , 1 4 0 ) , a n d the r e c o m b i n a n t h u m a n f a c t o r a l s o s t i m u l a t e s f o r m a t i o n o f e o s i n o p h i l c o l o n i e s (139) . 19 2.1.3. C l a s s HI Factors W i t h i n the context of the hemopoietic system, the major role of factors belonging to t h i s c l a s s i s to synergize w i t h one or more of the hemopoietic growth f a c t o r s d e s c r i b e d above. C l a s s III factors have no i n t r i n s i c c o l o n y - s t i m u l a t i n g a c t i v i t y b u t can, by poorly u n d e r s t o o d m e c h a n i s m s , enhance c o l o n y f o r m a t i o n i n response to the CSFs. Some of these f a c t o r s are now de s c r i b e d , a l t h o u g h the l i s t w i l l l i k e l y grow as more s y n e r g i s t i c activities are discovered. Interleukin-1 (JX-1). IL-1 is produced p r i m a r i l y by monocytes (141) and endothelial c e l l s (142), a n d Is a key m e d i a t o r of the host response to i n f e c t i o u s , i n f l a m m a t o r y and immunologic challenges. Murine (143,144) and h u m a n (145) IL-1 have both been cloned, have s i m i l a r activities, and are both cross-reactive. Two forms of the molecule (IL-1 a and IL-lp) have been described; both are 22 k D a polypeptides, b i n d to the same receptor, and share most if not a l l biological activities. IL-1 induces the proliferation of activated T cells as w e l l as m a t u r a t i o n a n d f u n c t i o n a l a c t i v a t i o n of T helper c e l l s , cytotoxic T cells , and B cells (146). In a d d i t i o n to s t i m u l a t i n g the secretion of acute phase proteins, it induces the m o b i l i z a t i o n a n d a c t i v a t i o n of n e u t r o p h i l s , a n d appears to be i d e n t i c a l to endogenous pyrogen (thus c o n t r i b u t i n g to fever, headaches and body pa i n s commonly accompanying infections) (147). K n o w n to i n d u c e the s e c r e t i o n of platelet-derived growth factor (PDGF) an d nerve growth factor (NGF) from various mesenchymal cell types, IL-1 has recently been shown to s t i m u l a t e the s e c r e t i o n of G M -CSF a n d G-CSF f r o m f i b r o b l a s t s a n d e n d o t h e l i a l c e l l s (64-69). F u r t h e r m o r e , IL-1 synergizes w i t h GM-CSF and M-CSF i n s t i m u l a t i n g colony formation (148), perhaps by increasing cell surface expression of receptors for these factors. Also, it is now known that I L - l a is identical to hemopoietin 1 (125), a previously described s y n e r g i s t i c f a c t o r w h i c h i n c r e a s e s the r e s p o n s i v e n e s s of stem c e l l s to 11-3 a n d of early-progenitor cells to later-acting C S F s (149). Interleukin-4 (JX-4). Produced by activated T cells, IL-4 was ori g i n a l l y identified on the b a s i s of i t s a b i l i t y to s t i m u l a t e M H C c l a s s II e x p r e s s i o n as w e l l as IgG a n d IgE 2 0 p r o d u c t i o n i n B c e l l s , a n d to a c t a s a g r o w t h f a c t o r f o r T c e l l s a n d m a s t c e l l s ( 1 5 0 ) . A l s o c a l l e d B c e l l s t i m u l a t i n g f a c t o r - 1 ( B S F - 1 ) , i t h a s a n a p p a r e n t m o l e c u l a r m a s s o f 1 5 - 2 0 k D a , a n d t h e g e n e s f o r b o t h m u r i n e ( 1 5 1 ) a n d h u m a n ( 1 5 2 ) f o r m s h a v e b e e n c l o n e d . R e c e n t s t u d i e s h a v e s h o w n t h a t I L - 4 c a n s y n e r g i z e w i t h 11-3 i n s t i m u l a t i n g t h e p r o l i f e r a t i o n o f p r i m i t i v e p r o g e n i t o r c e l l s ( 1 5 3 ) , a n d w i t h G - C S F i n t h e f o r m a t i o n o f g r a n u l o c y t e c o l o n i e s (148) . Interleukin-6 (IL-6). I L - 6 h a s p r e v i o u s l y b e e n r e f e r r e d to b y a n u m b e r o f d i f f e r e n t n a m e s : i n t e r f e r o n P2 (IFNP2) b e c a u s e o f i t s a n t i v i r a l a c t i v i t y (154) a n d c o - p u r i f i c a t i o n w i t h i n t e r f e r o n (3-^  ( 1 5 4 ) , h y b r i d o m a g r o w t h f a c t o r ( H G F ) b e c a u s e o f i t s g r o w t h s t i m u l a t i n g effect o n c e r t a i n B c e l l h y b r i d o m a s a n d p l a s m a c y t o m a s ( 1 5 5 ) , a n d B c e l l s t i m u l a t o r y f a c t o r - 2 ( B S F - 2 ) b e c a u s e o f i t s o r i g i n a l l y d e f i n e d r o l e a s a B c e l l d i f f e r e n t i a t i o n f a c t o r ( 1 5 6 ) . T h e s e a c t i v i t i e s w e r e a l l f o u n d to r e s i d e i n t h e s a m e p o l y p e p t i d e , w h i c h w a s r e c e n t l y n a m e d I L - 6 . T h i s 2 6 k D a f a c t o r i s p r o d u c e d b y T c e l l s , f i b r o b l a s t s , a n d a c t i v a t e d m o n o c y t e s . T h e g e n e s f o r b o t h m u r i n e (157) a n d h u m a n (156) I L - 6 h a v e b e e n c l o n e d . H u m a n I L - 6 h a s r e c e n t l y b e e n s h o w n to s y n e r g i z e w i t h 11-3 i n s t i m u l a t i n g t h e f o r m a t i o n o f b l a s t c e l l c o l o n i e s f r o m m o u s e s p l e e n c e l l s o r b o n e m a r r o w ( 1 5 8 ) , p o s s i b l y b y s h o r t e n i n g t h e G ^ p h a s e o f t h e c e l l c y c l e i n c e l l s s t i m u l a t e d b y 11-3. TC-1 synergistic activity. A f a c t o r r e c e n t l y i s o l a t e d f r o m a m u r i n e m a r r o w a d h e r e n t c e l l l i n e , T C - 1 . d i s p l a y s t h e a b i l i t y to s y n e r g i z e w i t h M - C S F i n sof t a g a r c u l t u r e to p r o d u c e g i a n t m a c r o p h a g e c o l o n i e s ( 1 5 9 ) . T h i s f a c t o r a l s o a p p e a r s to s y n e r g i z e w i t h m u r i n e 11-3 a n d p a r t i a l l y p u r i f i e d G M - C S F , a l t h o u g h i t h a s n o c o l o n y - s t i m u l a t i n g a c t i v i t y i t s e l f . It a p p e a r s t o b e a g l y c o p r o t e i n s i n c e i t b i n d s t o C o n c a n a v a l i n A , a n d h a s a n a p p a r e n t m o l e c u l a r m a s s o f 6 0 k D a a s d e t e r m i n e d b y S e p h a r o s e G 1 0 0 g e l f i l t r a t i o n c h r o m a t o g r a p h y . Mesenchymal cell activators. A l t h o u g h n o t d i r e c t l y s y n e r g i s t i c , s e v e r a l p o l y p e p t i d e m o l e c u l e s l a c k i n g i n t r i n s i c c o l o n y - s t i m u l a t i n g a c t i v i t y c a n i n d i r e c t l y e n h a n c e c o l o n y f o r m a t i o n i n t h e h e m o p o i e t i c s y s t e m . T h e s e f a c t o r s a r e o f t e n p r o d u c e d d u r i n g i n f l a m m a t o r y r e a c t i o n s , a n d a c t i v a t e v a r i o u s m e s e n c h y m a l c e l l t y p e s t o p r o d u c e a n d 2 1 s e c r e t e C S F s . T h e a b i l i t y o f I L - 1 to f u n c t i o n a s a m e s e n c h y m a l c e l l a c t i v a t o r h a s b e e n d e s c r i b e d a b o v e ; o t h e r f a c t o r s w i t h t h i s p r o p e r t y i n c l u d e p l a t e l e t - d e r i v e d g r o w t h f a c t o r ( P D G F ) a n d t u m o r n e c r o s i s f a c t o r - a ( T N F - a ) . P D G F i s a g l y c o p r o t e i n h e t e r o d i m e r o f 3 0 k D a s u b u n i t s , o n e o f w h i c h ( t h e B c h a i n ) i s i d e n t i c a l t o t h e c - s i s o n c o g e n e p r o d u c t ( 1 6 0 ) . E n d o t h e l i a l c e l l s a n d m a c r o p h a g e s a c t i v a t e d b y e n d o t o x i n , T N F - a , o r p h o r b o l e s t e r s p r o d u c e P D G F , a s d o p l a t e l e t s a f t e r a d h e s i o n . A m o n g i t s m a n y a c t i v i t i e s , P D G F i s t h o u g h t t o p l a y a k e y r o l e i n t h e w o u n d h e a l i n g p r o c e s s ( 1 6 1 ) . It i s m i t o g e n i c fo r c o n n e c t i v e t i s s u e c e l l s , w h i c h a r e t h o u g h t to b e t h e p r i m a r y t a r g e t o f P D G F a c t i o n . I n a d d i t i o n , P D G F h a s r e c e n t l y b e e n s h o w n to e n h a n c e i n v i t r o c o l o n y f o r m a t i o n b y s t i m u l a t i n g m e s e n c h y m a l c e l l s to s e c r e t e G M - C S F (162) . T u m o r n e c r o s i s f a c t o r - a , a l s o c a l l e d c a c h e c t i n , w a s o r i g i n a l l y i d e n t i f i e d b y i t s a b i l i t y to m e d i a t e h e m o r r h a g i c n e c r o s i s o f t u m o r s i n r e c i p i e n t a n i m a l s e x p o s e d to e n d o t o x i n (163) . A c t i v a t e d m o n o c y t e s a re t h e m a i n s o u r c e of T N F - a , a n d m o n o c y t e s a n d e n d o t h e l i a l c e l l s are t h e p r i n c i p a l t a r g e t c e l l s . T h e f a c t o r i s a g l y c o p r o t e i n w i t h a n a p p a r e n t m o l e c u l a r m a s s o l 17 k D a . A m o n g i t s m a n y a c t i v i t i e s ( 1 6 4 ) , T N F - a a p p e a r s t o m e d i a t e e n d o t o x i n - i n d u c e d s h o c k . It h a s a l s o r e c e n t l y b e e n s h o w n to i n d u c e m e s e n c h y m a l c e l l s to s e c r e t e G - C S F a n d G M - C S F ( 1 6 5 , 1 6 6 ) . I n t e r e s t i n g l y , i t h a s a l s o b e e n r e p o r t e d t h a t G M - C S F s t i m u l a t e s e n h a n c e d s e c r e t i o n o f T N F - a f r o m h u m a n m o n o c y t e s ( 1 6 7 ) , a n d t h a t h u m a n G M - C S F a n d 11-3 s t i m u l a t e m o n o c y t e c y t o t o x i c i t y t h r o u g h a t u m o r n e c r o s i s f a c t o r - d e p e n d e n t m e c h a n i s m (168) . 2 . 1 . 4 . C l a s s I V F a c t o r s T h e p o s s i b l e i n v o l v e m e n t o f i n h i b i t o r y f a c t o r s i n t h e h e m o p o i e t i c s y s t e m h a s b e e n p o s t u l a t e d f o r m a n y y e a r s b u t r e m a i n s a c o n t r o v e r s i a l i s s u e , a l t h o u g h i t s e e m s l i k e l y t h a t h e m o p o i e s i s i s a c c o m p l i s h e d b y a b a l a n c e o f p o s i t i v e a n d n e g a t i v e i n f l u e n c e s . A n u m b e r o l f a c t o r s h a v e b e e n i d e n t i f i e d t h a t h a v e p o t e n t i a l p h y s i o l o g i c a l r o l e s a s n e g a t i v e r e g u l a t o r s o l h e m o p o i e s i s , e i t h e r b y d i r e c t l y i n h i b i t i n g p r o g e n i t o r s o r b y m o d u l a t i n g C S F s e c r e t i o n S o m e y e a r s a g o , t h e g l y c o p r o t e i n l a c t o f e r r i n w a s i m p l i c a t e d i n t h e r e g u l a t i o n o f g r a n u l o p o i e s i s , a s i t w a s s h o w n t o h a v e a p o t e n t i n h i b i t o r y e f f e c t o n m o n o c y t i c C S F 2 2 p r o d u c t i o n i n v i t r o ( 1 6 9 ) . H o w e v e r , b e c a u s e t h e c o n c e n t r a t i o n r e q u i r e d f o r t h i s i n h i b i t i o n w a s m u c h l o w e r t h a n t h a t n o r m a l l y f o u n d i n p l a s m a , t h e s i g n i f i c a n c e o f l a c t o f e r r i n i n v i v o r e m a i n e d u n c l e a r ( 1 7 0 ) . H o w e v e r , a m o r e r e c e n t r e p o r t h a s d e m o n s t r a t e d t h a t t h e i n v i v o a d m i n i s t r a t i o n o f l a c t o f e r r i n d e c r e a s e s t h e n u m b e r o f m u l t i p o t e n t i a l , e r y t h r o i d , a n d g r a n u l o c y t i c p r e c u r s o r s i n m u r i n e m a r r o w a n d s p l e e n , a n d a r r e s t s t h e t u r n o v e r o f t h e s e n o r m a l l y c y c l i n g p r o g e n i t o r s (171) . E - t y p e p r o s t a g l a n d i n s ( P G E ) h a v e a l s o b e e n r e p o r t e d to i n f l u e n c e g r a n u l o c y t i c p r o g e n i t o r s , i n t h i s c a s e b y d e c r e a s i n g t h e r e s p o n s i v e n e s s o f t h e s e c e l l s t o G M - C S F ( 1 7 2 ) . P G E i s a l s o a p o t e n t i n h i b i t o r o f C S F - i n d u c e d m o n o c y t e p r o l i f e r a t i o n (173) . A s e r i e s o f r e p o r t s i n t h e l a t e 1 9 7 0 s s u g g e s t e d t h e e x i s t e n c e o f t w o m a r r o w f a c t o r s w i t h o p p o s i n g e f f e c t s , o n e i n h i b i t o r y a n d o n e s t i m u l a t o r y , o n t h e p r o l i f e r a t i v e a c t i v i t y o f C F U - S ( 1 7 4 - 1 7 6 ) . T h e i n h i b i t o r y f a c t o r , w i t h a n a p p a r e n t m o l e c u l a r m a s s i n t h e 5 0 - 1 0 0 k D a r a n g e , w a s i s o l a t e d f r o m m u r i n e m a r r o w i n w h i c h t h e m a j o r i t y o f C F U - S w e r e q u i e s c e n t . It w a s a l s o s h o w n to p r o t e c t C F U - S f r o m t h e l e t h a l e f f e c t s o f h i g h s p e c i f i c a c t i v i t y ^ H - t h y m i d i n e . T h e s t i m u l a t o r y a c t i v i t y , o n t h e o t h e r h a n d , w a s i s o l a t e d f r o m r e g e n e r a t i n g m u r i n e m a r r o w i n w h i c h a h i g h p r o p o r t i o n o f C F U - S w e r e i n S - p h a s e . F u r t h e r m o r e , t h i s s m a l l e r ( 3 0 - 5 0 k D a ) f a c t o r c o u l d r e v e r s e t h e e f f e c t s o f t h e i n h i b i t o r y m o l e c u l e . It w a s t h e r e f o r e s u g g e s t e d t h a t t h e r e l a t i v e c o n c e n t r a t i o n s o f t h e s e t w o f a c t o r s in v i v o c o u l d d e t e r m i n e t h e l e v e l o f s t e m c e l l p r o l i f e r a t i o n (177) . A l s o i m p l i c a t e d i n t h e r e g u l a t i o n o f p r i m i t i v e h e m o p o i e t i c c e l l s a r e i n t e r f e r o n - y (IFN-v) a n d t r a n s f o r m i n g g r o w t h f a c t o r - P ( T G F - P ) . I N F - y i s a l y m p h o k i n e w i t h a n a p p a r e n t m o l e c u l a r m a s s o f 3 8 - 8 0 k D a i n m o u s e a n d 2 0 - 2 5 k D a i n h u m a n s ( 1 7 8 ) . A m o n g i t s m a n y a c t i v i t i e s ( 1 7 8 ) , I F N - y a c t i v a t e s t h e c y t o c i d a l a c t i v i t y o f m a c r o p h a g e s a n d h a s b e e n c a l l e d m a c r o p h a g e a c t i v a t i n g f a c t o r ( M A F ) . M o r e r e c e n t l y , i t h a s b e e n s h o w n t o e x e r t a n i n h i b i t i n g e f fec t o n m y e l o p o i e s i s i n v i t r o a n d s t e m c e l l p r o d u c t i o n i n l o n g - t e r m m a r r o u ' c u l t u r e s ( 1 7 9 , 1 8 0 ) . T G F - P i s a 2 5 k D a h o m o d i m e r w h i c h is p r e s e n t i n m a n y t i s s u e s . It h a s a w i d e s p e c t r u m o f a c t i v i t i e s , a n d h a s b e e n i m p l i c a t e d i n t h e r e g u l a t i o n o f w o u n d h e a l i n g , b o n e f o r m a t i o n , e m b r y o g e n e s i s , c e l l g r o w t h , a n d e x p r e s s i o n o f r e c e p t o r s fo r c e l l a d h e s i o n 2 3 p r o t e i n s ( 1 8 1 ) . T w o f o r m s o f T G F - p w i t h s i m i l a r b i o l o g i c a l a c t i v i t i e s h a v e b e e n i d e n t i f i e d a n d h a v e b e e n c a l l e d TGF-P i a n d TGF-P 2 . A r e c e n t r e p o r t h a s d e m o n s t r a t e d t h a t c e r t a i n m I L - 3 - d e p e n d e n t c e l l l i n e s b e a r r e c e p t o r s f o r b o t h T G F - p ^ a n d T G F - p 2 ( 1 5 5 ) . I n a d d i t i o n , t h e f a c t o r - d e p e n d e n t p r o l i f e r a t i o n o f t h e s e c e l l s i s p o t e n t l y i n h i b i t e d b y e x p o s u r e to TGF-P, ( 1 5 5 ) . F u r t h e r e v i d e n c e f o r a n i n h i b i t o r y r o l e o f T G F - p i n h e m o p o i e s i s c o m e s f r o m t h e d e m o n s t r a t i o n t h a t TGF-P ± c a n d i r e c t l y a n d r e v e r s i b l y i n h i b i t t h e c y c l i n g o f p r i m i t i v e m y e l o i d p r o g e n i t o r c e l l s i n b o t h m o u s e a n d h u m a n b o n e m a r r o w (182) . T w o v e r y r e c e n t l y d e s c r i b e d i n h i b i t o r y p r o t e i n s i n c l u d e n e g a t i v e r e g u l a t o r y p r o t e i n ( N R P ) a n d C F U - S i n h i b i t o r y p e p t i d e A c S D k P . N R P i s a n a p p a r e n t l y p h y s i o l o g i c a l , r a p i d a n d r e v e r s i b l e i n h i b i t o r o f D N A s y n t h e s i s i n t h e e a r l y e r y t h r o i d p r o g e n i t o r s ( B F U - E ) o f m u r i n e m a r r o w (183) . A c S D k P a p p e a r s t o i n h i b i t D N A s y n t h e s i s o f m u r i n e p l u r i p o t e n t h e m o p o i e t i c s t e m c e l l s , b o t h i n v i v o a n d i n v i t r o (184) . It i s r e a d i l y a p p a r e n t f r o m t h e a b o v e d i s c u s s i o n t h a t a l a r g e n u m b e r o f m o l e c u l e s h a v e p o t e n t i a l r e g u l a t o r y a c t i v i t i e s w i t h i n t h e h e m o p o i e t i c s y s t e m , i n c l u d i n g f a c t o r s o t h e r t h a n t h e C S F s . H o w e v e r , a f u l l e r u n d e r s t a n d i n g o f t h e r e g u l a t i o n o f h e m o p o i e s i s b y t h e s e a n d a s y e t u n d i s c o v e r e d m o l e c u l e s w i l l r e q u i r e n o t o n l y a k n o w l e d g e o f t h e i r i n d i v i d u a l a c t i v i t i e s b u t a l s o a n a p p r e c i a t i o n o f t h e ef fec ts o f c o m b i n a t i o n s o f r e g u l a t o r y a g e n t s , a s i s l i k e l y t o b e t h e s i t u a t i o n i n v i v o . A l s o , l i t t l e m e n t i o n h a s b e e n m a d e o f f a c t o r s r e g u l a t i n g l y m p h o p o i e s i s . S i n c e m a n y o f t h e h e m o p o i e t i c g r o w t h f a c t o r s d e s c r i b e d a r e l y m p h o k i n e s , it s e e m s l i k e l y t h a t t h e r e g u l a t i o n o f h e m o p o i e s i s a l s o d e p e n d s o n t h e s t a t u s o f T c e l l p o p u l a t i o n s p r o d u c i n g t h e s e l y m p h o k i n e s . M o r e o v e r , m o s t o f t h e b i o l o g i c a l e f f e c t s d e s c r i b e d a b o v e h a v e b e e n d e m o n s t r a t e d u s i n g i n v i t r o s y s t e m s , w h i c h d o e s n o t p r o v e t h a t t h e f a c t o r s h a v e s i m i l a r r o l e s i n v i v o . W i t h t h e d e v e l o p m e n t o f r e c o m b i n a n t b a c t e r i a l l y s y n t h e s i z e d h e m o p o i e t i c g r o w t h f a c t o r s w i t h f u l l i n v i v o b i o l o g i c a c t i v i t y , m a n y i n v e s t i g a t i o n s i n t o t h e i n v i v o a c t i o n s o f t h e s e m o l e c u l e s h a v e b e e n i n i t i a t e d , a n d s o m e o f t h e s e s t u d i e s i n v o l v i n g C S F s a r e n o w d e s c r i b e d . 2 4 2.2. I n v i v o a c t i o n s o f t h e C S F s S t u d i e s i n t o t h e r o l e o f t h e C S F s i n v i v o w e r e i n i t i a l l y p e r f o r m e d u s i n g p a r t i a l l y p u r i f i e d o r p u r i f i e d n a t i v e m o l e c u l e s . T h e f i r s t h e m o p o i e t i c g r o w t h f a c t o r s h o w n t o h a v e a n e f fec t i n v i v o w a s e r y t h r o p o i e t i n . T h i s w a s d e m o n s t r a t e d b y s h o w i n g t h a t e r y t h r o i d c e l l p r o l i f e r a t i o n , h e m o g l o b i n a c c u m u l a t i o n , a n d d e v e l o p m e n t o f m a t u r e r e d b l o o d c e l l n u m b e r s i n t h e c i r c u l a t i o n w e r e r e l a t e d t o t h e i n j e c t e d l e v e l o f t h i s h o r m o n e ( 1 8 5 ) . S u b s e q u e n t l y , i t w a s s h o w n t h a t i n j e c t i o n o f p a r t i a l l y p u r i f i e d C S F f r o m h u m a n u r i n e i n t o n o r m a l a d u l t m i c e c a u s e d a m o d e r a t e i n c r e a s e i n g r a n u l o c y t e a n d m o n o c y t e f o r m a t i o n (186) . I n j e c t i o n o f p u r i f i e d C S F f r o m t h i s s o u r c e i n t o l e u k o p e n i c p a t i e n t s c a u s e d a s l i g h t l y a c c e l e r a t e d r e c o v e r y o f l e u k o c y t e l e v e l s (187) . L a t e r , i t w a s s h o w n t h a t t h e i n f u s i o n o f e x o g e n o u s p u r e 11-3 i n t o m i c e m a r k e d l y i n c r e a s e d t h e r a t e o f s t e m c e l l p r o l i f e r a t i o n a n d c a u s e d a n i n c r e a s e o f b o t h s t e m c e l l s a n d c o m m i t t e d p r o g e n i t o r c e l l s i n t h e s p l e e n (188) . M o r e r e c e n t l y , t h e a v a i l a b i l i t y o f r e c o m b i n a n t b a c t e r i a l l y s y n t h e s i z e d f a c t o r s i n l a r g e q u a n t i t i e s h a s i n t e n s i f i e d e f for t s t o d e t e r m i n e t h e b i o l o g i c a l e f fec t s o f t h e C S F s i n v i v o . In o n e s t u d y , c o n t i n u o u s i n f u s i o n o f r e c o m b i n a n t m I L - 3 r e s u l t e d i n a 2 - f o l d i n c r e a s e i n h e m o p o i e t i c p r o g e n i t o r c e l l s i n n o r m a l m i c e , a n d a 1 0 - f o l d i n c r e a s e o f t h e s e c e l l s i n s u b l e t h a l l y i r r a d i a t e d m i c e (73) . I n a d d i t i o n , m a t u r e h e m o p o i e t i c c e l l s w e r e i n c r e a s e d 2 to 1 0 - f o l d i n t h e s p l e e n s o f n o r m a l m i c e r e c e i v i n g m u l t i p l e i n j e c t i o n s o f r e c o m b i n a n t m I L - 3 ( 1 8 9 ) . F u r t h e r m o r e , i n m i c e p r e t r e a t e d w i t h l a c t o f e r r i n (to i n c r e a s e t h e s e n s i t i v i t y o f t h e m i c e t o C S F s ) , B F U - E a n d C F U - G E M M p r o g e n i t o r c e l l c o m p a r t m e n t s w e r e m o r e s e n s i t i v e to t h e e f f ec t s o f r e c o m b i n a n t m I L - 3 t h a n w a s t h e C F U - G M c o m p a r t m e n t ( 1 9 0 ) . I n c o n t r a s t , M - C S F ( p u r i f i e d n a t i v e m a t e r i a l ) a p p e a r e d to b e a m o r e effect ive s t i m u l a t o r o f C F U - G M t h a n o f B F U - E a n d C F U - G E M M , w h i l e r e c o m b i n a n t m u r i n e G M - C S F w a s m o r e e f f e c t i v e i n s t i m u l a t i n g C F U - G M t h a n C F U - G E M M a n d h a d n o e f f e c t o n B F U - E . C o n t i n u o u s i n t r a v e n o u s i n f u s i o n o f h u m a n r e c o m b i n a n t G M - C S F i n t o n o r m a l m a c a q u e m o n k e y s d r a m a t i c a l l y i n c r e a s e d t h e p e r i p h e r a l l e u k o c y t e c o u n t (191) . S i m i l a r r e s u l t s w e r e o b t a i n e d i n r h e s u s m o n k e y s (192 ) ; t h i s s t u d y a l s o d e m o n s t r a t e d t h a t r e c o m b i n a n t h u m a n G M - C S F a c t i v a t e s p e r i p h e r a l b l o o d n e u t r o p h i l s i n p r i m a t e s . T h u s G M - C S F a p p e a r s to b e a p o t e n t 2 5 s t i m u l a t o r o f n o r m a l p r i m a t e h e m o p o i e s i s . T h e i n v i v o e f f ec t s o f r e c o m b i n a n t h u m a n G -C S F i n p r i m a t e s h a v e a l s o b e e n e x a m i n e d ( 1 9 3 ) . T h i s f a c t o r i s a c t i v e i n n o r m a l m o n k e y s a n d c a u s e s a d o s e - d e p e n d e n t i n c r e a s e i n n e u t r o p h i l s . F u r t h e r m o r e , i t m a y s h o r t e n t h e p e r i o d o f n e u t r o p e n i a a f t e r s u b l e t h a l c h e m o t h e r a p y o r a f t e r l e t h a l t o t a l b o d y i r r a d i a t i o n f o l l o w e d b y a u t o l o g o u s b o n e m a r r o w t r a n s p l a n t a t i o n . T h e a b o v e r e s u l t s i n d i c a t e t h a t i n j e c t i o n o f n a t i v e a n d r e c o m b i n a n t C S F s i n t o a n i m a l s s i g n i f i c a n t l y s t i m u l a t e s m a n y o f t h e s a m e c e l l p o p u l a t i o n s a s a r e s t i m u l a t e d i n v i t r o b y t h e s e g r o w t h f a c t o r s . H o w e v e r , s i n c e m a n y o f t h e i n v i v o e f fec ts d e s c r i b e d r e q u i r e m u l t i p l e o r c o n t i n u o u s i n f u s i o n o f r e l a t i v e l y h i g h l e v e l s o f C S F s , t h e s e d a t a d o n o t p r o v i d e d i r e c t e v i d e n c e o f a p h y s i o l o g i c a l r o l e i n n o r m a l h e m o p o i e s i s . I n f ac t , t h e s e r u m h a l f - l i f e o f i n j e c t e d C S F i s s h o r t , w i t h a m u l t i p h a s i c c l e a r a n c e c o n s i s t i n g o f a n i n i t i a l d e c a y o f 5 - 1 5 m i n u t e s f o l l o w e d b y a s l o w e r p h a s e w i t h a h a l f - l i f e o f 1-7 h o u r s ( 7 2 , 1 0 3 ) . F u r t h e r m o r e , i n c o n t r a s t t o t h e a b o v e e x p e r i m e n t a l c o n d i t i o n s , 11-3 i s n o r m a l l y n o t d e t e c t a b l e i n t h e c i r c u l a t i o n ( 1 8 8 ) . O n t h e o t h e r h a n d , e r y t h r o p o i e t i n , M - C S F , G - C S F , a n d G M - C S F c a n b e d e t e c t e d i n s e r u m ( 1 8 8 ) . M o r e o v e r , t h e l e v e l s o f t h e l a t t e r t h r e e c a n b e d r a m a t i c a l l y i n c r e a s e d b o t h i n f a c t o r - p r o d u c i n g t i s s u e s a n d i n t h e c i r c u l a t i o n w h e n a n i m a l s a r e t r e a t e d w i t h b a c t e r i a l l i p o p o l y s a c c h a r i d e s s u c h a s e n d o t o x i n , o r w i t h t h e p r o d u c t s o f a c t i v a t e d m o n o c y t e s , s u c h a s I L - 1 a n d T N F - a (73) . T h i s h a s l e d to t h e p r o p o s a l t h a t t h e r e a r e t w o t y p e s o f t i s s u e c o n t r o l o f h e m o p o i e t i c c e l l p o p u l a t i o n s b y t h e C S F s ( 7 3 , 1 0 3 ) . I n t h e f i r s t , b a s a l h e m o p o i e s i s i s p r o b a b l y m a i n t a i n e d b y t h e l o c a l p r o d u c t i o n o f C S F s f r o m f i x e d s t r o m a l c e l l s ( e n d o t h e l i a l c e l l s , f i b r o b l a s t s , m o n o c y t e s ) a n d T l y m p h o c y t e s i n t h e b o n e m a r r o w . T h i s s y s t e m c a n a p p a r e n t l y r e s p o n d t o t h e t u r n o v e r o f h e m o p o i e t i c c e l l p o p u l a t i o n s b y r e p o p u l a t i n g t h e v a r i o u s c e l l u l a r c o m p a r t m e n t s . N e w e v i d e n c e f o r s u c h l o c a l c o n t r o l m e c h a n i s m s c o m e s f r o m t h e v e r y r e c e n t f i n d i n g t h a t h e p a r i n s u l p h a t e , t h e m a j o r s u l p h a t e d g l y c o s a m i n o g l y c a n o f t h e m a r r o w E C M , c a n a d s o r b b o t h m I L - 3 a n d m u r i n e G M - C S F , a n d c a n p r e s e n t t h e m t o h e m o p o i e t i c c e l l s i n b i o l o g i c a l l y a c t i v e f o r m s (194) . 26 The second type of control system Is called into play during periods of stress. This system involves tissues throughout the body and appears to be activated most commonly by exposure to micro-organisms and their products, including endotoxin. The large increases i n circulating CSFs (mentioned above) that result from infections, or following injection of endotoxin (103), occur within minutes, and peak by 3-6 hours. When infections are resolved, the CSF levels promptly return to normal. The rise in CSF may therefore be regarded as a self-limiting defense mechanism designed to respond to acute periods of hemopoietic stress, such as a host infection. Indeed, the major function of the elevated CSF levels in the circulation and tissues under these conditions may not be to regulate the proliferation and development of progenitor cells, but to activate pre-existing mature cells such as neutrophils and macrophages (73,103,185). With regard to the mechanism by which tissues are induced to rapidly increase production of CSFs, it appears that monocytes may play a central role (73). These cells are known to produce IL-1 (141) and TNF-a (195) in response to endotoxin, and these monokines may in turn induce circulating T cells or tissue fibroblasts and endothelial cells to produce CSFs (64-69,165,166). Another purpose for investigating the i n vivo role of the CSFs, in addition to confirming the results of in vitro analyses, is to determine the therapeutic potential of these agents. A number of clinical trials with CSFs have now been completed or are in progress. Recombinant erythropoietin has been used very successfully in the treatment of anemia in end stage renal d i a l y s i s patients (196). Phase I and II studies are i n progress for recombinant G-CSF and GM-CSF (197). These two CSFs appear to be relatively non-toxic, with the main side effects being phlebitis at sites of infusion and bone pain during infusion. Both of these agents cause a marked dose-dependent increase in peripheral leukocyte counts when administered over a period of days to months to various normal or diseased individuals. G-CSF stimulates predominantly an increase in neutrophils, while GM-CSF stimulates increased neutrophils at low doses, and increased eosinophils and monocytes a t higher doses. Enhanced mature granulocyte and monocyte functions have also been documented (197). In addition, it has been reported that G-CSF and GM-CSF can shorten 27 the period of neutropenia i n patients treated with chemotherapy, including patients receiving autologous bone marrow transplants for various forms of leukemia (148). In patients with acquired immunodeficiency syndrome (AIDS), GM-CSF given subcutaneously over a period of 6 months was effective in elevating the peripheral leukocyte count in these patients (198). Furthermore, when 8 patients with myelodysplastic syndrome were treated with GM-CSF over an 8-32 week period, they all showed marked increases in peripheral leukocyte counts and three of eight patients had increases i n platelet counts and erythrocytes (199). Initial results from phase I and II trials with erythropoietin, G-CSF, and GM-CSF therefore provide strong indications that the CSFs will find therapeutic applications in the treatment of hematological dysfunction in a number of different disease settings. Further c l i n i c a l t r i a l s are required to test other CSFs and cytokines, and to study potential synergistic effects with different combinations of hemopoietic growth factors. 2.3. Colony-stimulating factors and myeloid leukemia Myeloid leukemias are clonal malignancies in which the normally rigorous control of survival, proliferation and development of myeloid stem cells or progenitors is lost. Since studies both i n vitro and in vivo support the concept that the CSFs are key regulators within the hemopoietic system, it is of interest to determine if derangements in CSF-mediated regulation are involved in myeloid leukemogenesis. The conversion of cells from a normal to a malignant state is thought to occur through a series of compounding genetic events, rendering primary cells immortal (with unlimited proliferative potential) and conferring malignant characteristics on populations of immortalized cells. It appears that these genetic events lead to alterations in the nature or expression of a number of genes controlling cellular growth and development. In recent years, a series of viral oncogenes have been discovered that have the ability to immortalize or f u l l y transform cells into which they are transfected (200). It has further been demonstrated that many viral transforming genes have normal cellular homologues. 28 possibly controlling cell growth and development (201,202). It is therefore postulated that in naturally arising tumors, genetic lesions are incurred in cellular genes (proto-oncogenes) that f u n c t i o n a l l y overlap with v i r a l oncogenes and which are important i n growth regulation. Moreover, the release of premalignant cells from dependence on exogenous growth factors for growth and development has been suggested as an important stage in tumor development. This might arise if, through the expression of certain oncogenes, cells aberrantly produced either; i) growth factors which stimulated these cells to grow; ii) growth factor receptors that were constitutively active, even in the absence of growth factors; or iii) altered components of intracellular signal transduction pathways which were not subject to normal control mechanisms (and thus were constantly turned on). This concept has been termed the autocrine hypothesis (203). In support of this model, certain transformed cells have been shown to both synthesize and respond to tumor growth factors (TGFs) (204,205). It has also been shown that the sis oncogene of Simian sarcoma virus (SSV) encodes a product that is identical to the B-chain of PDGF (206,207), and that autocrine production of this growth factor results in continuous cellular proliferation of infected cells by stimulation of the PDGF receptor (208,209). In addition, the protein encoded by the v-erbB oncogene of A v i a n erythroleukemia v i r u s (AEV) has been demonstrated to be a truncated form of the epidermal growth factor (EGF) receptor (210). with a constitutively.activated intracellular tyrosine kinase domain (211). Furthermore, the products of the three ras oncogenes (v-Harvey-ras, v-Kirsten-ras, and N-ras), are now known to be 21 kDa GTP-binding proteins with GTPase activities that are decreased compared with normal cellular GTP-binding proteins (212,213). Since GTP-binding proteins have been implicated in normal growth factor-stimulated signal transduction pathways (see below), it has been postulated that the reduced GTPase activity of ras proteins contributes to continuous mitogenic signaling and unregulated growth of cells transformed by the action of these oncogenes (214). It should be noted that active ras oncogenes have been isolated from a number of human cancers (215). 29 Within the hemopoietic system, it is unlikely that autonomous production of CSFs would by itself lead to the emergence of a leukemic clone. Firstly, these cells would still be subject to the normal regulatory influences of the surrounding microenvironment. Secondly, a feature of primary leukemias in humans and in mice is that the leukemic cells remain absolutely dependent on exogenous CSFs for survival and continuous proliferation in vitro (216). Thirdly, the amount of CSF produced by an emerging leukemic clone would likely be insignificant compared with that produced by surrounding stromal cells or that reaching the cells via the circulation (103). Furthermore, there is no sequence homology between the CSFs and the known oncogene products, and It has not been demonstrated that leukemic cells are responding to abnormal CSFs (86). Despite the above arguments, however, several experiments using previously immortalized myeloid cell lines have linked constitutive CSF production with leukemic transformation. Using mIL-3-dependent, non-leukemogenic cell lines, two groups have reported the isolation of spontaneously arising mutants that grow independently of added mIL-3 (217,218). In both cases it was shown that the mutant cells secreted mIL-3 and that they were tumorigenic when injected into syngeneic animals. Moreover, when the mIL-3 or GM-CSF-dependent cell line, FDC-P1, was transfected with a recombinant retrovirus containing the gene for murine GM-CSF, infected cells grew independently of exogenous growth factors and produced tumors in syngeneic mice (219). These cells were shown to synthesize and secrete GM-CSF. The fact that the growth of transformed cells was unaffected by the presence of antibodies to GM-CSF suggested to the investigators that the growth factor need not be secreted to stimulate the cells (i.e., it may be capable of activating receptors within cells) (219). Another line of evidence for autocrine stimulation comes from the study of chicken macrophages immortalized by the v-myc oncogene. Secondary infection of these factor-dependent cells with the v-mil oncogene resulted in the emergence of leukemic cells which synthesized, secreted, and were dependent on an avian form of M-CSF (220). There is also evidence that in the leukemic murine cell line WEHI-3B. which 30 secretes mIL-3, the mIL-3 gene has at some stage come under the influence of a viral promoter sequence (221). Recent studies have demonstrated that the v-fms oncogene of the McDonough strain of feline sarcoma virus encodes a transmembrane protein that can bind M-CSF (222). It was subsequently shown that the product of the c-fms proto-oncogene is the surface receptor for M-CSF (223). Both the c-fms protein and the v-fms protein have intracellular tyrosine kinase domains (224), but the v-fms product appears to harbour a constitutively active kinase domain (possibly due to a deletion of an important carboxyterminal regulatory site). Therefore, as i n the case of the v-erbB gene and the EGF receptor, the v-fms gene may encode a truncated version of the M-CSF receptor. This is as yet the only known example of a proto-oncogene encoding a CSF or the receptor for a CSF. When mIL-3- or GM-CSF-dependent cell lines such as FDC-P1 are transfected with v i r u s c o n t a i n i n g the v-abl or v-myc oncogenes, these cells become growth factor independent and leukemic (220,225). The growth autonomy is not due to growth factor production or altered expression of growth factor receptors (188,220,225,226). Rather, it appears that expression of v-myc or v-abl has bypassed the requirement for growth factor stimulation of these cells. Presumably, factor-independence is achieved by the v-myc product (a nuclear protein (201) which appears to bind to DNA (227)) or the v-abl product (a tyrosine kinase associated with the cytoplasmic face of the plasma membrane (228)) acting as constitutive mediators of intracellular signal transduction. This may then lead to the activation of common downstream targets involved in mitogenesis. Taken together, data from the above studies have demonstrated the existence of a number of mechanisms by which hemopoietic cells may have their dependence on exogenous growth factors abrogated, and by which these cells may concomitantly become tumorlgenic. Therefore, although some primary human leukemic cells do appear capable ol autonomous production of CSFs (229), it is unlikely that factor secretion and subsequent autostimulation by these factors are by themselves sufficient for the emergence of a leukemic clone. A more likely possibility is that leukemic cells possess an intrinsic 31 abnormality s u c h that C S F s t i m u l a t i o n of these cells (by exogenous or autocrine-produced CSFs) r e s u l t s i n a n abnormally high ratio of self-renewal divisions versus div i s i o n s leading to d i f f e r e n t i a t i o n . In t h i s model, the i n t r i n s i c defect, p o s s i b l y c a u s e d by the a c t i o n of an oncogene p r o d u c t , w o u l d l e a d to f a c t o r - i n d e p e n d e n c e or a b e r r a n t r e s p o n s e s to C S F stimulation. Clearly the role of the C S F s i n myeloid leukemogenesis is complex and m u ch remains to be learned i n order to determine how these molecules may influence the development of m a l i g n a n c y i n the h e m opoietic system. S e v e r a l a d d i t i o n a l p o i n t s , however, s h o u l d be emphasized. F i r s t , the apparent CSF-dependency of myeloid l e u k e m i a c e l l p r o l i f e r a t i o n m u s t be kept i n m i n d when C S F s are a d m i n i s t e r e d c l i n i c a l l y . Second, the role of C S F s w h i c h are able to influence the d i f f e r e n t i a t i o n of l e ukemic c e l l s (such as G-CSF) m u s t be further evaluated for possible therapeutic applications i n this regard, and s u c h t r i a l s have b e e n i n i t i a t e d (73). T h i r d , G M - C S F h a s b e e n s h o w n to s t i m u l a t e b o t h n o r m a l a n d transformed non-hemopoietic cells to proliferate (230). C a u t i o n must therefore be exercised before a d m i n i s t e r i n g C S F s to reduce the period of neutropenia following chemotherapy or r a d i o t h e r a p y . F o u r t h , a complete u n d e r s t a n d i n g of the events t h a t l e a d to the l o s s of g r o w t h c o n t r o l i n l e u k e m i a f i r s t r e q u i r e s the e l u c i d a t i o n of the b i o c h e m i c a l p athways u n d e r l y i n g t h e s t i m u l a t i o n of g r o w t h i n n o r m a l c e l l s . T h e n a t u r e of t h e s e s i g n a l t r a n s d u c t i o n p athways an d how they may be activated by the C S F s i s the subject of the following section. C. M E C H A N I S M OF ACTION OF POLYPEPTIDE GROWTH FACTORS Hemopoietic progenitor cells receive regulatory signals from their environment i n the form of hemopoietic growth factors. Interactions between the appropriate factors and cells r e sults i n the conversion of external signals into a complex series of internal c e l l u l a r events w h i c h ultimately lead to changes i n the proliferative or differentiative states of the cells. As i n other systems, the hemopoietic growth factors, i n c l u d i n g the CSFs, appear to f u n c t i o n 32 via binding to cell surface receptors (231). However, only limited data are currently available on the functional activities of receptors for hemopoietic growth factors, and even less is known about the i n t r a c e l l u l a r biochemical pathways that are activated as a consequence of receptor binding. One strategy for gaining insight into these molecular processes is to compare the mechanisms of action of the hemopoietic growth factors with those of well-characterized growth factors such as EGF, PDGF, insulin, insulin-like growth factors (IGFs), and fibroblast growth factor (FGF). These latter polypeptides are classical mitogens i n that they stimulate DNA synthesis and cell division in quiescent target cells such as the Swiss 3T3 fibroblast cell line. It seems likely that at least some of the molecular signaling mechanisms utilized by these growth factors are also enlisted when hemopoietic cells are mitogenically stimulated by the CSFs. However, while fibroblast survival appears to be independent of EGF, PDGF, and FGF, hemopoietic cells maintain an absolute dependence on the CSFs for survival as well as for proliferation (232). Therefore, any model which attempts to delineate the mechanism of action of the CSFs must also identify the vital molecular events which enable the CSFs to act as survival factors (the CSFs also appear to stimulate differentiation of target cells, but this complex activity remains very poorly understood and will not be discussed further). It should also be kept in mind that while many classical mitogens stimulate maximal growth of fibroblasts only in combination with other mitogens, the CSFs are often maximally potent in stimulating cell division in the absence of any other synergistic factors. The following section summarizes current concepts of signal transduction involving the regulation of c e l l growth by polypeptide growth factors. 1. Cellular Responses to Growth Factors Dissection of the molecular events intervening between growth factor binding to cell surface receptors and initiation of DNA synthesis has become one of the major objectives of cell biology. In view of the l i n k that has been made between growth factors or their receptors and oncogene products (233), elucidation of growth factor signal transduction 33 m e c h a n i s m s may prove to be a c r u c i a l p r e r e q u i s i t e for u n d e r s t a n d i n g the u n c o n t r o l l e d growth of cancer cells. M u c h of the c u r r e n t knowledge regarding the m o l e c u l a r events i n i t i a t e d by growth factors h a s been obtained from stu d y i n g target c e l l responses to EGF, PDGF, i n s u l i n , a nd other w e l l - k n o w n polypeptide mitogens. The f i r s t step is the b i n d i n g of these factors to s p e c i f i c h i g h - a f f i n i t y receptors. M a n y receptors t h e n u ndergo r a p i d p h o s p h o r y l a t i o n , followed by r e d i s t r i b u t i o n i n the plasma membrane a n d endocytosis of the receptors (234). Receptor a c t i v a t i o n leads to the generation of a number of early s i g n a l s i n the membrane and cyt o s o l , i n c l u d i n g changes i n i o n fluxes and c y c l i c n u c l e o t i d e levels, a c t i v a t i o n of a number of key enzymes, cytoskeletal changes, metabolism of membrane phospholipids, and pr o t e i n p h o s p h o r y l a t i o n s (235). W i t h i n minutes, the mitogenic s i g n a l i s received by the n u c l e u s , r e s u l t i n g i n the a c t i v a t i o n of v a r i o u s genes. These m u l t i p l e events e v e n t u a l l y converge into a common f i n a l pathway leading, after 10-24 hours, to the i n i t i a t i o n of DNA synthesis and cell division. 1.1. Ion Fluxes One of the earliest consequences of growth factor b i n d i n g i s a n increase i n the fluxes of N a + , K +, and H + across the p l a s m a membrane (236,237). Mitogens for S w i s s 3T3 cells have been shown to sti m u l a t e a n elect r i c a l l y silent N a + / H + antiport, r e s u l t i n g i n entry of N a + into cells and ex t r u s i o n of H + with concomitant cytoplasmic a l k a l i n i z a t i o n (236,238). C h a n g e s i n the a c t i v i t y of t h i s i o n p u m p may be i m p o r t a n t i n the i n i t i a t i o n of DNA s y n t h e s i s i n some c e l l s , as a m u t a n t c e l l l i n e l a c k i n g the N a + / H + e x c h a n g e r has been described w h i c h f a i l s to grow i n response to growth factors n o r m a l l y active on the parent c e l l line (239). Increases i n cytoplasmic N a + secondarily stimulates N a + / K + pump action, t h u s i n c r e a s i n g i n t r a c e l l u l a r K + levels a nd restoring the electrochemical gradient for Na + (237). The a b i l i t y to alter i on f l u x e s appears to be a general property of mitogens, but it r e m a i n s to be determined whether these changes play a permissive or triggering role (or both) i n m i t o g e n e s i s . A n o t h e r i o n whose i n t r a c e l l u l a r l e v e l s are a l t e r e d by mi t o g e n i c stimulation is calcium. Growth factors such as PDGF and EGF stimulate Ca2+ efflux from quiescent fibroblasts within 15 seconds of stimulation (240). This response is elicited even i n the absence of extracellular C a 2 + , suggesting that C a 2 + is being released from intracellular stores. In fact, the earliest response observed is a very transient increase in cytoplasmic C a 2 + (241). This apparently promotes C a 2 + efflux by activation of the plasma membrane Ca 2 +-dependent Ca 2 +-ATPase (235). Calcium ions have long been recognized as second messengers within cells, being intimately involved in processes such as muscle contraction and activation of various Ca 2 +-dependent enzymes (242), and thus may similarly be important in mitogenic pathways. Ion fluxes which are altered in response to mitogens are summarized in Fig. 2A. 1.2. GTP-binding proteins and receptor-effector coupling Increases i n other second messenger molecules have also been correlated with mitogenic stimulation. These include cyclic AMP (cAMP), produced from ATP by the action of membrane bound adenylate cyclase, and inositol trisphosphate (IP3) and diacylglycerol (DAG), both of which are produced by the hydrolysis of phosphoinositide in the plasma membrane by the action of phospholipase C (235,242,243). The observation that certain growth factors stimulate the accumulation of cAMP but not IP3 and DAG, or vice versa, suggested that specific regulatory elements are interposed between extracellularly oriented receptors and intracellular effector enzymes (such as adenylate cyclase) that in turn produce second messenger molecules. This concept has been confirmed by the discovery and characterization of a family of membrane-associated proteins known as the G proteins, so called because they are activated by the binding of guanosine triphosphate (GTP) (244). It is now believed that these proteins, by regulating the function of intracellular effector enzymes, act as receptor-effector coupling agents in a variety of transmembrane signaling systems, including those triggered by certain growth factors (245). A number of G proteins have now been identified (243-245), including G s (which activates adenylate cyclase leading to an increase in cAMP), Gj (which inhibits adenylate cyclase leading to a decrease in cAMP), G T or t r a n s d u c i n (which c o u p l e s r h o d o p s i n to a c y c l i c G M P - s p e c i f i c 35 phosphodiesterase i n retinal rod outer segments), G 0 (a G protein of unknown function which is abundant i n brain tissue), and Gp (a recently discovered GTP-binding protein of apparent molecular mass 21 kDa, which is of unknown function and is present in placenta and platelets (246)). Involvement of G proteins has also been implicated in the regulation of phosphoinositide turnover by phospholipase C (see below), and i n the control of K + channels in membranes (247). G proteins are heterotrimeric (244), consisting of a 40-46 kDa a subunit, a 35-37 kDa P subunit and 8-10 kDa y subunit, although this does not appear to be the case for Gp (247). While the Py complex probably serves to anchor the trimer to the cytoplasmic face of the plasma membrane, it is the cc-chain that binds GTP and determines the specificity of the protein for its detector and effector. In fact, it has recently been demonstrated that the presence of conserved sequences in certain receptors allows for the Interaction with specific G protein a-chains (248). The a-chain also harbours an intrinsic GTPase activity, which cleaves GTP to GDP and maintains the G protein in its inactive state (G-GDP). The proposed sequence of reactivation (246), is schematically represented in Fig. 2B. On stimulation by a receptor protein, G-GDP dissociates GDP and binds GTP. In its GTP-bound conformation, the G protein can interact with and stimulate an effector molecule. Hydrolysis of bound GTP to GDP then terminates the regulatory effect of the G protein. G proteins can also be regulated non-physiologically or pathologically by bacterial toxins, which catalyze the NAD-dependent ADP-ribosylation of sites in a subunits. ADP-ribosylation of G s is catalyzed by cholera toxin resulting in activation of the G protein, while Gj and G 0 are substrates for pertussis toxin (islet-activating protein). In the case of Gj, the modification results in an impaired ability to interact with receptors and impaired ability to exchange GTP for the bound GDP (249). The products of the ras proto-oncogenes are 21 kDa proteins that bind GTP, have GTPase activity, and are associated with the plasma membrane (250). These properties, along with their significant sequence homology with G proteins (251), have led to the suggestion that ras proteins are related to G proteins and may participate i n signal transduction. There is preliminary evidence that these proteins can couple receptors to 36 effectors, p a r t i c u l a r l y growth factor receptors to a p h o s p h a t i d y l i n o s i t o l - s p e c i f i c phospholipase C (252). It is as yet unclear if the previously mentioned 21 kDa Gp molecule is a member of the ras protein family. Further evidence for a role of the ras proteins in growth regulation is the demonstration that the products of the transforming ras oncogenes have decreased GTPase activity, a property which is presumed to render the proteins constitutively active, thereby contributing to their transforming abilities (253). Possible roles of the ras proteins in signal transduction are discussed in a subsequent section. 1.3. Elevation of cAMP Levels The role of cAMP as a second messenger in the control of cell proliferation has been and remains a controversial issue. Earlier views held that cAMP had an inhibitory function in modulating proliferation of fibroblasts (254). It is now known that sustained increases in intracellular levels of cAMP constitute a mitogenic signal for Swiss 3T3 cells (255). Both PDGF and FGF induce large increases in cAMP when added to cells i n combination with inhibitors of cAMP phosphodiesterase (235). However, other mitogens such as EGF. insulin, vasopressin, and activators of protein kinase C do not elevate cAMP levels. In fact, agents which increase cAMP levels act synergistically, but not additively, with EGF, insulin, and vasopressin in promoting growth of Swiss 3T3 cells (235,255). Insulin appears to activate the G protein Gj in some cells, resulting in decreased cAMP levels, but it is not known if this activity plays a role in insulin-stimulated cell proliferation (256). The ability of PDGF (and FGF) to elevate cAMP occurs through an indirect mechanism (235). As indicated i n Fig. 2C, PDGF elicits a striking intracellular release of arachidonate from membrane phospholipids, and this fatty acid is converted into E-type prostaglandins. These latter molecules then leave the cell and bind to their own cell surface receptors, leading to, i n turn, activation of G s, activation of adenylate cyclase, and elevation of intracellular cAMP. It is assumed, but unproven, that the action of cAMP as a putative second messenger in mitogenesis is brought about by the activation of cAMP-dependent 37 protein k i nase A (257), with subsequent serine and/or threonine p h o s p h o r y l a t i o n of as yet unidentified c e l l u l a r substrates. 1.4. Phosphoinositide Hydrolysis and the Activation of Protein Kinase C In recent years, convincing evidence has accumulated indicating that the metabolism of m e m b r a n e p h o s p h o l i p i d s p l a y s a n active role i n c o n t r o l l i n g c e l l u l a r r e s p o n s e s to a v a r i e t y of s t i m u l i , i n c l u d i n g g r o w t h - p r o m o t i n g agents. It i s now r e c o g n i z e d t h a t the turn o v e r of a quantitatively m i n o r membrane pho s p h o l i p i d , p h o s p h a t i d y l i n o s i t o l (PI), is a p r i m a r y event i n m a n y s i g n a l t r a n s d u c t i o n systems (258). PI c a n be p h o s p h o r y l a t e d by specific PI ki n a s e s to form phosphatidylinositol-4,5-bisphosphate (PIP2). w h i c h i n t u r n is hydrolyzed by PIP 2-specific phospholipase C (PLC) (243,259). There is now compelling evidence to suggest that the activation of PLC is coupled to receptors (such as growth factor receptors) by an intervening G protein. P L C activation is enhanced i n the presence of GTP and its non-hydrolyzable analogs (244). Also, P I P 2 h y d r o l y s i s is b l o c k e d i n some cells by the presence of p e r t u s s i s t o x i n (244). F u r t h e r m o r e , NIH-3T3 c e l l s w h i c h over-express normal p21 ras proteins have increased P I P 2 breakdown compared with control cells (252). It h a s therefore been proposed that r a s proteins directly or i n d i r e c t l y couple receptors to PLC, a l t h o u g h t h i s i s s u e r e m a i n s to be proven. P I P 2 h y d r o l y s i s r e l e a s e s two r a p i d l y degraded s e c o n d messengers, inositol-1,4,5-trisphosphate (IP3) a n d 1,2-diacylglycerol (DAG), as indicated i n Fig. 2D. IP3 stimulates the release of C a 2 + from in t r a c e l l u l a r stores, namely the endoplasmic r e t i c u l u m (260). This, then, may at least par t i a l l y account for the rise i n C a 2 + seen when ce l l s are m i t o g e n i c a l l y s t i m u l a t e d . There is some evidence that f u r t h e r m e t a b o l i t e s of IP3 also have f u n c t i o n a l a c t i v i t i e s w i t h i n c e l l s (243). D A G i s an endogenous a c t i v a t o r of a p h o s p h o l i p i d - a n d C a 2 + - d e p e n d e n t s e r i n e / t h r e o n i n e protein kinase, protein kinase C (PKC) (261). T h i s enzyme exists i n its inactive form as an 80 kDa cytoplasmic protein. In response to agents that increase DAG levels, PKC is translocated to the inner face of the p lasma membrane (possibly by increases i n the level of C a 2 + (262)). w h e r e i t i s a c t i v a t e d b y f o r m i n g a m e m b r a n e - a s s o c i a t e d c o m p l e x w i t h at l e a s t 4 38 phosphatidylserine molecules, one molecule of DAG, and one C a 2 + ion (242). PKC is autophosphorylated on activation, which may lead to i t s release from the plasma membrane (242). Complementary DNA clones of PKC have recently been obtained (263), allowing for the complete amino acid sequence of the protein to be determined. Sequence analysis reveals that PKC has both a regulatory and a catalytic domain (263). Further studies suggest that there is a family of PKC-related genes, each of which is located on a distinct chromosome (264). This finding may help to explain the diversity of responses observed when cells are treated with agents that induce PKC activation. Protein kinase C has been shown to be a major receptor for the tumour promoters o f the phorbol ester family (261). The enzyme is also activated by the synthetic diacylglycerol analogue, l-oleoyl-2-acetyl-glycerol (OAG). Since phorbol esters and OAG can act a s mitogens for quiescent cells, PKC may play a role in the production of a mitogenic response (235). In fact, many peptide or polypeptide growth factors have been shown to stimulate Pn°2 metabolism and appear to activate PKC in target cells. Growth stimuli known t o induce PIP2 breakdown include PDGF, FGF, interleukin-2 (IL-2), thrombin, bombesin, vasopressin, and prostaglandin F 2 a (235,243,259,265). Several of these agents (PDGF. FGF, IL-2, and thrombin) also stimulate translocation of PKC to the plasma membrane w i t h activation of the enzyme, and it is presumed that PKC plays an integral role in t h e mitogenic response to growth factors whose mechanism of action involves PIP2 metabolism (261). Strategies for assessing the role of PKC i n stimulating cell proliferation include testing-the ability of OAG or PKC-activating phorbol esters such as 12-O-tetradecanoyl-phorbol-12,13-acetate (TPA) to substitute for a particular mitogen in promoting growth of target cells. In some cells, including T-lymphocytes (266), t h e addition o f agents w h i c h elevate intracellular C a 2 + (such as calcium ionophores) is required in combination w i t h PKC activators for a mitogenic response. This suggests that there are synergistic interactions between the I P 3 / C a 2 + and DAG/PKC pathways (243). Another method to test if a mitogen requires the activation of PKC to elicit a response is to exploit the fact t h a t chronic exposure to phorbol esters leads to a marked decrease in measurable PKC activity 3 9 E. EGF I. EGF receptor (inactive tyrosine kinase) active tyrosine kinase lipocortin P L A 2 (inhibited) lipocortin s P L A 2 (active) ararhirionate 1 phospholipids | prostaglandins Figure 2A-E. Figure legends on following page. 40 Figure 2. Schematic representation of proposed mechanisms of early signals in the mitogenic response. Binding of a polypeptide mitogen to its specific surface receptor induces a number of rapid biochemical changes in the cytosols and membranes of target cells. Possible mechanisms have been proposed for some of the observed cellular changes. A. Changes in ion fluxes; activation of protein kinase C (PKC - see D) results in increases in calcium concentration; (by unknown mechanisms (X) or by activation of PKC) sodium/hydrogen antiport activity and potassium fluxes. B. G protein activation; receptor binding results in activation of the G protein (to form G aGTP) allowing it in turn to activate the effector protein E (to form E ); hydrolysis of GTP by the intrinsic GTPase activity of G a causes release of deactivated E. C. Increased intracellular cyclic AMP (cAMP) in response to PDGF; PDGF binding results in a c t i v a t i o n of ph o s p h o l i p a s e C (PLC), w h i c h metabolizes membrane phospholipids to diacylglycerol (DAG), which is further metabolized to form prostaglandins; E-type prostaglandins then bind to extracellular receptors, with subsequent activation of adenylate cyclase. D. Activation of PKC; G protein activation of PLC, with formation of DAG and inositol trisphosphate ( I P 3 ) from phosphatidylinositol -4 ,5-bisphosphate ( P I P 2 ) . results in increased calcium levels and activation of PKC by translocation of the protein from the cytosol to the membrane and subsequent complex formation with DAG and other membrane phospholipids. E. Increased prostaglandins in response to EGF; tyrosine phosphorylation of lipocortin by the activated EGF receptor frees phospholipase A 2 ( P L A 2 ) of the inhibitionsby non-phosphorylated lipocortin; P L A 2 subsequently releases prostaglandin precursors from membrane phospholipids. 41 in cells, presumably due to increased degradation of the enzyme (267). Hence, not only can TPA or OAG mimic the mitogenic activity of PDGF on Swiss 3T3 cells, but chronic exposure of these cells to TPA reduces the stimulation of DNA synthesis by low levels of PDGF (235). Hence, PKC activation constitutes one of the multiple signaling pathways utilized by PDGF. Similar studies with EGF and i n s u l i n have led to the conclusion that the mitogenic potential of these agents does not involve PIP2 breakdown/PKC activation (235,243,259); therefore, PKC activation does not appear to constitute an obligatory event in mitogenesis. Insulin does not detectably increase PIP2 hydrolysis in fibroblasts and EGF does so only to a minor degree (259,268). although EGF is known to induce PIP2 breakdown in a cell line (A431 human epidermoid vulval carcinoma cells) whose proliferation is inhibited by usual concentrations of EGF (269). Very recent evidence suggests that insulin binding stimulates an insulin-specific phospholipase C, which cleaves a glycosyl-phosphoinositide precursor in the plasma membrane (268,270). This results in the release of an inositol-phosphoglycan into the cytoplasm, leaving DAG in the membrane. The function of these membrane-derived second messenger molecules in EGF- and insulin-induced cell proliferation remains to be determined. The mechanism by which activated PKC relays growth-promoting signals in stimulated cells is presumed to involve serine/threonine phosphorylation of protein substrates. As with cAMP-dependent protein kinase A, however, few of these substrates have been identified. In intact cells, PKC appears to phosphorylate a number of proteins including an unknown 80 kDa polypeptide, various contractile and cytoskeletal proteins such as vinculin, the ribosomal S6 protein (known to be phosphorylated in response to mitogens (271)), pp60 c" s r c, and several metabolic enzymes (261). PKC also phosphorylates and activates the N a + / H + antiport in intact cells and therefore may be involved in cytoplasmic alkalinization observed after mitogenic stimulation (272). Furthermore. PKC phosphorylates the glucose transport protein i n vitro and i n vivo (273). Covalent modification of this protein by PKC may thus, in part, underlie the ability of phorbol esters and certain growth factors to stimulate glucose uptake into cells. Another possible 42 m e c h a n i s m for the ac t i o n of P K C w h i c h has come into focus more recently (274), involves the t r a n s l o c a t i o n of the enzyme to the n u c l e u s where it may regulate gene expression. F u t u r e s t u d i e s s h o u l d h e l p to i d e n t i f y k ey P K C - c a t a l y z e d r e a c t i o n s i n the m i t o g e n i c response. A c t i v a t i o n of P K C also appears to be involved i n c l a s s i c a l regulatory feedback loops s t i m u l a t e d by ligand-receptor i n t e r a c t i o n s . W h e n a i-adrenergic agonists, p-adrenergic agonists, a n d t r a n s f e r r i n b i n d to their specific receptors, they s t i m u l a t e PIP2 m e t a b o l i s m a n d a c t i v a t i o n of PKC; the k i n a s e t h e n c a t a l y z e s receptor p h o s p h o r y l a t i o n , l e a d i n g to d e s e n s i t i z a t i o n of the receptors to t h e i r respective l i g a n d s a n d d a m p e n i n g the agonist-i n d u c e d response (275). The m e c h a n i s m of receptor d e s e n s i t i z a t i o n by p h o s p h o r y l a t i o n has not been elucidated i n these systems. It has also been recently shown that activators of P K C c a n down-modulate the binding-capacity of vario u s c e l l types for t u m o u r necrosis factor-a (276). Moreover, receptors for E G F (277), i n s u l i n (278), a n d IGF-I (279), each of w h i c h h a r b o u r s a n i n t r i n s i c t yrosine k i n a s e a c t i v i t y (see below), are p h o s p h o r y l a t e d on serine and/or threonine residues by PKC i n certain cell types i n response to ligand binding. T h i s covalent m o d i f i c a t i o n r e s u l t s i n decreased l i g a n d b i n d i n g as w e l l as i n h i b i t i o n of t y r o s i n e k i n a s e a c t i v i t y of these r e c e p t o r s (275). PKC, then, a p p e a r s to f u n c t i o n i n a negative feedback loop i n these growth factor systems. The enzyme performs a s i m i l a r role i n the t r a n s m o d u l a t i o n of E G F receptors by PDGF. B i n d i n g of P D G F to its own receptors o n n o r m a l f i b r o b l a s t s c o - e x p r e s s i n g r e c e p t o r s f o r E G F s t i m u l a t e s P K C - c a t a l y z e d p h o s p h o r y l a t i o n of E G F receptors at the same site (threonine 654) as i s i n d u c e d by E G F (275). P D G F therefore induces heterologous desensitization of fibroblasts to E G F (275) by a m e c h a n i s m i n v o l v i n g the a c t i o n of PKC. In view of these r e s u l t s , a n d the f i n d i n g that chronic exposure of cells to TPA and other activators of PKC leads to enhanced degradation of P K C (264), a n o t h e r h y p o t h e s i s c a n be p u t f o r t h for the a b i l i t y of P K C a g o n i s t s to s t i m u l a t e mitogenesis. Namely, it may be that the g r o w t h - s t i m u l a t i n g a c t i v i t y of these agents i s a consequence of the removal of PKC from cells with subsequent loss of growth-inhibitory regulation. 43 1.5. Tyrosine-speciflc Protein Phosphorylation The p h o s p h o r y l a t i o n of p r o t e i n s i n c e l l s most c ommonly involves e s t e r i f i c a t i o n of phosphate to serine a n d threonine residues. In 1979, however, the fir s t reports emerged i d e n t i f y i n g t y r o s i n e as a n a d d i t i o n a l site for the a c t i o n of p r o t e i n k i n a s e s , i n t h i s case p r o t e i n t y r o s i n e k i n a s e s (PTKs) (280). P h o s p h o t y r o s i n e (P-tyr) was f i r s t d e t e c t e d i n hydrolysates of v i r a l transforming proteins labeled by i n c u b a t i n g immunoprecipitates with 3 2 P - l a b e l e d ATP (280,281). It is now known that a l l a n i m a l cells contain low levels of P-tyr, a l t h o u g h linkage to tyrosine represents only 0.1% of total phosphoamino aci d s i n normal cells (282). T h i s increases 20-fold i n transformed cells (282). Moreover, the products of the v-src, v-yes, v-fgr, v-fps, v-fes, v-abl, v-ros, v-erb B and v-fms retroviral oncogenes (200), as w e l l as the p r o d u c t of the H ER-2/neu oncogene i s o l a t e d f r o m p r i m a r y h u m a n br e a s t cancers (283), the product of the kit oncogene (284), and p 5 6 l c k isolated from L S T R A cells (285), a l l have i n t r i n s i c PTK activities a nd are capable of a u t o p h o s p h o r y l a t i o n on tyrosine residues. D e t a i l e d analyses of some of these t r a n s f o r m i n g proteins indicate that the PTK activities are necessary for transformation of cells (286), although the essential substrates have not been definitively identified. These findings have led to the hypothesis that tyrosine p h o s p h o r y l a t i o n is involved i n growth regulation. A s mentioned, the receptors for EGF, i n s u l i n a n d IGF-I have i n t r i n s i c cytoplasmic PTK domains w h i c h undergo ac t i v a t i o n upon ligand b i n d i n g (287). This property is also shared by the P D G F receptor (287) as well as the r e c e p t o r f o r M-CSF (224). More r e c e n t l y , it h a s been r e p o r t e d t h a t the r e c e p t o r s for b o mbesin (288) and growth hormone (289) are tyrosine phosphorylated on ligand binding. It r e m a i n s to be determined if the receptors for the lat t e r two factors have i n t r i n s i c PTK activity, or are associated with proteins bearing this activity. The cytoplasmic PTK domains of the E G F , i n s u l i n , IGF-I, a n d P D G F rec e p t o r s are homologous to s i m i l a r d o m a i n s i n re t r o v i r a l t r a n s f o r m i n g PTK products (286). Moreover, the v-erbB p r o d u c t a n d the v-fms p r o d u c t a p p e a r to be t r u n c a t e d f o r m s of the E G F a n d M-CSF r e c e p t o r s , r e s p e c t i v e l y (210,211.222-224). There is also sequence s i m i l a r i t y between the P D G F receptor and the k i t oncogene (284), a n d it has been suggested t h a t the neu proto-oncogene, c-erb-2. 44 encodes a p r o d u c t (already k n o w n to possess t y r o s i n e k i n a s e a c t i v i t y (290)) w h i c h Is a receptor for a n as yet u n k n o w n l i g a n d (287). Hence, there i s a n expanding l i s t of growth f a c t o r r e c e p t o r s a n d r e l a t e d oncogene p r o d u c t s w h i c h have b e e n s h o w n to p o s s e s s associated PTK activity. M a n y detailed s t u d i e s of receptor tyrosine k i n a s e s have been performed u s i n g the E G F receptor (a single polypeptide c h a i n of 170 kDa) and the i n s u l i n receptor (composed of two 135 k D a i n s u l i n - b i n d i n g a-chains, each of which i s disulphide-bonded to a 95 k D a f3-c h a i n b e a r i n g a n i n t r a c e l l u l a r PTK domain). In both cases, the growth factor-dependent tyrosine kinase activity of the cytoplasmic domain is regarded as the primary, though not n e c e s s a r i l y exclusive, m e c h a n i s m for the generation of i n t r a c e l l u l a r second messengers (287). G r o w t h f a c t o r a c t i v a t i o n of receptor P T K s r e q u i r e s s a t u r a t i n g c o n c e n t r a t i o n s of ligand, and is manifested principally as a n increase i n the V m a x of the kinase activity (291). However, the m e c h a n i s m of ligand-induced activation remains controversial. Two models have been c o n s i d e r e d for the E G F system (287). The " f l u s h c h a i n " model proposes that l i g a n d b i n d i n g effects slight changes i n the transmembrane region, the end result being a c o n f o r m a t i o n a l change i n and a c t i v a t i o n of the k i n a s e domain. Evidence for t h i s model inc l u d e s the observation that allosteric activation of the receptor kinase by E G F occurs via a n i n t r a m o l e c u l a r m e c h a n i s m (292), as w e l l as the f i n d i n g that a point m u t a t i o n i n the t r a n s m e m b r a n e re g i o n c o n s t i t u t i v e l y a c t i v a t e s the t y r o s i n e k i n a s e a c t i v i t y of the E G F recep t o r (293). The " c l u s t e r " model proposes that E G F b i n d i n g i n d u c e s aggregation of receptor monomers a n d that t h i s c l u s t e r i n g , i n v o l v i n g at least the f o r m a t i o n of dimers. leads to PTK activation. The E G F receptor is known to form dimers on ligand b i n d i n g (294), and it has been reported that receptor dimerization leads to an increased affinity for E G F as well as increases i n kinase activity (295). However, this model fails to explain the ability of E G F to s t i m u l a t e the P T K a c t i v i t y of m o n o m e r i c E G F r e c e p t o r m o l e c u l e s (287). The r e s o l u t i o n of t h i s i s s u e awaits f u r t h e r study. In the i n s u l i n system, experiments u s i n g kinase-activating monoclonal a n t i - i n s u l i n receptor antibodies suggest that intramolecular c r o s s l i n k i n g of af} heterodimers to form tetramers is sufficient for PTK activation (296). 45 T h e E G F a n d i n s u l i n r e c e p t o r s , l i k e o t h e r P T K s s t u d i e d to d a t e , r a p i d l y a u t o p h o s p h o r y l a t e o n c y t o p l a s m i c t y r o s i n e r e s i d u e s u p o n a c t i v a t i o n ( 2 8 6 ) . A u t o p h o s p h o r y l a t i o n of the E G F receptor appears to occur by a n i n t r a m o l e c u l a r reaction (287,292). a l t h o u g h evidence for a n intermolecular reaction has also been reported (295). M u t a n t E G F receptors l a c k i n g the p r i m a r y carboxy-terminal a u t o p h o s p h o r y l a t i o n sites, p r i n c i p a l l y tyrosine- 1173, exhibit reduced PTK activity at non-saturating levels of E G F and are s i g n i f i c a n t l y less effective at m e d i a t i n g c e l l growth i n response to E G F (297). It has been suggested that the autophosphorylation sites act as competitive i n h i b i t o r s of the PTK domain for exogenous substrates during the unstimulated state, t h u s decreasing undesired s u b s t r a t e p h o s p h o r y l a t i o n s ( 2 9 7 ) . U p o n e n z y m e a c t i v a t i o n b y E G F b i n d i n g , a u t o p h o s p h o r y l a t i o n o c c u r s leading to a co n f o r m a t i o n a l change i n the carboxy-terminal domain, w h i c h , i n t u r n , allows access of exogenous s u b s t r a t e s to the active site. In the case of the i n s u l i n receptor, on the other hand, a u t o p h o s p h o r y l a t i o n a p p ears to play a direct role i n activating the PTK activity of the enzyme (298). In fact, t h i s reaction appears to alter the V m a x of the enzyme and renders it independent of i n s u l i n for f u l l PTK activity (299). In both cases, therefore, a u t o p h o s p h o r y l a t i o n appears to have a b i o l o g i c a l role i n regulating receptor activity. A s mentioned earlier, the E G F and i n s u l i n receptors are also phosphorylated on serine and/or threonine residues following l i g a n d b i n d i n g , r e s u l t i n g i n decreased receptor affinity, reduced PTK activity, a nd receptor i n t e r n a l i z a t i o n (275). T h i s does not appear to be an autophosphorylation reaction since the serine/threonine kinase is lost o n receptor p u r i f i c a t i o n (286), and has i n fact been a t t r i b u t e d to the a c t i o n of P K C (276-279). Direct evidence that the PTK activity of the E G F and i n s u l i n receptors is required for post-receptor effects, i n c l u d i n g mitogenesis, h a s come f r o m s i t e - d i r e c t e d m u t a g e n e s i s s t u d i e s . A p o i n t m u t a t i o n at the A T P - b i n d i n g s i t e ( l y s i n e 721) of the E G F r e c e p t o r a b o l i s h e s s i g n a l t r a n s d u c t i o n i n E G F - t r e a t e d c e l l s (300). E G F b i n d i n g to these c e l l s , c ompared to n o r m a l cells, does not induce receptor autophosphorylation, changes i n ion fluxes, phosphoinositide metabolism, receptor in t e r n a l i z a t i o n , changes i n c-myc a n d c-fos 46 e x p r e s s i o n (see below), or s i g n i f i c a n t ^ H - t h y m i d i n e i n c o r p o r a t i o n i n t o DNA. The observation that phosphoinositide breakdown is abrogated when the receptor PTK activity i s defective suggests that either P I P 2 hydrolysis or P I P 2 formation is somehow dependent on tyrosine phosphorylation. S i m i l a r studies u s i n g i n s u l i n receptors l a c k i n g ATP b i n d i n g sites i n d i c a t e t h a t i n m u t a n t cell s , i n s u l i n f a i l s to induce receptor a u t o p h o s p h o r y l a t i o n , d e o x y g l u c o s e u p t a k e , S 6 k i n a s e a c t i v a t i o n ( a n d s u b s e q u e n t S 6 p h o s p h o r y l a t i o n ) , e n d o g e n o u s s u b s t r a t e p h o s p h o r y l a t i o n , g l y c o g e n s y n t h e s i s , a n d ^ H - t h y m i d i n e i n c o r p o r a t i o n into D N A (301). In ne i t h e r of these s t u d i e s (300,301) was l i g a n d b i n d i n g capacity affected by receptor mutation. Despite the putative importance of tyrosine phosphorylation i n s i g n a l t r a n s d u c t i o n , few substrates for the growth factor receptor PTKs have, as yet, been identified. Even less is k n o w n regarding the f u n c t i o n a l significance of the substrates that have been identified. In a d d i t i o n to the E G F receptor itself, several proteins r a p i d l y phosphorylated on tyrosine have been detected i n cells treated w i t h E G F (286,287). These i n c l u d e a n 81 k D a protein c a l l e d ezrin, members of the l i p o c o r t i n family (34-39 k D a membrane-associated proteins also k n o w n as c a l p a c t i n s ) , a n d a 42 k D a p r o t e i n (p42). The l i p o c o r t i n s are of interest because they have been reported to in h i b i t the activity of phospholipase A 2. Upon tyrosine p h o s p h o r y l a t i o n of the l i p o c o r t i n s , t h e i r a b i l i t y to i n h i b i t t h i s p h o s p h o l i p a s e i s lost or dimi n i s h e d (302). According to th i s scheme (indicated i n Fig. 2E), tyrosine phosphorylation of the lipocortins would be expected to lead to increases i n prostaglandins (which have been reported to o c c u r i n some EGF-treated cells (287)). The phosphorylation of p42, a protein of u n k n o w n s t r u c t u r e or f u n c t i o n , i s also of i n t e r e s t as it i s p h o s p h o r y l a t e d r a p i d l y in r e s p o n s e to s e v e r a l m i t o g e n i c agents, i n c l u d i n g E G F a n d P D G F (303). A n u m b e r of proteins are tyrosine phosphorylated when target cells are treated with i n s u l i n , i n c l u d i n g the i n s u l i n receptor, a 46 k D a membrane protein (304), a 15 k D a soluble protein (305), and the GDP-bound a sub u n i t of t r a n s d u c i n (306). Again, the physiological importance of these phosphorylation events remains to be determined. It is obvious from t h i s d iscussion, then, that a ful l e r understanding of the mechanism by which tyrosine phosphorylation mediates 47 the actions of certain growth factors w i l l require the id e n t i f i c a t i o n and funct i o n a l characterization of crucial PTK substrates. 1.6. Changes in Gene Expression Growth factor stimulation of quiescent cells brings about transcriptional activation of a number of genes. Treatment of fibroblasts with PDGF and other mitogens that stimulate phosphoinositide hydrolysis induces a transient, 40-fold increase in the expression of c-fos and c-myc (235,243,307). The enhanced expression of c-fos mRNA occurs within 20-45 minutes after PDGF addition, while increased c-myc expression is observed within two hours (235). These changes i n gene expression also occur i n response to treatment of fibroblasts with phorbol esters (307). This observation, coupled with the demonstration that down-regulation of the PKC pathway by prolonged exposure to TPA reduces the induction of c-myc by PDGF (308), has implicated the PKC pathway in the activation of c-myc. This may also be the case for c-fos, although it should be noted that EGF, which does not significantly increase PKC activity, also induces c-fos gene transcription (309). Since c-fos and c-myc both encode nuclear proteins (310), their transient expression may play a role i n the transduction of the mitogenic signal in the nucleus. It will be important to establish how increased expression of these proto-oncogenes results, many hours later, in the initiation of DNA synthesis. Other genes are also induced by growth factors, including P- and y-actin by EGF (311), and genes coding for three mRNAs of unknown function, KC. JE, and JC, by PDGF (312). It remains to be determined if these genes are involved in the regulation of cell growth. 2. Signal Transduction Pathways Involved in Mitogenesis The above discussion has focused on early cellular events elicited by the binding of polypeptide growth factors and other mitogens to quiescent target cells. In many cases, however, only some of the described c e l l u l a r changes can be demonstrated i n cells responding to a particular mitogen. For example, not all growth factor receptors have 48 associated PTK activity, and not a l l growth factors stimulate P I P 2 hydrolysis, PKC activation, or cAMP accumulation. It is therefore important to determine if the observed biochemical alterations in mitogenically stimulated cells can be regarded as components of distinct signal transduction pathways. In fact, one model has proposed that multiple signaling pathways exist which can, depending on the interacting growth factor, be utilized by the cell to deliver a proliferative stimulus to the nucleus (235,243). Growth factors such as EGF and insulin, whose receptors have Intrinsic or associated PTK activity are thought to stimulate tyrosine phosphorylation of c e r t a i n proteins, leading to activation of downstream events involved in DNA synthesis and cell division. Other mitogens, such as bombesin and a-thrombin, are thought to influence cell growth by inducing G protein-mediated activation of PLC, leading to P I P 2 hydrolysis and activation of PKC. A third potential signaling pathway involves the activation of adenylate cyclase by G s, leading to cAMP accumulation, activation of cAMP-dependent protein kinase A, and serine/threonine phosphorylation of c r u c i a l substrates. A number of studies support the concept of alternate signaling pathways for the control of cell proliferation. Cholera toxin (which stimulates G s and hence adenylate cyclase) acts synergistically with EGF to stimulate DNA synthesis i n Swiss 3T3 cells, but has no effect i n combination with prostaglandin E j (235,255). Microinjection of antibodies to PIP 2 abolishes the mitogenic response of NIH-3T3 cells to bombesin and PDGF (both known to induce phosphoinositide breakdown in these cells), but not to EGF, i n s u l i n , or FGF (313). This result suggests that P I P 2 hydrolysis is not required for NIH-3T3 cells to respond to the growth-promoting effects of EGF, i n s u l i n and FGF. In addition, pertussis toxin blocks the mitogenic response of Chinese hamster lung fibroblasts to bombesin and a-thrombin, but not to EGF, FGF, insulin, and IGF-I (314). A similar observation has been reported for Swiss 3T3 cells, where pertussis toxin inhibits bombesin- but not PDGF-induced mitogenicity (315). Since both factors stimulate PIP 2 breakdown in these cells. PDGF either utilizes a pertussis-insensitive G-protein to couple its receptor to PLC, or is capable of acting through an alternative pathway (i.e., tyrosine phosphorylation) in these cells. Another line of evidence to suggest 4 9 t h a t a t l e a s t t w o s e p a r a t e t r a n s d u c t i o n p a t h w a y s c a n b e u t i l i z e d b y t h e s a m e m i t o g e n c o m e s f r o m s t u d i e s w i t h t h e M D A - 4 6 8 h u m a n b r e a s t c a n c e r c e l l l i n e ( 3 1 6 ) . T h e e f fec t s o n M D A - 4 6 8 c e l l g r o w t h o f h i g h c o n c e n t r a t i o n s o f E G F c a n b e b l o c k e d w i t h p e r t u s s i s t o x i n , b u t t h e t o x i n d o e s n o t b l o c k E G F - i n d u c e d t r a n s c r i p t i o n a l a c t i v a t i o n o f c - m y c a n d c - fo s n o r d o e s i t i n h i b i t g r o w t h e f fec t s a t l o w c o n c e n t r a t i o n s o f E G F . T h i s a g a i n s u g g e s t s t h a t E G F c a n a c t i v a t e a l t e r n a t e p a t h w a y s i n t h e s e c e l l s . A n o t h e r p o i n t t h a t i s a p p a r e n t f r o m t h e a b o v e s t u d i e s i s t h a t i t m a y n o t b e p o s s i b l e to g r o u p d i f f e r en t m i t o g e n s s t r i c t l y a c c o r d i n g to t h e s i g n a l p a t h w a y s t h e y a c t i v a t e , s i n c e t h e r e a p p e a r s t o b e m u c h t a r g e t c e l l t y p e v a r i a t i o n i n t h i s r e g a r d . F o r e x a m p l e , w h i l e E G F a p p e a r s t o a c t i n d e p e n d e n t l y o f PIP2 h y d r o l y s i s i n t h e s t i m u l a t i o n o f f i b r o b l a s t s ( p r e s u m a b l y a c t i n g t h r o u g h t y r o s i n e p h o s p h o r y l a t i o n ) ( 2 3 5 , 3 1 4 ) , t h e p h o s p h o i n o s i t i d e p a t h w a y c a n b e u t i l i z e d i n M D A - 2 6 8 b r e a s t c a n c e r c e l l s ( 3 1 6 ) . S i m i l a r l y , b o t h P D G F a n d b o m b e s i n b i n d to r e c e p t o r s a s s o c i a t e d w i t h t y r o s i n e k i n a s e s ( 2 8 7 , 2 8 8 ) a n d a l s o i n d u c e P I P 2 h y d r o l y s i s w i t h a c t i v a t i o n o f P K C ( 2 3 5 ) . H o w e v e r , w h i l e t h e l a t t e r p a t h w a y a p p e a r s to be c r i t i c a l f o r b o t h b o m b e s i n - a n d P D G F - s t i m u l a t e d m i t o g e n e s i s i n s o m e c e l l s ( 3 1 3 ) , i t c a n a p p a r e n t l y b e b y - p a s s e d i n o t h e r c e l l t y p e s r e s p o n d i n g t o P D G F ( 3 1 5 ) . A d d i n g e v e n m o r e c o m p l e x i t y t o t h e c o n c e p t o f m u l t i p l e s i g n a l i n g p a t h w a y s i n t h e c o n t r o l o f c e l l p r o l i f e r a t i o n a r e r e c e n t f i n d i n g s s u g g e s t i n g p o t e n t i a l l i n k s ( o r " c r o s s - t a l k " ) b e t w e e n t h e d i f f e r e n t p a t h w a y s . T h e t y r o s i n e p h o s p h o r y l a t i o n o f t h e l i p o c o r t i n s b y t h e E G F r e c e p t o r a n d o t h e r P T K s m a y l e a d , t h r o u g h i n c r e a s e d p r o s t a g l a n d i n p r o d u c t i o n b y PLA2. t o c A M P a c c u m u l a t i o n . T h e r e i s a l s o e v i d e n c e f o r c r o s s - t a l k b e t w e e n t h e P K C a n d c A M P p a t h w a y s . It h a s r e c e n t l y b e e n s h o w n t h a t p h o r b o l e s t e r - a c t i v a t e d P K C c a n p h o s p h o r y l a t e a n d a c t i v a t e t h e c a t a l y t i c s u b u n i t of b o v i n e b r a i n a d e n y l a t e c y c l a s e ( 3 1 7 ) . E v i d e n c e f o r a l i n k b e t w e e n t h e P T K a n d PIP2 p a t h w a y s i n c l u d e s t h e o b s e r v a t i o n t h a t t h e P T K a c t i v i t y o f t h e E G F r e c e p t o r i s r e q u i r e d for E G F - s t i m u l a t e d PIP2 b r e a k d o w n ( 3 0 0 ) , a n d t h e d e m o n s t r a t i o n t h a t i n s u l i n b i n d i n g s t i m u l a t e s t h e r e l e a s e i n t o t h e c y t o p l a s m o f a n i n o s i t o l - p h o s p h o g l y c a n w i t h c o n c o m i t a n t g e n e r a t i o n o f d i a c y l g l y c e r o l i n t h e m e m b r a n e ( 2 6 8 , 2 7 0 ) . I n a d d i t i o n , i n s u l i n i s k n o w n to i n d u c e t y r o s i n e p h o s p h o r y l a t i o n o f t h e G p r o t e i n , t r a n s d u c i n (306) , a n d t h e r e f o r e m a y a l s o 50 stimulate phosphorylation of other G proteins. Considerable interest was i n i t i a l l y generated by reports that purified PTKs, including the EGF receptor (318), insulin receptor (319), and v-src (320) and v-ros (321) oncogene products, had inositol lipid kinase activity. Although these activities now appear to represent contaminating proteins (287), it Is known that cells transformed with src, ros, and abl show enhanced turnover of inositol lipids (243). It has also been demonstrated that polyoma virus middle T antigen (MTAg), which is closely associated with the c-src product i n infected cells, stimulates the activity of a phosphatidylinositol kinase (PI kinase) (322). If either intrinsic to PTKs or proximally related, the significance of such associated PI kinase activities could be manifold, as phosphorylation of phosphatidylinositol appears to be obligatory for its turnover (286). Interestingly, it has recently been reported that PDGF as well as a complex consisting of the MTAg/c-src product both stimulate the tyrosine phosphorylation of an 85 kDa protein In NIH-3T3 cells, and that this phosphorylation in turn stimulates the PI kinase activity of the 85 kDa protein (323). Future studies will help to unravel the possible implications of these intriguing findings. Rozengurt has put forth the following model in an attempt to provide a framework for understanding the obviously complex network of signaling mechanisms by which diverse extracellular factors regulate cell proliferation (235). This model does not dismiss the notion of multiple signaling pathways in target cells, but rather divides the key events elicited by activation of the signaling pathways into two broad categories: regulatory signals and obligatory events. Regulatory signals can be regarded as i n t r a c e l l u l a r processes that are initiated by the binding of specific classes of mitogens, such as those that induce PIP 2 metabolism. Regulatory signals are crucial for the action of a given factor, but can be bypassed by the regulatory signals generated by another group of factors. Obligatory events, on the other hand, are factor-stimulated biochemical events that must take place in order for DNA synthesis and cell division to occur, regardless of which regulatory events lead to their initiation. Furthermore, while certain regulatory signals are elicited by some mitogens but not by others, any mitogen or combination of mitogens 51 stimulating cell proliferation should stimulate all obligatory events. This may help to explain why some mitogens stimulate proliferation of target cells on their own. while other mitogens do so only in certain combinations. For example, while EGF or phorbol esters alone induce little or no DNA synthesis in Swiss 3T3 cells, the combination of both is very effective in stimulating growth of these cells (235). PDGF, on the other hand, does induce cell proliferation in these cells in the absence of other mitogens (235). Since PDGF stimulates cAMP accumulation, PIP2 metabolism, and PTK activity in these cells, while EGF stimulates only PTK activity, it may be that certain combinations of regulatory signals are required to elicit a common set of obligatory events leading to DNA synthesis. Moreover, since EGF in combination with agents that increase cellular cAMP concentration also promotes cell proliferation in Swiss 3T3 cells, it appears that there are multiple combinations of regulatory events that are capable of generating the required obligatory events for initiating cell growth. Therefore, PIP2 metabolism, PKC activation, cAMP accumulation, and certain tyrosine phosphorylations may represent regulatory signals. It remains to be determined if the activation of c-fos, c-myc, and other genes fall into the category of obligatory events in this model. One candidate as an obligatory event is the activation of cellular ras proteins. The genes encoding this family of proteins have attracted attention because they are frequently mutated in. spontaneous tumours (212-214). Recently, by microinjection of an antibody (Y13-259) that neutralizes c-ras proteins, it was shown that p 2 1 r a s is required for the initiation of DNA synthesis in all cell types tested (324). It was subsequently demonstrated that inhibition of cell proliferation by Y13-259 could not be overcome by TPA (which activates PKC) or prostaglandin F 2 a (which is produced by PLA2 a n c * activates G s); this suggests that ras proteins act downstream of PKC, PLA2- a n o - adenylate cyclase (325). Furthermore, other studies have reported that growth factors and oncogene products related to receptor tyrosine kinases require ras proteins for mitogenic induction (326). A role for phosphatidic acid, itself a mitogen for fibroblasts (327), as a link between signaling pathways and ras protein activation has recently been proposed (325). An overview o f 52 mitogenic signal transduction pathways, incorporating a possible role for ras protein activation as an obligatory event, is presented schematically ln Fig. 3. While the model of regulatory signals and obligatory events in the control of cell proliferation has many obvious gaps, it may be superior to the more established competence-progression model of Pledger (328). The latter model divides mitogens into competence factors (typified by PDGF), which render cells competent to initiate DNA synthesis, and progression factors (typified by EGF), which allow cells to progress through G 0 / G j and to subsequently synthesize DNA. Competence factors, then, are necessary to prime cells for the action of progression factors. However, this model does not account for the ability of some mitogens, including the CSFs, to stimulate the proliferation of target cells in the absence of other factors. Further studies, some of which have been done and are described in later chapters, will help to elucidate which aspects of these or other models accurately describe the complex molecular control of cell growth. 3. Mechanism of Action of the CSFs When the present study was initiated, very limited information was available on the biochemical events occurring when CSFs stimulate hemopoietic target cells to pass through a cell cycle and divide. In the past several years, a number of reports have begun to yield insight into the nature of CSF receptors and the mechanism of signal transduction in response to the CSFs. Since the present study has contributed directly to this field, some of the more recent data from the literature will be described in later sections in the context of the findings presented in this thesis. Binding experiments have established that each CSF binds to its own receptor with absolute specificity (329). This binding occurs rapidly with high affinity, and is essentially irreversible over a period of 24 hours (330-332). These studies have also revealed that the numbers of CSF receptors on target cells are generally low. Scatchard analysis indicates that cells binding GM-CSF and G-CSF have 100-500 receptors/cell (330,333), while cells binding 11-3 and M-CSF have somewhat higher levels of 2,000-16,000 receptors/cell 53 (332 ,333) . In the case of m I L - 3 , F D C - P 1 a n d 3 2 D - c l o n e 2 3 m I L - 3 - d e p e n d e n t ce l l s were repor ted to have 1500 -2500 a n d 4 0 0 0 - 5 0 0 0 r ecep to r s / ce l l , respect ively, w i t h d i s soc i a t i on cons tan t s of 10-60 p M (332). It h a s also been de termined that occupa t ion of on ly 5-10% of C S F receptors i s sufficient for m a x i m a l b io log ica l act ivi ty (330-332). F u r t h e r m o r e , b i n d i n g s t ud i e s at 3 7 ° C have revealed a h i e r a r c h i c a l a b i l i t y of m u r i n e C S F - r e c e p t o r complexes to down-modula te u n o c c u p i e d receptors for other C S F s (329). T h u s , m I L - 3 decreases receptor s i tes for G M - C S F , G - C S F , a n d M - C S F , whi le G M - C S F decreases the b i n d i n g of G - C S F and M - C S F . A l t h o u g h the m e c h a n i s m for down-modu la t i on r ema ins unclear , it does not appear to i nvo lve d i r ec t c o m p e t i t i o n for b i n d i n g s i tes . It h a s b e e n p r o p o s e d t ha t the a b i l i t y of c e r t a in C S F s , s u c h as m I L - 3 a n d G M - C S F , to m i m i c the ac t ions of o ther C S F s m a y resu l t f rom d o w n - m o d u l a t i o n , a n d consequent ac t iva t ion , of u n o c c u p i e d receptors (329). S tud ies u s i n g r a d i o l a b e l e d C S F s a n d c h e m i c a l c r o s s - l i n k i n g agents have recen t ly a l l owed for the i d e n t i f i c a t i o n of the m u r i n e C S F receptors a n d d e t e r m i n a t i o n of t h e i r m o l e c u l a r weights (see Chap te r s 3 a n d 6). R e m o v a l of growth factors from hemopoiet ic target cel ls does not resu l t i n w i t h d r a w a l of these ce l l s in to a quiescent state, as is the case w i t h f ibroblasts a n d the i r g rowth factors. M u r i n e 11-3 d e p l e t i o n f rom m I L - 3 - d e p e n d e n t ce l l s r e su l t s , after a 6 h o u r l ag , i n a loss of v i a b i l i t y w h i c h f o l l o w s f i r s t o r d e r k i n e t i c s (87); h a l f of t he c e l l s d ie eve ry 1-6 h o u r s , depend ing o n the ce l l l ine . T h i s proper ty of CSF-dependen t cel ls c a n be explo i ted to s tudy C S F a c t i o n b y a s k i n g wha t happens w h e n a p a r t i c u l a r C S F is w i t h d r a w n a n d wha t is the effect of i t s r e - a d d i t i o n . T h i s type of s t u d y h a s b e e n u s e d to d e m o n s t r a t e tha t , i n the absence of m I L - 3 , g lucose up t ake fal ls , g lyco lys i s is r educed , a n d there i s a s teady fal l i n in t r ace l lu l a r A T P levels i n F D C - P 2 cells (334,335). These effects o n p r i m a r y m e t a b o l i s m are a l l r e v e r s e d i f m I L - 3 i s r e - a d d e d before the c e l l s d ie . T h u s , the a b i l i t y o f m I L - 3 , a n d p e r h a p s o t h e r C S F s , to ac t a s a s u r v i v a l f ac to r m a y s t e m f r o m i t s effects o n p r i m a r y m e t a b o l i s m . T h e d e m o n s t r a t i o n tha t a n A T P r e g e n e r a t i n g s y s t e m ( c o m p o s e d of A T P , creat ine phosphate , a n d creatine phosphokinase) c o u l d par t ia l ly replace the growth 54 Mitogen prostaglandins activation of | cytosolic kinases growth stimulation Figure 3. Signal transduction pathways in mitogen-stimulated cell proliferation. This figure incorporates many of the biochemical events outlined in Fig. 2A-E into an overview of possible pathways activated during the mitogenic response. A role is shown for ras protein activation (to ras-GTP) as an obligatory event in the induction of cytosolic kinases involved in growth stimulation. In this scheme, first proposed by Stacey and co-workers (see text), DAG is metabolized to phosphatide acid by DAG kinase. Increased levels of phosphatidlc acid result in the activation of ras proteins. PL = phospholipids; (+) = activation. 55 requirement of these cells for mIL-3, suggests that mIL-3 may also mediate at least some of its effects on proliferation by regulating Intracellular ATP levels (334). Of the limited studies on the molecular mode of CSF action, it has been shown that M-CSF stimulates tyrosine-specific autophosphorylation of the M-CSF receptor (the c-fms product) (223,224). Tyrosine phosphorylation of cellular substrates by the PTK activity of this receptor may then mediate, at least in part, the growth-promoting ability of M-CSF. Aside from this, there is essentially no information on functional activities that may be associated with CSF receptors. Changes in the levels of phosphorylation of certain cellular proteins have been reported for purified hemopoietic progenitor cells exposed to GM-CSF (89, 103), although the kinases involved are unknown. In addition, it has recently been reported that binding of mIL-3 to intact cells transiently increases the association of PKC with the plasma membrane (336). This finding, coupled with the demonstration that the combination of a phorbol ester and a calcium ionophore could partially replace the requirement of proliferating FDCP-Mix cells for mIL-3 (337,338) suggests that the PKC system may be involved in mIL-3 signal transduction. The previously mentioned ability of v-myc and v-abl oncogenes, when transfected into mIL-3-dependent cells, to abrogate the growth requirement of these cells for mIL-3 may also provide clues to the mechanism of action of mIL-3 (220,225). A constitutively activated myc gene may bypass the necessity for factor-induced activation, and the PTK of the v-abl product may take the place of endogenous, regulated PTKs involved in signal transduction. In summary, we have only a sketchy knowledge of the mechanisms involved in CSF-regulated growth. At least some aspects of the potential signaling pathways involved appear to be similar to those mediating the regulation of fibroblast growth. If common mechanisms of growth regulation are involved in hemopoietic cells and fibroblasts, it will be important to determine if the ability of CSFs to act as survival factors is independent of or inherent to their growth-promoting activities. It will also be of considerable interest to 56 determine whether all CSFs stimulate a common pathway of growth regulation within the hemopoietic system, in which specificity of a target cell for a growth stimulus is conferred only by the specificity of its CSF receptors, or whether different CSFs utilize different biochemical pathways. It is hoped that answers to these and other questions regarding signal transduction in the hemopoietic system will help to elucidate the mechanism of normal growth regulation, as well as to determine which events lead to loss of growth control and the development of malignancy in the hemopoietic system. D. THESIS OBJECTIVES A biological function for polypeptide growth factors in the growth and development of normal cells is firmly established. In addition, it is becoming increasingly apparent that these regulatory molecules play a role in transformation and maintenance of the neoplastic state. In spite of this, details of the signal transduction pathways activated in growth factor-stimulated cells have only recently begun to appear in the literature. This is especially true in the field of hemopoiesis, where extensive knowledge has accumulated on the biological activities of a set of hemopoietic growth factors that remain relatively uncharacterized in terms of their molecular modes of action. Murine 11-3 appears to be a key regulator of hemopoiesis in the mouse. Elucidating the components of signal transduction in cells responding to this growth factor may therefore provide insights into the design and use of pharmacological agents that can mimic the biological effects of mIL-3 on hemopoiesis. Furthermore, such knowledge may lead to a greater understanding of molecular events underlying the development of malignancy in the hemopoietic system. The overall objective of my work, then, was to gain insight into the mechanism of action of mIL-3. The specific objectives were to: 1. Characterize the binding of mIL-3 to mIL-3-dependent target cells. 57 2. 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I n t e r l e u k i n 3 st i m u l a t e s p r o l i f e r a t i o n via p r o t e i n k i n a s e C activa t i o n without inc r e a s i n g i n o s i t o l l i p i d turnover. Proc Natl Acad Sc i U S A 85: 3284, 1988. 79 C H A P T E R H MATERIALS AND METHODS A. MATERIALS P u r i f i e d r e c o m b i n a n t m u r i n e 11-3 (1) a n d r e c o m b i n a n t m u r i n e G M -CSF (2) were k i n d l y s u p p l i e d by Biogen, Geneva, Switzerland, as were recombinant h u m a n GM-CSF and recombinant h u m a n interleukin-1(3 (11-1(3). Recombinant h u m a n i n t e r l e u k i n - 2 (11-2) was p u r c h a s e d f r o m A m g e n B i o l o g i c a l s , T h o u s a n d Oaks, C a l i f o r n i a , USA, a n d r e c o m b i n a n t m u r i n e i n t e r l e u k i n - 4 (11-4) f r o m / Genzyme, Boston, M a s s a c h u s e t t s , USA. Recombinant m u r i n e i n t e r l e u k i n - 5 (11-5) was g e n e r o u s l y p r o v i d e d by Dr. V. P a e t k a u , U n i v e r s i t y of A l b e r t a . E d m o n t o n , A l b e r t a , C anada. R e c o m b i n a n t h u m a n i n t e r I e u k i n - 6 (11-6) was a g e n e r o u s g i f t f r o m Dr. L. A. A a r d e n , U n i v e r s i t y of A m s t e r d a m , A m s t e r d a m , Th e N e t h e r l a n d s . H u m a n u r i n a r y e r y t h r o p o i e t i n (Ep) was p u r i f i e d i n o u r l a b o r a t o r y as described (3). I n s u l i n was p u r c h a s e d from the Sigma C h e m i c a l Company, while i n s u l i n -l i k e g r o w t h f a c t o r - I (IGF-I) was p u r c h a s e d f r o m B a c h e m F i n e C h e m i c a l s , T o r r a n c e , C a l i f o r n i a , USA. R a b b i t a n t i p h o s p h o t y r o s i n e a n t i s e r u m a n d a f f i n i t y p u r i f i e d r a b b i l a n t i p h o s p h o t y r o s i n e a n t i b o d i e s (4), at a c o n c e n t r a t i o n of 350 ug/ml, were g e n e r o u s l y s u p p l i e d by Dr. Tom Deuel, J e w i s h H o s p i t a l , W a shington U n i v e r s i t y M e d i c a l Center, St. L o u i s , M i s s o u r i . T h e c r o s s - l i n k i n g a g e n t s , s u l p h o s u c c i n i m i d y 1 - 2 - (p -' a z i d o s a l i c y l a m i d o ) - 1 , 3 ' - d i t h i o p r o p i o n a t e (SASD), d i s u c c i n i m i d y l s u b e r a t e (DSS) a n d d i t h i o b i s ( s u c c i n i m i d y l p r o p i o n a t e ) (DSP) were p u r c h a s e d f r o m the P i e r c e C h e m i c a l Company. [ 3 2P]-orthophosphate (8 mCi/ml; carrier-free) i n acid free solution, [methyl-^H] t h y m i d i n e (1 m C i / m l ; 2 Ci/mmole), a n d N a [ 1 2 5 I ] (100. m C i / m l ; carrier-free) were from Amersham. A l l other reagents, i n c l u d i n g TPA and l-oleoyl-2-acetyl-sn-glycerol (OAG), were purchased from the Sigma Chemical Company unless otherwise indicated. 80 B. CELLS The o r i g i n a l mIL-3 dependent ce l l line, B6SUtA, k i n d l y s u p p l i e d by Dr. Joel Greenberger, was derived from a Friend virus infected culture of C57B1.S bone marrow cells (5) and was propagated in our laboratory in RPMI 1640 supplemented with 2 0 % fetal calf serum (FCS) and 5 % Pokeweed mitogen stimulated spleen cell conditioned medium (PWM-SCCM) (6). The clone, BeSUtA,, was obtained by plucking an individual B6SUtA colony growing in methylcellulose. These cells were shown to be mycoplasma-free by the method of Chen (7). The mIL-3 dependent cell lines, FDC-P1, obtained from Dr. Peter Quesenberry, and 32D-clone 23, from Dr. Joel Greenberger, were maintained in RPMI 1640 containing 2 0 % FCS and 5 % PWM-SCCM. The P815 murine mastocytoma cell line was grown in RPMI 1640 supplemented with 1 0 % FCS, while the EL-4 murine T-cell lymphoma cell line was maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 1 0 % FCS. C. CELL PROLIFERATION ASSAYS To measure factor-induced proliferation, B6SUtA or BeSUtA^  cells were washed twice with unsupplemented RPMI 1640 and then incubated at 2 x l 0 5 cells/ml with 2 0 % FCS in RPMI 1640 i n the presence and absence of various factors in a total volume of 0.1 ml in Linbro U-shaped microtitre plate wells. After 22 h at 37°C in a humidified atmosphere of 5 % C 0 2 and 9 5 % air, 20 ul of 3H-thymidine stock containing 50 uCi/ml (2 Ci/mmole) in RPMI 1640 was added to each well to give a final 3H-thymidine concentration of 1 uCi/well. After a further 2 h incubation at 37°C, the well contents were harvested onto glass fiber filters using a microharvester (Richter Scientific, Vancouver, Canada) and assayed for radioactivity using a Beckman LS7500 liquid scintillation counter. In some experiments, the effect on cell viability of short term incubation with various factors was determined. Cells were seeded at 2 x l 0 5 cells/ml in RPMI 1640 + 2 0 % F C S containing the indicated agents, and counted after 24 h (day 1) or 48 h (day 2) in culture. 81 Viable cell counts were determined by Trypan blue dye exclusion, and represent the means of 2 cultures for each data point. D. IODINATION OF mil-3 The iodination of mIL-3 was performed using the chloramine T method (8). Briefly, aliquots of mIL-3 containing 1-2 ug of the growth factor were resuspended i n a total of 50 ul of O.l M sodium phosphate, pH 7.0, containing 1 0 % dimethyl sulphoxide and 100 ug/ml polyethylene glycol 6000. To this was added 10 ul of Na 1 2 5 I (lmCi), followed by 10 ul of freshly prepared 1 mg/ml chloramine T. This solution was incubated for 30 min at 4°C, after which 10 ul of freshly prepared 3 mg/ml sodium metabisulfite and 10 ul of 0.1 M potassium iodide were added. After a 5 minute Incubation at room temperature, 10 ul of 10 mg/ml bovine serum albumin (BSA) were added as carrier protein. To separate iodinated mIL-3 from unbound 1 2 ^ I , samples were chromatographed on 4 ml P2 columns (Bio-Rad) equilibrated with phosphate-buffered saline (PBS) containing 0.02% Tween 20 (Bio-Rad). The void volume fractions were then pooled, made 0.01% BSA, and frozen in aliquot at -70°C. Following iodination, mIL-3 retained >95% of its biological activity as assessed by the ability of material to stimulate ^H-thymidine incorporation in B6SUtA cell proliferation assays. E. BINDING STUDIES WITH 1 2 5 I - m i l - 3 Cells to be tested for 1 2^I-mIL-3 binding were washed three times in binding buffer, which consisted of PBS containing 0.1% BSA and 100 ug/ml ba c i t r a c i n (to prevent internalization of the ligand-receptor complex (8)). Cells were then resuspended in binding buffer at a concentration of 1.2 x 1 0 6 - ,1.2 x 10 7/ml (B6SUtA and B6SUtAj cells) or 1.2 x 10 7/ml (FDC-P1 and P815 cells) and transferred to 0.5 ml Eppendorf microtubes in 100 ul aliquots. To half the samples were added 10 ul of binding buffer while the remainder 82 received 10 u.1 of a 20-fold excess of unlabeled recombinant mIL-3 (to account for non-specific binding) in binding buffer. The binding reaction was initiated by the addition of 10 ul aliquots of binding buffer containing various concentrations of 1 2^I-mIL-3. Following incubation of the microtubes at 37°C for 1 hr (or various times as indicated) in a Queue Orbital Shaker, 100 ul aliquots from each microtube were layered over 250 ul of a 1.5:1 mixture of dibutyl phthalate and dioctyl phthalate, respectively, and centrifuged at 13,000 i nc g for 1 min i n a Beckman microfuge. The oil phase excludes the aqueous unbound l z o I -mIL-3 while allowing cells to sediment, thereby separating bound from free 1 2^I-mIL-3 (8). The microtubes were frozen and the tips excised and counted in a Beckman Gamma 5500 counter. Results have been corrected for non-specific binding (specific binding = total b i n d i n g - binding i n the presence of a 20-fold excess of unlabeled mIL-3), and the duplicates were within ± 5% of the S.E.M. F. RADIOLABELING OF THE mil-3 RECEPTOR USING SASD 1 2^I-SASD was prepared by dissolving SASD in acetone (to 20 umoles/ml) in a glass tube i n the dark, and then mixing a 100 ul aliquot (2 umoles) with 20 ul of 10 mg/ml chloramine T (0.75 umoles), prepared in acetone, and 5 ul (2.5 x 10" 4 umoles) N a 1 2 ^ I (17 mCi/ug, 100 mCi/ml, Amersham, carrier free) for 2 min at 23°C. The 1 2 5I-SASD-mIL-3 was formed by incubating 12 ug pure recombinant mIL-3 (16) i n 90 ul 0.1 M sodium phosphate buffer, pH 9.0 + 0.02% Tween 20 with 10 ul of 1 2 5 I - S A S D in acetone plus 6 ul DMSO for 30 min at 23°C. This solution was then passed through a 5 ml 200-400 mesh P-2 column (Bio-Rad) equilibrated with PBS, pH 7.4 + 0.02% Tween 20 and eluted with the same buffer. Two hundred ul fractions were collected and assayed for radioactivity using a Beckman Gamma 5500 gamma counter. The void volume peak, consisting of 1 2^I-SASD-mIL-3, was pooled, yielding on average 400,000-800,000 cpm in 1 ml of solution. Identification of the mIL-3 receptor using 1 2 5I-SASD-mIL-3 was carried out by preparing experimental test samples as follows, and then analyzing them by SDS-83 pol y a c r y l a m i d e gel electrophoresis. One h u n d r e d u l a l i q u o t s of the pool (40,000-80.000 cpm) were c o m b i n e d with either 50 ul of PBS, p H 7.4 + 0.02% Tween 20 (see C h a p t e r III, F i g u r e 7. l a n e s A, B, a n d D-I), or 50 u l of P B S p H 7.4 + 0.02% Tween 20 c o n t a i n i n g a 20-fold excess of unlabeled recombinant mIL-3 (lane C). For samples cont a i n i n g cells, 150 p i aliquots of the above were used to resuspend 2 x 10^ B 6 S U t A cells (lanes A-C and G) or 2 x 1 0 6 P815 cells (lanes D and H) previously washed 3x w i t h PBS, p H 7.4, containing 800 u g / m l b a c i t r a c i n (to prevent i n t e r n a l i z a t i o n of l i g a n d - r e c e p t o r c o m p l e x e s (8)). These samples p l u s control samples containing no cells (lanes E, F, and I) were th e n incubated at 37°C for 1 h, with a fi n a l concentration of 100 ug/ml bacitracin, i n a Queue O r b i t a l Shaker at 180 r p m to effect ligand-receptor b i n d i n g . A l l the above procedures were c a r r i e d out under dim (40w) red light and a l l reaction vessels were covered i n a l u m i n u m foil. Following the i n c u b a t i o n , samples to be irradiated (lanes B-D a n d F-I) were placed i n L i n b r o 96 well r o u n d bottom trays and exposed to 460 u w / c m 2 U V i r r a d i a t i o n u s i n g a Blak-Ray UVL-21 lamp at 14 c m f r o m the source for 30 m i n at 23°C. Other samples (lanes A a n d E) were kept i n the d a r k for 30 m i n at 23°C. After the 30 m i n period, the samples c o n t a i n i n g cells were w a s h e d 2 x w i t h PBS, p H 7.4. T h e n a l l s a m p l e s were b o i l e d f o r 2 m i n i n f i n a l c o n c e n t r a t i o n s of 3 % SDS, 1 0 % glycerol and 3 % p-mercaptoethanol (fi-ME) (lanes A-F) or 3 % S D S a n d 1 0 % glycerol (lanes G-I). Two lanes between lanes F a n d G c o n t a i n e d only S D S sample buffer w i t h fJ-ME (next to lane F) and without P-ME (next to lane G) a n d have been omitted from Figure 7 for the sake of brevity. Labeled proteins were then separated by o n e - d i m e n s i o n a l gel e l e c t r o p h o r e s i s (as de s c r i b e d below) u s i n g a 5 - 1 5 % g r a d i e n t SDS-polyacrylamide gel. P r e p a r a t i o n of r a d i o l a b e l e d mIL-3 r e c e p t o r s a m p l e s f o r t w o - d i m e n s i o n a l gel electrophoresis was as follows. B 6 S U t A cells (4 x 10^ cells) were incubated i n PBS, pH 7.4 con t a i n i n g 100 ug/ml b a c i t r a c i n and 0.02% Tween 20 for 1 h at 37°C i n the dark with 200 pi 1 2 5 I - S A S D - m I L - 3 (212,000 cpm) i n a to t a l volume of 300 ul. Following i r r a d i a t i o n and washing as described above, cells were suspended i n 1 m l 10 mM T r i s - C l , pH 7.4, 0.1 mM phenylm e t h y l s u l p h o n y l fluoride (PMSF) and allowed to swell for 5 m i n at 23°C. They were 84 then passed through a 26 gauge needle 6 times to lyse the cells and the released nuclei were pelleted at 800 g for 5 min. The supernatant was microfuged (Eppendorf microfuge #5412) at top speed for 30 min at-4°C to pellet the larger membrane fragments and these fragments were then dissolved by adding 10 ul of 2 % SDS, 5 % fj-ME and boiling for 1 min. Eight volumes of a buffer containing 9 M urea, 4% NP40, 2 % ampholines (1:1.5 mixture of 3-10 (Bio-Rad) and 5-8 (Pharmacia)) and 2 % p-mercaptoethanol were then added and the sample electrophoresed as described below. G. ONE- AND TWO-DIMENSIONAL GEL ELECTROPHORESIS Samples for one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were adjusted to 2 % SDS. 5 % p-mercaptoethanol, 1 0 % glycerol and boiled for 1 min prior to electrophoresis at 20 mA/gel on one-dimensional 10%, 5-15% or 8-20% gradient SDS-polyacrylamide gels, as described by Laemmli (9). In some experiments, samples were analysed by two-dimensional gel electrophoresis using the method of O'Farrell (10), with the following modifications. Cytosolic samples to be analyzed by two-dimensional gel electrophoresis (see Chapter IV) were first passed through a 10 ml Sephadex G25 column (Pharmacia) to remove digitonin. Proteins were then acetone precipitated and redissolved in 9 M urea, 4% NP40, 2 % ampholines (a 1:1.5 mixture of 3-10 (Bio-Rad) and 5-8 (Pharmacia), and 2 % P-mercaptoethanol (isoelectric sample buffer) for electrophoresis. Membrane samples (see Chapter IV) were solubilized in SDS sample buffer, boiled for 1 min, and diluted by the addition of 8 volumes of isoelectric sample buffer. Samples from immunoprecipitation experiments using antiphosphotyrosine antibodies (see Chapter V) to be analyzed by two-dimensional gel electrophoresis were prepared by the addition of 8 volumes of isoelectric sample buffer, except that only Bio-Rad 3-10 ampholines were used. A l l samples were then microfuged for 15 min prior to electrophoresis. They were then loaded onto 2 mm diameter isoelectric gels consisting of 8 M urea, 3.54% acrylamide (from a stock containing 3 0 % total acrylamide, 5.3% cross-85 linker), 2 % NP40, and 2 % ampholines. Samples were then overlayed with a solution consisting of 5 M urea, 2 % NP40 and 2 % ampholines. Isoelectric focusing was then performed for 16 h at 400 volts, followed by 2 h at 800 volts. The electrode solutions consisted of 25 mM H 3 P O 4 and 50 mM NaOH. After isoelectric focusing, gels were equilibrated i n SDS sample buffer for 15 min at 23°C. The second dimension consisted of SDS-PAGE on 8-20% gradient gels (Chapter IV) or 1 0 % gels (Chapter V). Gels were then dried and exposed to XAR-5 film (Kodak) at -70°C. To determine pH gradients in isoelectric gels, 1-cm slices were sealed and shaken in scintillation vials containing 5 ml of degassed double distilled water for 2 h at 23°C and the pH of each fraction measured. H. LABELING OF CELLS WITH ( 3 2P) ORTHOPHOSPHATE Cells were grown to near confluence and then washed twice with unsupplemented RPMI 1640. Cells were then resuspended and grown overnight at 37°C in RPMI 1640 supplemented with 2 0 % FCS and 0.5% PWM-SCCM, with no loss of viability as assessed by Trypan blue dye exclusion. The culture medium was then changed to RPMI 1640 containing 1 0 % FCS and incubated a further 3 h at 37°C, again with no loss of viability. Cells were then washed twice with phosphate-free RPMI 1640 and incubated in phosphate-free RPMI 1640 containing 0.25 mCi/ml carrier-free [ 3 2 P ] orthophosphate for 60-90 minutes at 37°C. I. STIMULATION WITH FACTORS AND SUBCELLULAR FRACTIONATION After 3 2 P - l a b e l i n g , 0 . 2 - 5 x l 0 7 cells were resuspended at 2 x l 0 7 c e l l s / m l and incubated at 37°C with or without mIL-3, mGM-CSF, TPA. or OAG in PBS + 0.1% BSA for various time intervals as indicated. Examination of cytosolic phospho-proteins was carried out by digitonin extraction of stimulated and unstimulated cells as described (11,12). 86 Briefly, after i n c u b a t i o n with factors, 200 ul cel l aliquots (4x10^ cells) were q u i c k l y added to the upper phase of a two phase system consisting of 800 p i of 4 mg/ml di g i t o n i n i n 10 m M T r i s - H C l , p H 7.4, 50 m M NaF, 10 mM EDTA, 5 m M EGTA, 200 m M sucrose, 1 m M freshly a d d e d PMSF, l a y e r e d over 200 p i of 90:110 bromodecane:bromododecane ( A l d r i c h a n d Sigma, respectively). After 10 sec, samples were microfuged for 1 minute at 14,000 rpm i n a n Eppendorf #5415 microfuge. A n aliquot of the upper phase was t h e n analyzed by two-d i m e n s i o n a l g e l e l e c t r o p h o r e s i s . C y t o s o l i c p r o t e i n s were a l s o i s o l a t e d u s i n g more c o n v e n t i o n a l methods. S p e c i f i c a l l y , 3 2 P - l a b e l e d c e l l s were homogenized on ice by 30 strokes of a Wheaton homogenizer, passed 3x through a 26 gauge needle, a n d centrifuged at 8 0 0 g to remove n u c l e i a n d u n b r o k e n c e l l s . S u p e r n a t a n t s were t h e n c e n t r i f u g e d at 100,000 g for 1 h to pellet the particulate material. Cytosolic phosphoproteins were then acetone-precipitated from the resulting supernatants, and analyzed by two-dimensional gel electrophoresis. F o r a n a l y s i s of membrane proteins, membranes from 0 . 2 - 5 x l 0 7 c e l l s were prepared by f i r s t a d d i n g 5 volumes of ice-cold P B S to al i q u o t s of s t i m u l a t e d or u n s t i m u l a t e d cells a n d t h e n centrifuging for 5 minutes at 250 g. The cel l pellets were resuspended i n ice-cold h o m o g e n i z a t i o n b u f f e r c o n s i s t i n g of 10 m M T r i s - H C l , p H 7.4, 25 m M NaF. 100 u M a m m o n i u m m e t a v a n a d a t e , 1 m M p h e n y l p h o s p h a t e , 1 m M Z n C l 2 > 10 m M (3-glycerol phosphate, 10 m M s o d i u m pyrophosphate, 100 k a l l i k r i e n i n h i b i t o r u n i t s / m l a p r o t i n i n , 40 ug/ml l e u p e p t i n , 1 m M f r e s h l y added P M S F a n d homogenized on ice by 30 s t r o k e s of a Wheaton homogenizer. The homogenates were then passed 3x through a 26 gauge needle a n d c e n t r i f u g e d at 800 g for 5 m i n u t e s at 2°C to pellet n u c l e i a n d u n b r o k e n ce l l s . The supernatants were layered onto 4 1 % sucrose c u s h i o n s a n d centrifuged at 50,000 g for 1 h at 4°C u s i n g a n SW41 rotor i n a B e c k m a n L8-80M u l t r a c e n t r i f u g e . The m a t e r i a l at the interface was t h e n ce n t r i f u g e d at 80,000 g for 1 h at 4°C, a n d the r e s u l t i n g p e l l e t s were analyzed by one- or two-dimensional gel electrophoresis. In some experiments, the method of B e l s h a m et a l (13) was u s e d to determine i f samples e n r i c h e d s p e c i f i c a l l y for p l a s m a membranes gave r e s u l t s s i m i l a r to samples prepared as above. The pellet from the above 87 80,000 g spin was resuspended i n 500 u.1 of 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 5 mM EDTA, 0.5 mM freshly added PMSF (extraction buffer) and diluted in 8 ml of a 7:1:32 volumetric ratio of Percoll (Pharmacia; d=1.13)/80 mM Tris HCI, pH 7.4, 2 M sucrose, 8 mM EDTA. 0.5 mM freshly added PMSF/extraction buffer. The suspension was centrifuged at 10,000 g for 30 min at 4°C in a fixed angle rotor using a Beckman J2-21 centrifuge. The plasma membrane band, clearly visible at the top of the gradient (13), was then diluted with 5 volumes of ice-cold 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl. 1 mM EGTA, 0.5 mM freshly added PMSF and centrifuged at 80,000 g for 1 h at 4°C. The r e s u l t i n g pellet was subsequently analyzed by gel electrophoresis. J. PHOSPHOAMINO ACID ANALYSIS The analysis of phosphoamino acids was based on the method of Hunter and Sefton (14). Phosphoproteins of interest were localized by autoradiography of unfixed gels dried onto cellulose acetate sheets. Phosphoproteins were electro-eluted from gel slices into 12.5 mM Tris, 96 mM glycine, 0.05% SDS, dialyzed against 0.01% SDS i n distilled water, and lyophilized. Residues were dissolved in 300 ul of 6 M HCI, boiled for 1.5 h at 110°C under N 2 i n sealed tubes, diluted with 2 ml distilled H 20 and lyophilized a further 2 times. The dried residues were then dissolved i n 5 0 % ETOH containing 1 mg/ml phosphoserine, phosphothreonine, and phosphotyrosine standards and applied to cellulose thin-layer plates. The plates were wetted evenly with a solution consisting of either 1:10:189 pyridine/acetic acid/water or 1:10:189 formic acid/acetic acid/water. Electrophoresis was performed at 1000 V for 60 min at room temperature using either solvent system. The 3 2 p -labeled phosphoamino acids and the standards were detected by autoradiography and ninhydrin staining, respectively. 88 K. AFFINITY PURIFICATION OF ANTIPHOSPHOTYROSINE ANTIBODIES Rabbit antiphosphotyrosine antibodies were obtained from Dr. Tom Deuel (Jewish Hospital. Washington University Medical Centre, St. Louis. Missouri) (4), either in the form of affinity purified antibodies, at a concentration of 350 ug/ml in normal saline, or as rabbit antiserum. Both sources were from rabbits immunized with the phosphotyrosine analog, azobenzophosphonate. In the case of rabbit a n t i s e r u m received from Dr. Deuel, antiphosphotyrosine antibodies were purified using a phosphotyrosine-linked Sepharose 4B column. This column was prepared by first dissolving 100 mg of phosphotyrosine in 10 ml of 0.1 M NaHC0 3, 0.5 M NaCl , pH 8.3. This solution was then added to 5 ml of swollen cyanogen bromide-activated Sepharose 4B beads (Pharmacia), and shaken overnight at 4°C. Remaining binding sites were then blocked by incubation of beads with 10 ml of 1 M ethanolamine for 2 h at 23°C. Beads were then washed with alternating 50 ml solutions of 0.1 M NaHC0 3, 0.5 M N a C l , pH 8.3, and 0.1 M sodium acetate. 0.5 M N a C l , pH 5.0, a total of 2 times for each solution. Washed beads were then placed in a 10 ml Pharmacia column. Prior to being loaded on to the column, rabbit antiphosphotyrosine antiserum was adjusted to 50 mM NaF, 1 mM N a 3 V 0 4 and allowed to stand at 4°C for 30 min. The antiserum was then centrifuged at 10.000 g for 15 min at 4°C. After equilibration of the phosphotyrosine column with 10 mM sodium phosphate, 200 mM N a C l , pH 7.4. the sample was applied and allowed to cycle through the column 5 times at 4°C. The column was then washed successively with 8 ml 10 mM sodium phosphate, 200 mM NaCl, pH 7.4, 8 ml 200 mM sodium phosphate, pH 7.4, and 8 ml 50 mM HEPES, pH 7.4, all at 4°C. Antiphosphotyrosine antibodies were then eluted with 8 ml of a solution containing 50 mM HEPES, 200 mM p-nitrophenylphosphate, pH 7.4 at 23°C. Fractions of 0.7 ml were collected and analyzed for protein content by the Coomassie protein assay; active fractions were then pooled and dialyzed extensively against PBS + 0.05% NaN 3. The presence of immunoglobulin was confirmed by SDS-PAGE. 89 L. WESTERN BLOT ANALYSIS OF CYTOSOLIC AND MEMBRANE PROTEINS WITH  ANTIPHOSPHOTYROSINE ANTIBODIES Western blot analysis was-performed as described by Burnette (15), with slight modifications. Cytosolic and membrane fractions from B6SUtAi cells incubated with or without mIL-3, mGM-CSF or TPA were prepared as described above, except that cells were not 3 2 P - l a b e l e d . Samples were electrophoresed on 8-15% polyacrylamide gels and electrophoretically transferred onto 0.45 pm nitrocellulose (Schleicher and Schuell) at 150 mA for 16 h at room temperature. Filters were washed with 50 mM Tris-HCl, pH 7.4, 0.15 M N a C l and then incubated overnight In 50 mM T r i s - H C l , pH 7.4, 0.15 M N a C l , 0.1% NP40. 5 % BSA at 4°C. Filters were then incubated for 2 h with 3.5 ug/ml affinity purified antiphosphotyrosine antibodies or 4 ug/ml protein A-sepharose purified rabbit IgG i n the above Tris-HCl/BSA buffer at room temperature. After extensive washing with the Tris-H C l / B S A buffer, the filters were incubated for 4 h i n T r i s - H C l / B S A buffer containing 5x10^ cpm/ml 1 2 ^ I - l a b e l e d protein A (Amersham). The filters were washed extensively with Tris-HCl/BSA buffer, air-dried, and subjected to autoradiography at -70°C. M. IMMUNOPRECIPITATION OF PHOSPHOPROTEINS WITH ANTIPHOSPHOTYROSINE  ANTIBODIES BeSUtAn cells were labeled with [ 3 2P] orthophosphate and incubated with or without mIL-3 for various times at 37°C as described above. Cells were washed and solubilized at 4°C for 20 minutes i n a solution containing 50 mM HEPES. pH 7.4, 1% NP40, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 2 mM sodium orthovanadate, 4 mM EDTA, 1 mM (freshly added) PMSF, 100 kallikrien inhibiting units/ml aprotinin, and 40 ug/ml leupeptin (lysis buffer). Samples were then centrifuged for 15 minutes at 15,000 g at 2°C to remove insoluble material and then dialyzed at 4°C against lysis buffer without aprotinin and leupeptin. Phosphotyrosine-containing proteins were immunoprecipitated from dialyzed whole-cell extracts by the addition of antiphosphotyrosine antibodies (1:100 90 final dilution) for 2 hours at 4°C, with subsequent incubation of the samples for 1.5 hours at 4°C with protein A-sepharose beads to immobilize the antibody-antigen complexes. After the precipitates were washed three times with lysis buffer containing 0.1% SDS and 150 mM NaC l at 4°C, phosphotyrosine-containing proteins were eluted from beads by addition of a solution containing 10 mM p-nitrophenyl phosphate, 0.1% NP40, 1.0 mM (freshly added) PMSF, and 25 mM HEPES, pH 7.4. The eluates were then analyzed by one-dimensional SDS-PAGE on 10% SDS-polyacrylamide gels. N. ANTIPHOSPHOTYROSINE ANTIBODY IMMUNOPRECIPITATION AFTER CHEMICAL  CROSS-LINKING OF 1 2 5 I - m i l - 3 TO INTACT CELLS B e S U t A ^ cells were incubated overnight i n growth factor-depleted medium as described i n section H. After a 3 h incubation i n RPMI 1640 + 1 0 % FCS, cells were resuspended i n PBS + 0.1% B S A at 2 x l 0 7 cells/ml. Aliquots of 1 2 5 I - m I L - 3 ( 9 x l 0 4 cpm/ng) were then added to give a final concentration of 1x10^ cpm/2xl0^ cells. After a 15 min incubation at 37°C, cells were washed with ice-cold PBS and resuspended in PBS + 1 mM MgCl2> again at 2 x l 0 7 c e l l s / m l . To the c e l l s u s p e n s i o n was then added disuccinimidyl suberate (DSS) or dithio (succinimidyl propionate) (DSP) to give a final concentration of 1 mM crosslinking agent. Crosslinking was allowed to proceed for 30 min on ice, after which the cells were washed with 20 mM Tris-HCl. 150 mM N a C l , pH 7.4, c o n t a i n i n g 1 mM EDTA. S o l u b i l i z a t i o n of c e l l s and i m m u n o p r e c i p i t a t i o n with a n t i p h o s p h o t y r o s i n e antibodies was then performed as described i n se c t i o n M. Immunoprecipitated proteins were analyzed by SDS-PAGE on 1 0 % SDS-polyacrylamide gels. 91 O. FLUORESCEINATION OF mIL-3 M u r i n e IL-3 was incubated with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester ( F L U O S ) at a m o l e r a t i o of 1:100 i n 2 0 0 m M N a H C 0 3 , p H 8, 0.2% T w e e n 20. A f t e r i n c u b a t i o n at 23°C for 1.5 h, unreacted F L U O S was hydrolyzed by the ad d i t i o n of 1 M Tris-C l , p H 7.5. F l u o r e s c e i n a t e d m I L - 3 ( F l - m I L - 3 ) w a s t h e n s e p a r a t e d f r o m f r e e c a r b o x y f l u o r e s c e i n by gel f i l t r a t i o n t h r o u g h a G-25 Sephadex c o l u m n e q u i l i b r a t e d w i t h PBS, 0.01% gelatin. Preparations of Fl-mIL-3 (2 ug/ml) retained > 9 0 % of t h e i r b i o l o g i c a l activity, assessed as for iodinated mIL-3. P. BIOTINYLATION OF mIL-3 M u r i n e IL-3 and s u l f o s u c c i n i m i d y l 6-(biotinamido) hexanoate (NHS-LC-Biotin), at a mole ratio of 1:1000, were allowed to react i n 100 m M N a H C 0 3 , pH 8, 0.2% Tween 20 for 5 h at 23°C. The r e a c t i o n was th e n t e r m i n a t e d by the a d d i t i o n of £ M T r i s - C l , p H 7.5 and biotinylated mIL-3 (Bi-mIL-3) was separted from free b i o t i n by gel f i l t r a t i o n chromatography as d e s c r i b e d above. Bi-mIL-3 p r e p a r a t i o n s (2 pg/ml) r e t a i n e d > 9 0 % of t h e i r b i o l o g i c a l activity. Q. AFFINITY PF<£CIPITATION OF 3 2 P - L A B E L E D PROTEINS USING F L U O R E S C E I N A T E D  A N D BIOTINYLATED mIL-3 3 2 P - l a b e l e d B 6 S U t A i cells were suspended i n PBS, 1 % B S A at 2 x 1 0 7 c e l l s / m l and aliq u o t s c o n t a i n i n g 1 0 7 cells i n c u b a t e d at 37°for 5 m i n with 50 pi of either Fl-mIL-3, B i -mIL-3, or control buffers as indicated i n the text. Cells were th e n washed with Ice-cold PBS, s o l u b i l i z e d i n the above described l y s i s buffer c o n t a i n i n g 0.5% NP40 i n s t e a d of 1 % NP40 an d c e n t r i f u g e d at 16,000 g for 30 m i n at 4°C. S u p e r n a t a n t s (cell lysates) were d i l u t e d 2-fold w i t h l y s i s b u f f e r m i n u s NP40, a n d pre-cleared by i n c u b a t i n g for 1 h at 4°C w i t h 92 glycine blocked Affi-Gel 10 beads (Bio-Rad), which were subsequently removed by centrifugation. Pre-cleared cell lysates from cells treated with Fl-mIL-3 were then incubated with 5.4 ug/ml mouse anti-fluorescein antibody (IgG) (which binds fluorescein) for 2 h at 4°C. Antibody-antigen complexes were immobilized by addition of goat anti-mouse IgG antibodies coupled to Affi-Gel 10 beads (Bio-Rad) for 1.5 h at 4°C. Beads were washed 3 times with lysis buffer containing 0.25% NP40, 150 mM NaCl and bound 3 2 P - l a b e l e d proteins eluted by incubation in 0.2 M citrate, pH 3.5, 150 mM NaCl for 1 hr at 4°C. Pre-cleared lysates from Bi-mIL-3-treated cells were incubated with streptavidin-agarose beads for 2 h at 4°C and beads washed 3 times with washing buffer. Bound 3 2 P - l a b e l e d proteins were eluted as described above. Eluted proteins were analyzed by SDS-PAGE using 1 0 % polyacrylamide gels. 93 R. REFERENCES 1. Kindler V, Thorns B, de Kossodo S et al. Stimulation of hematopoiesis in vivo by recombinant bacterial murine interleukin 3. 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In s i t u detection of mycoplasma co n t a m i n a t i o n i n c e l l c u l t u r e s by fluorescent Hoechst 33258 stain. Exp Cell Res 104: 255, 1977. 8. P a l a s z y n s k i EW, Ihle JN. Evidence for specific receptors for i n t e r l e u k i n 3 on lymphokine dependent cell lines established from long-term bone marrow cultures. J Immunol 132: 1872, 1984. 9. Laemmli VK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680, 1970. 10. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007, 1975. 11. J a n s k i AM, Cornell NW. Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes. Biochem J 186: 423, 1980. 12. Garrison JC. Measurement of hormone-stimulated protein phosphorylation in intact cells. Methods of Enzymology 99: F, 20. 1983. 13. Belsham GJ, Denton RM, Tanner MJA. Use of a novel rapid preparation of fat-cell plasma membranes employing Percoll to investigate the effects of i n s u l i n and adrenaline on membrane protein phosphorylation in intact cells. Biochem J 192: 457, 1981. 14. H u n t e r T. S e f t o n BM. T r a n s f o r m i n g gene p r o d u c t of Rous s a r c o m a v i r u s phosphorylates tyrosine. Proc Natl Acad Sci USA 77: 1311, 1980. 15. Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195, 1981. 94 C H A P T E R III IDENTIFICATION OF T H E MURINE INTERLEUKIN-3 R E C E P T O R USING A C L E A V A B L E , PHOTOREACTIVE CROSSLINKING A G E N T A. INTRODUCTION M u r i n e IL-3 is a lymphokine that s t i m u l a t e s the p r o l i f e r a t i o n a n d di f f e r e n t i a t i o n of both pluripotent hemopoietic stem cells a nd their committed progeny. However, very little is k n o w n about the mechanism by w h i c h t h i s growth factor el i c i t s i t s effects on responsive c e l l populations. Since characterization of the cel l surface receptor for mIL-3 s h o u l d yield i m p o r t a n t i n f o r m a t i o n c o n c e r n i n g the m e c h a n i s m of a c t i o n of mIL-3 a n d w h e t h e r i t s r e c e p t o r h a s t y r o s i n e k i n a s e , l i p i d k i n a s e , or other a c t i v i t i e s , s t u d i e s were i n i t i a t e d to isolate the receptor and study its properties. The f i r s t step i n the c h a r a c t e r i z a t i o n of a c e l l surface receptor is to demonstrate its expression o n target cel l populations. In the first part of t h i s chapter, the b i n d i n g of 1 2 ^ I -labeled, bioactive mIL-3 to v a r i o u s mIL-3-dependent c e l l l i n e s i s described. These studies demonstrate that a B 6 S U t A cell subline, derived i n our laboratory from the parent B 6 S U t A li n e (itself derived f r o m a F r i e n d v i r u s infected c u l t u r e of m u r i n e C57B1.S bone marrow c e l l s (1)), e x p r e s s e s u n u s u a l l y h i g h l e v e l s of mIL-3 s u r f a c e r e c e p t o r s . B 6 S U L A c e l l s therefore served as a r i c h source of mIL-3 receptors i n subsequent studies. The next step i n receptor characterization i s to determine the m o l e c u l a r mass of the receptor. C h e m i c a l c r o s s l i n k i n g agents have been used to identify cell surface receptors in various ligand-receptor systems (2-7). Preliminary studies i n our laboratory suggested that s u l f o s u c c i n i m i d y l 2 ( p - a z i d o s a l i c y l a m i d o ) - 1 , 3 ' - d i t h i o p r o p i o n a t e ( S A S D ) , a new heteroblfunctional, iodinatable, cleavable, photoreactive c r o s s l i n k i n g agent, c o u l d be used. S A S D h a s several advantages over more c o n v e n t i o n a l c r o s s l i n k i n g agents. F i r s t l y , the 95 heteroblfunctionality of SASD allows each of the two reactive moieties to be linked to proteins under different conditions. Secondly, it can be radiolabeled with 1 2 £ T such that cleavage of the ligand-receptor complex, once formed, results i n the labeled moiety remaining with the receptor. Therefore, an accurate determination of the molecular mass of the receptor itself, as opposed to the ligand-receptor complex, can be made. The second part of this chapter describes the identification of the mIL-3 receptor on B6SUtA cells using SASD and other crosslinking agents. B. RESULTS 1. Binding of 1 2 5I-mIl-3 to mIl-3-Dependent Cell Lines A number of murine cell lines were screened for their ability to bind 1 2 ^ I - l a b e l e d mIL-3 under equilibrium binding conditions. For this purpose, pure recombinant mIL-3 was 1 2^I-labeled using the chloramine T method (8). This resulted in no detectable loss of biological activity as determined by 3H-thymidine incorporation into B6SUtA cells (9). Binding experiments were performed by incubating 1x10^ cells with 9x10^ cpm of 1 2 5 i -labeled bioactive mIL-3 for 1 h at 37°C as described in Chapter II. Table 2 indicates that the mIL-3-dependent cell lines. B6SUtA, FDC-P1, and 32D-clone 23, all demonstrated specific binding of ^ 2^I-mIL-3. while specific binding could not be demonstrated for the mlL-3-independent P815 murine mastocytoma and EL-4 murine T-cell lymphoma cell lines. Table 2 further indicates that the B6SUtA cell line grown in our laboratory (see Chapter II) bound higher levels of 1 2 5I-mIL-3 per cell than did either FDC-P1 or 32D-clone 23 cells. These data suggested that this B6SUtA cell subline expresses increased levels of mIL-3 surface receptors in comparison to FDC-P1 and 32D-clone 23 cells. To further characterize the binding of 1 2^I-mIL-3 to B6SUtA cells, a time course analysis was performed. Binding was carried out at 37°C as before, except that cells were incubated with 1 2^I-mIL-3 for varying time intervals ranging from 0 min to 180 min. As 96 TABLE 2 - Binding of 1 2 5I-mIL-3 to Murine Hemopoietic Cell Lines CELL LINE SPECIFIC BINDING B6SUtA FDC-P1 32D-C123 P815 EL-4 129,817+ 1593 2888 ± 152 2449 ± 362 0± 312 0± 248 Specific Binding = (Total 1 2 5 I counts per minute bound) - ( 1 2 5 I counts per minute bound in presence of 20-fold excess of unlabeled mIL-3). Data represents binding of l x l O 6 cpm of 1 2 5I-mIL-3 to l x l O 6 cells. 97 Time (min) Figure 4. Time course of binding of mIL-3 to B6SUtA cells. Aliquots of B6SUtA cells (1 x 1 0 6 cells) were incubated with 9 x 1 0 5 cpm 1 2 5I-mIL-3 (5 nmol/L) for different times at 37°C. Total binding, non-specific binding and specific binding were calculated as described in Chapter II. with results representing the means of 3 determinations ± S.E.M. 98 shown i n Figure 4, binding was rapid, with 8 5 % of maximal levels attained within 15 min. Maximal binding was attained within 60 min, and remained stable for up to 3 h. 2. Scatchard Analysis of the Binding of 1 2 5I-mIl-3 to B6SUtA and FDC-P1 Cells To more rigorously test if the B6SUtA subline grown i n our laboratory does possess higher mIL-3 receptor levels than other mIL-3-dependent cell lines studied to date, Scatchard analyses were performed (10). Cells were incubated for 1 h at 37°C with increasing concentrations of 1 2 5I-mIL-3 (i.e., 1.4, 2.8, 5.6, 11.2, 22.4, and 44.7 ng/ml, respectively), and then cell-associated radioactivity was determined as described in Chapter II. From Figure 5A it can be seen that the B6SUtA subline expresses approximately 115,000 receptors/cell, and that the receptors are of a single affinity class with a dissociation constant (KQ) of 3.1 nM. For comparison. Figure 5B shows a Scatchard analysis of FDC-P1 cells, indicating a more typical receptor density of approximately 1600 receptors/cell with a K D of 1.1 nM. These latter data are i n good agreement with those reported by others (8). Thus, the B6SUtA subline grown i n our laboratory appears to be a rich source of mIL-3 receptors, and was therefore used i n subsequent studies to identify the mIL-3 receptor. 3. Identification of the mIl-3 Receptor Using SASD The mIL-3 receptor was i d e n t i f i e d u s i n g SASD, a novel h e t e r o b i f u n c t i o n a l crosslinking agent that is both iodinatable and cleavable. The scheme used for crosslinking mIL-3 to receptors present on B6SUtA cells is shown i n Figure 6. A l l steps up to the photolysis stage were carried out under dim (40 w) red light with reaction vessels covered in aluminum foil. SASD was first iodinated at the 2(p-azidosalicylamido) moiety using N a 1 2 ^ I and chloramine T. The product, 1 2 5I-SASD, was then incubated for 30 min at 23°C with either pure recombinant mIL-3 or pure mIL-3 isolated from conditioned medium obtained following culture of spleen cells i n the presence of pokeweed mitogen. This resulted i n the covalent linkage of mIL-3 to 1 2 ^ I - S A S D through an acylation step with loss of the 99 sulphosuccinlrnidyl moiety. Preliminary studies with bovine serum albumin showed that this covalent linkage was best achieved under alkaline conditions, i.e., at pH >9. 1 2 ^ I -SASD-mIL-3 was then separated from free 1 2 ^ I and 1 2 5I-SASD by passage through a 5 ml P2 column (Bio-Rad), with the protein-iodinated crosslinker complex eluting i n the void volume. In a preliminary experiment, this void volume peak was treated with 5 % p-ME and rechromatographed on P2. This resulted i n less than 2 % of the original 1 2 5 I - l a b e l e d material eluting i n the P2 void volume, compared with greater than 9 8 % i n control (non-reduced) samples, thus indicating that the original void volume peak was indeed 1 2 ^ I -SASD-mIL-3 and not 1 2 5I-mIL-3 (which might result if significant free 1 2 5 I remained after iodination of SASD). Aliquots of the void volume peak were then incubated for 60 min at 37°C with B6SUtA cells. Following this incubation period, crosslinklng of bound mIL-3 to its receptor, through photolysis of the azide bond, was initiated by exposing the cells to long-wave UV light at room temperature (5). Cells were then washed and prepared for one-dimensional or two-dimensional gel electrophoresis. The advantage of using 1 2^I-SASD is that, as indicated i n Figure 6. cleavage of the crosslinker with P-ME results i n the labeled moiety remaining with the receptor. The labeled receptor can then be visualized, free of the ligand, using autoradiography. The autoradiogram (Figure 7) shows the banding patterns obtained with solubilized B6SUtA cells and P815 cells in the presence and absence of p-ME, along with various controls. In the absence of exposure to UV light (lane A), no bands are seen with B6SUtA cells. In the presence of P-ME and UV light (lane B), a band is seen at an apparent molecular mass of 67 kDa. The intensity of this band is markedly reduced when 20-fold excess unlabeled recombinant 11-3 (lane C) is included with 1 2 5I-SASD-mIL-3, suggesting that the binding of 1 2^I-SASD-mIL-3 to its receptor is specific and thus susceptible to competitive inhibition. However, i n a preliminary experiment, 1 2^I-SASD-mIL-3 was added to the same number of cells 5 min prior to a 1 h incubation of these cells with excess unlabeled mIL-3. In t h i s case, the intensity of the 67 kDa band was diminished by only 4 0 % as determined by densitometer tracings of the autoradiogram. 100 0.06 r 200 Bound (pM) B) 0.024 r 0 4 8 12 16 20 24 Bound (pM) Figure 5. Scatchard analysis of the binding of 1 2 5I-mIl-3 to B6SUtA and FDC-P1 cells. Aliquots of 1 x 10 5 B6SUtA cells (A) or 1 x 10 6 FDC-P1 cells (B) were incubated with increasing concentrations of 125I-mIl-3 (i.e.. 1.4. 2.8. 5.6. 11.2, 22.4. and 44.7 ng/ml. respectively) for 1 hr at 37°C. Specific binding was then determined as described in Chapter II. K^: dissociation constant. 101 T h i s suggests that s a t u r a t i o n b i n d i n g is very r a p i d at 37°C, consistent w i t h the data shown i n F i g u r e 4, a n d f u r t h e r i n d i c a t e s t h a t mIL-3 d i s s o c i a t e s s l o w l y f r o m i t s r e c e p t o r , as documented elsewhere (8). A n o t h e r p o s i b i l i t y that c a n not be r u l e d out i s that after the i n i t i a l b i n d i n g step, the mIL-3 receptor i s no longer accessible to competitive ligands. In the absence of [j-ME (lane G), a sharp b a n d is seen at a molecular weight of 56 kDa. That t h i s band, w h i c h t h e o r e t i c a l l y represents mIL-3 s t i l l c r o s s l i n k e d to i t s receptor, s h o u l d migrate faster t h a n the receptor itself, l i k e l y reflects the presence of i n t r a c h a i n disulphide b r i d g e s w i t h i n the receptor molecule. U p o n r e d u c t i o n of these d i s u l p h i d e bridges, the receptor m i ght u n f o l d to assume a more rod-like shape a n d con s e q u e n t l y migrate more sl o w l y i n a n e l e c t r o p h o r e t i c f i e l d . S o l u b i l i z e d P815 c e l l s i n the presence or absence of re d u c i n g agent showed no specific b a n ding patterns (lanes D and H) as expected for non-target c e l l s . Moreover, l a n e s c o n t a i n i n g o n l y 1 2^I-SASD-mIL-3 (i.e., no cel l s ) , whether exposed (lane F) or not exposed (lane E) to U V light a n d whether i n the presence (lanes E a n d F) or absence (lane I) of r e d u c i n g agent d i d not reveal b a n d s c o r r e s p o n d i n g to those seen i n lanes B and G. However, lane F, which represents 1 2^I-SASD-mIL-3 exposed to UV light a n d subjected to electrophoresis u n d e r r e d u c i n g c o n d i t i o n s , c o n t a i n s a b a n d at 15 kDa, w h i c h corresponds to the molecular weight of the unglycosylated recombinant form of mIL-3 (11). Thus, t h i s b and probably represents mIL-3 to w h i c h the labeled moiety of i 2 ^ l -S A SD has been l i n k e d either intramolecularly ( 1 2 ^ l - S A S D l i n k e d through its other reactive moiety to the same mIL-3 molecule) or intermolecularly (labeled moiety donated by another 1 2 5 I - S A S D - m I L - 3 molecule) d u r i n g exposure to UV light. W i t h o u t UV li g h t (lane E) t h i s b a n d i s not seen, as expected, since, after reduction, it w o u l d be expected that the 1 2 ^ I -S A S D moiety w o u l d be released. In lane I, the sample was p r e p a r e d as for lane F, but electrophoresis was carried out under nonreducing conditions. The increased radiodensity seen at the top of t h i s lane l i k e l y represents m u l t i m e r i c complexes of c r o s s l i n k e d i 2 ^ l -SASD-mIL-3. Id e n t i c a l r e s u l t s were obtained when pure mIL-3 from pokeweed mitogen sple e n c e l l c o n d i t i o n e d m e d i u m was used i n s t e a d of r e c o m b i n a n t mIL-3 except that the band i n lane F was 28 kDa, reflecting the molecular weight of the fully glycosylated mIL-3 102 1 2 S l - Labe l l ing of the mIL-3 Receptor Figure 6. Scheme for the cross-linking of mIL-3 to its B6SUtA cell surface receptor. SASD is iodinated as described in Chapter II. Then, under alkaline conditions mIL-3 (presumably through terminal amino groups) d i s p l a c e s the sulfosuccinimidyl moiety, forming ^2^I-SASD-mIL-3. This is separated from 1 2 5 I and 1 2 5I-SASD-mIL-3 by passage through a P-2 column, with the proteinaceous material eluting in the void volume. The 12^I-SASD-mIL-3 peak is then pooled and aliquots are incubated with B6SUtA cells to effect binding of 12^I-SASD-mIL-3 to the cell surface receptor. All the above steps are carried out in the dark or under dim red light. Cells are then exposed to long-wave UV light (460 microwatts/cm 2 at 14 cm from source) to initiate photolysis of the azide bond and covalent linkage of 1 2^I-SASD-mIL-3to the mIL-3 receptor. Cleavage of the disulfide bond within the cross-linker using |3-ME and subsequent solubilization of cell plasma membranes by SDS yields the iodinated mIL-3 receptor, which can be separated from other proteins by SDS-polyacrylamide gel electrophoresis. 103 LANE A B C D E F G H I NR/R R R R R R R NR NR NR B6SUtA P815 + + + + NC NC + + NC mIL-3 UV — + + + — + + + + 200-92.5-69-„ 46-30-14.3-Figure 7. Autoradiogram showing the incorporation of l Z 0 l from i Z DI-SASD-mIL-3 into B6SUtA or P815 cells with subsequent one-dimensional SDS-polyacrylamide gel electrophoresis. Experimental test samples were prepared as described in Chapter II. One-dimensional SDS-polyacrylamide gel electrophoresis was carried out using 5-15% gradient SDS-polyacrylamide gels as described. R. reduced (exposed to p-ME); NR, nonreduced (not exposed to p-ME); B6SUtA, B6SUtA cells either present (+) or absent (-); P815, P815 cells either present (+) or absent (-); NC, no cells i n sample; mIL-3, presence (+) or absence (-) of 20-fold excess of unlabeled recombinant mIL-3; UV, exposed (+) or not exposed (-) to 460 microwatts/cm 2 UV light at 14 cm from light source. Arrows indicate bands of interest as discussed in the text. 104 from this source (12). These data therefore suggest that the mIL-3 receptor on B6SUtA cells consists of a single 67 kDa polypeptide chain. As can be seen i n lane B of Figure 7, incubation of B6SUtA cells with 1 2 5I-SASD-mIL-3 also led to the incorporation of label into a minor band at approximately 50 kDa (<2% of the 67 kDa band when compared by densitometry). Since this band was also reduced in intensity in the presence of an excess of unlabeled mIL-3 (Figure 7. lane C), the 50 kDa species may represent a minor but specific site for mIL-3 binding. The SASD crosslinking procedure was then used to analyze the migration of the mlL-3 receptor on O'Farrell two-dimensional gels. Specifically, B6SUtA cells were exposed to 1 2^I-SASD-mIL-3 as above and, following UV catalyzed crosslinking, the cells were lysed and plasma membranes prepared as described in the legend to Figure 8. O'Farrell two-dimensional gel electrophoresis of the solubilized membranes demonstrated a radioactive protein smear at 67 kDa with a pi of 6.0 to 6.4 (Figure 8A). The corresponding protein band i n the silver stained gel is indicated in Figure 8B. These data suggest that the mIL-3 receptor has a pi of approximately 6.2, although this assignment may be erroneous since modification by SASD may have altered the migration of the receptor. 4. Receptor Identification Using Glutaraldehyde and DSS as  Crosslinking Agents To test whether the 67 kDa species is indeed the mIL-3 receptor using more conventional techniques, recombinant 1 2^I-mIL-3 was crosslinked to B6SUtA cells using either glutaraldehyde or disuccinimidyl suburate (DSS) as crosslinking agent(13). With glutaraldehyde-crosslinked cells, SDS-PAGE revealed one major band at approximately 83 kDa, as shown i n Fig. 9A. Subtracting the molecular weight of 15 kDa for recombinant mIL-3, this suggests that the receptor has an apparent molecular mass of approximately 68 kDa, in close agreement with our results using 1 2^I-SASD. However, when DSS was used as the crosslinking agent, an additional labeled band was detected at 155kDa (Fig. 9B). 105 suggesting that 1 2^I-mIL-3 was being crosslinked to both 67 and 140 kDa proteins with this crosslinking agent. Therefore, these data provide evidence for an additional 140 kDA mIL-3 binding protein. It remains to be determined why the 67 kDa receptor protein is observed using SASD, glutaraldehyde and DSS, while the 140 kDa molecule is detected only with DSS as the crosslinking agent. C. DISCUSSION We have analyzed the binding of recombinant bioactive 1 2^I-labeled mIL-3 to an mlL-3-dependent B6SUtA cell subline grown in our laboratory. Binding is rapid at 37°C, with 8 5 % of maximal levels attained within 15 min. Furthermore, it was shown by Scatchard analysis that this subline expresses unusually high numbers of specific mIL-3 surface receptors, i.e., approximately 115,000 receptors per cell with a KQ of 3.1 nM. For comparison, mIL-3-dependent FDC-P1 cells were shown to possess a more typical receptor density of approximately 1600 receptors per cell with a KQ of 1.1 nM. This latter result is in good agreement with previous studies i n which FDC-P1 cells were shown to have 1500-2500 receptors per cell (8). In addition, other mIL-3-dependent cell lines have been reported to express 1000-5000 receptors per cell (8,14). The B6SUtA subline therefore appears to.have a substantially higher mIL-3 surface receptor density than other mlL-3-dependent cells tested to date. We have therefore used this cell line as a source of starting material for characterizing the mIL-3 receptor. The basis of the increased receptor expression i n the B6SUtA subline remains to be determined, and is currently under investigation in our laboratory (9). In order to investigate the biochemical properties of a growth factor receptor, it is necessary to first identify the receptor in terms of its molecular mass. A variety of chemical crosslinking agents such as glutaraldehyde, disuccinimidyl suberate (DSS) and dithiobis succinimidyl propionate (DSP) have been used to crosslink radiolabeled growth factors to their cell surface receptors (2-7). Labeled proteins have then been identified by SDS-PAGE. Figure 8A. Figure legend on following page. 2 0 0 -92.5-69-46 -3 0 -7.4 l 6.8 I PH 6.3 L _ 5.6 4.8 _1 1_ Figure 8B. Autoradiogram (a) and silver stain (b) of a two-dimensional O'Farrell gel of plasma membranes from B6SUtA cells labelled with 1 2^I-SASD-mIL -3 . Experimental test samples were prepared as described in Chapter II. Two dimensional O'Farrell gel electrophoresis, with isoelectrofocussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second dimension using an 8-20% SDS-polyacrylamide gel, was carried out as described. Silver staining was performed according to published methods. The arrow in (b) points to the region of radioactivity indicated in (a). The low molecular weight material on the autoradiogram likely represents 1 2^I-SASD-mIL-3 and is present because of incomplete covalent cross-linking to the mIL-3 receptor followed by incomplete reduction of the SASD disulfide bridge. o 0^ 108 A disadvantage of t h i s technique i s that, aside from the growth factor itself, only complexes of the growth factor and its receptor are visualized autoradiographically. T h i s often leads to difficulties i n assigning accurate molecular weights to respective receptors. We have used a new c r o s s l i n k i n g agent, SASD. to identify the mIL-3 receptor on B 6 S U t A cells. SASD is not only h e t e r o b i f u n c t i o n a l a n d cleavable, but it i s also iodinatable w i t h i n one of i t s reactive moieties. Therefore, it c a n be u t i l i z e d to tag growth factor b i n d i n g proteins (or proteins i n close p r o x i m i t y to receptor molecules) w i t h 1 2 ^ I , as i n d i c a t e d s c h e m a t i c a l l y for the mIL-3 receptor i n Figure 6. O u r findings using SASD cro s s l i n k i n g analysis suggest that the mIL-3 receptor on B 6 S U t A cells is a single polypeptide w i t h a n apparent m o l e c u l a r m a ss of 67 k D a a n d a p i of a p p r o x i m a t e l y 6.2. We also observed a weak i n t e r a c t i o n between i 2 5 T _ SASD-mIL-3 and a 50 k D a protein w h i c h was not observed when excess u n l a b e l e d mIL-3 was also added (Figure 7, lanes B and C). F u r t h e r studies are necessary to determine the relationship between this protein and mIL-3 receptor species. W hen B 6 S U t A cells were exposed to 1 2^I-SASD-mIL-3 and samples processed under non-reducing conditions, a 56 k D a b a n d was detected after SDS-PAGE (Figure 7, lane G). Since t h i s b a n d theoretically represents a radiolabeled complex of mIL-3 and its receptor, the observation that the complex migrates more rapidly d u r i n g SDS-PAGE suggests that it is being constrained i n a more globular conformation under non-reducing conditions. This likely reflects the presence of disulphide bridges w i t h i n the 67 k D a receptor molecule. S i m i l a r data were obtained when we used glu t a r a l d e h y d e as a c r o s s l i n k i n g agent, w i t h 1 2 ^ I - m I L - 3 being c r o s s l i n k e d to a 68 k D a p r o t e i n on B 6 S U t A c e l l s . These f i n d i n g s have recently been confirmed by others. A putative antibody against the mIL-3 receptor was reported to recognize a p r o t e i n w i t h a p i of 5.7-6.2 i n a n u m b e r of mIL-3-dependent c e l l l i n e s (15). In addition, values of 50-70 k D a (15), 65-70 k D a (16), 72.5 k D a (17), and 60-75 k D a (18) have been reported for the m o l e c u l a r mass of the mIL-3 receptor u s i n g a variety of techniques. 109 1 2 3 Figure 9A. Glutaraldehyde crosslinking of 1 2 t > I - m I L - 3 to the mIL-3 receptor on B6SUtA cells. 1 2 5 I - m I L - 3 was bound to B 6 S U t A cells at 37°C for 1 hr. Cells were then washed and crosslinking effected by addition of 2.5% glutaraldehyde in b inding buffer for 30 minutes at 4°C (lane 3). Lane 1: no glutaraldehyde added; lane 2: 20-fold excess of unlabeled mIL-3 added with 1 2 ^ I -mIL-3 . 110 1 2 Figure 9B. DSS crosslinking of 1 2 5I-mIL-3 to the mIL-3 receptor on B6SUtA cells. Binding was carried out as in Figure 8A. Crosslinking was performed by adding ImM DSS in binding buffer for 30 minutes on ice (lane 2). Lane 1 20-fold excess of unlabeled mIL-3. I l l However, one of the above studies also reported that, in addition to a 72.5 kDa protein, 12^I-mIL-3 could be crosslinked to a protein of apparent molecular mass 115 kDa in intact FDC-P2 cells using DSS (17). We also observed an additional higher molecular weight receptor protein on B6SUtA cells. Specifically, when we crosslinked 12^I-mIL-3 to these cells using DSS, we detected both a 67 kDa and a 140 kDa mIL-3 binding protein. A number of possibilities exist as to the nature of the 140 kDa protein. Firstly, it may represent an additional rnIL-3 binding protein that, possibly due to physical constraints, is only detected when DSS is used as the crosslinking agent. However, Scatchard analysis reveals only a single affinity class of mIL-3 receptors on B6SUtA cells (Fig.5), indicating that, if the 67 kDa and 140 kDa proteins are two distinct mIL-3 binding proteins, they have very similar affinities for mIL-3. A second possibility is that the 140 kDa molecule is in fact the major mIL-3 binding protein on these cells, and the 67 kDa protein is a proteolytic cleavage product of the larger molecule. Consistent with this possibility is the finding that only the lower molecular weight binding protein is detected when 12^I-mIL-3 is DSS-crosslinked to purified membranes from B6SUtA cells. A third explanation for the above findings is that the 155 kDa band detected in Fig.8B represents a complex between the 15 kDa recombinant mIL-3, the 67 kDa receptor, and a third protein with an apparent molecular mass of approximately 60 kDa. In view of the data to be presented in Chapter VI, we currently favour the second possibility and the concept that the 140 kDa protein represents a distinct mIL-3 binding protein. The findings of the present study therefore suggest that mIL-3 binds to a 67 kDa receptor on B6SUtA cells and that this polypeptide has a pi of approximately 6.2. Moreover, this molecule likely contains intrachain disulphide bridges. In addition, we provide evidence that mIL-3 also binds to a 140 kDa protein on B6SUtA cells. These data should be useful in the isolation and purification of the mIL-3 receptor, and should facilitate the detection of receptor-associated changes, such as by protein phosphorylation, that may be involved in the mechanism of action of mIL-3. 112 D. REFERENCES 1. Greenberger JS, Sakakeeny MA, Humphries RK et al. Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines. Proc Natl Acad Sci USA 80: 2931, 1983. 2. J i TH. Crosslinking of lectins and receptors in membranes with heterobifunctional crosslinking reagents. J Biol Chem 252: 1566, 1977. 3. Yip CC, Yeung CWT, Moule ML. Photoaffinity labeling of insulin receptor of rat adipocyte plasma membrane. J Biol Chem 253: 1743, 1978. 4. J i I, J i TH. Macromolecular photoaffinity labeling of the lutropin receptor on granulosa cells. Proc Natl Acad Sci USA 77: 7167, 1980. 5. J i TH, J i I. Macromolecular photoaffinity labeling with radioactive photoactivable heterobifunctional reagents. Anal Biochem 121: 286, 1982. 6. Kudlow JE , Leung Y. Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase. Biochem J 220: 677, 1984. 7. Brenner MB, Trowbridge IS, Strominger JL. Cross-linking of human T cell receptor proteins: association between the T cell idiotype beta subunit and the T3 glycoprotein heavy subunit. Cell 40: 183, 1985. 8. Palaszynski EW, Ihle J N . Evidence for specific receptors for interleukin 3 on lymphokine dependent cell lines established from long-term bone marrow cultures. J Immunol 132: 1872, 1984. 9. Murthy SC, Sorensen PHB, Mui AL-F, Krystal G. Interleukin-3 down-regulates its own receptor. Blood 73: 1180, 1989. 10. Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51: 660, 1949. 11. Kindler V, Thorns B, de Kossodo S et al. Stimulation of hematopoiesis in vivo by recombinant bacterial murine interleukin 3. Proc Natl Acad Sci USA 83: 1001, 1986. 12. Ihle JN, Keller J , Oroszlan S et al. Biologic properties of homogeneous interleukin-3. J Immunol 131: 282, 1983. 13. Sahyoun N, Hoch RA, Hollenberg MD. Insulin and epidermal growth factor-urogastone: affinity crosslinking to specific binding sites in rat liver membranes. Proc Natl Acad Sci USA 75: 1675, 1978. 14. Crapper RM, Clark-Lewis I, Schrader JW. Analysis of the binding of a hemopoietic growth factor, P-cell-stimulating, to a cell surface receptor using quantitative adsorption of bioactivity. Exp Hematol 13: 941, 1985. 15. Palacios R, Neri T, Brockhaus M. Monoclonal antibodies specific for interleukin-3-sensitive murine cells. J Exp Med 163: 6652, 1986. 16. May WS, Ihle JN . Affinity isolation of the interleukin 3 surface receptor. Biochem Biophys Res Commun 135: 870. 1986. 113 Park LS, Friend D, Gillis S, Urdal DL. Characterization of the cell surface receptor for a multi-lineage colony-stimulating factor (CSF-2a). J Biol Chem 261: 205. 1986. Nicola NA, Peterson L. Identification of distinct receptors for two hemopoietic growth factors (granulocyte colony-stimulating factor and multipotential colony-stimulating factor) by chemical cross-linking. J Biol Chem 261: 12384, 1986. 114 C H A P T E R IV INTERLEUKIN-3, GM-CSF AND ACTIVATORS OF PROTEIN KINASE C INDUCE DISTINCT PHOSPHORYLATION EVENTS IN MURINE MULTIPOTENTIAL HEMOPOIETIC CELLS A. INTRODUCTION When polypeptide growth factors bind to their specific cell surface receptors, they trigger a cascade of biochemical events ultimately resulting in a change in the proliferative state of the target cell. How receptor occupancy transduces a signal for the cell to grow has been the subject of intensive investigation i n recent years. To date, three major biochemical pathways, all involving the reversible phosphorylation of proteins, have been implicated i n the early stages of growth factor mediated cell proliferation (1,2). These include the activation of receptor-associated tyrosine kinases (3), the hydrolysis of membrane phospholipids leading to the activation of protein kinase C (PKC) (4), and the stimulation of cAMP-dependent protein kinase A by cAMP in response to ligands which activate adenylate cyclase (5). It is not clear at present whether these pathways represent synergistic or alternative mechanisms of signal transduction. Because mIL-3 acts as both a mitogen and a survival factor for target cells (i.e., mIL-3-dependent cells die within 12-24 h in the absence of mIL-3 (6)), it is of interest to determine which of the above pathways is utilized by mIL-3, or if other pathways are also involved. In order to gain insight into the mechanism of action of mIL-3, a number of polypeptide growth factors and known low molecular weight mitogens were tested for their ability to mimic the growth-promoting activity of mIL-3 on a cloned B6SUtA cell line designated as B6SUtA!. In this chapter we demonstrate that these cells also respond to murine granulocyte-machrophage colony-stimulating factor (mGM-CSF), a hemopoietic growth factor with similar biological properties to those of mIL-3, but which appears to be 115 more restricted in the spectrum of marrow cell types that it stimulates (7). In addition, we show that the phosphotyrosine phosphatase inhibitor, sodium orthovanadate (3,8). and the phorbol ester, TPA, an activator of PKC (9), can partially substitute for mIL-3 in stimulating DNA synthesis in B6SUtA^ cells. In contrast, however, when TPA is added in combination with mIL-3 or mGM-CSF, it markedly inhibits the stimulatory effects of either growth factor. In view of these results, we investigated the role of protein phosphorylation in the mechanism of action of mIL-3. Specifically, we examined phosphorylation events induced by the short term incubation of B6SUtAj cells with mIL-3, mGM-CSF and TPA and have used the resulting data to delineate those phosphorylation events common to proliferating B6SU1AJ cells and those unique to the action of mIL-3. B. RESULTS 1. Effects of Various Agents on the Proliferation of mIl-3-dependent BSSUtA^ Cells To examine the mechanism of action of mIL-3, we first tested a number of known growth factors and low molecular weight mitogenic agents for their ability to mimic the proliferative activity of mIL-3 on B6SUtAi cells (Table 3). These cells were derived by plucking and expanding an individual B6SUtA colony growing in methylcellulose, and were subsequently shown by Scatchard analysis to express similar mIL-3 receptor levels as the B6SUtA subline described in Chapter III. All agents in Table 3 were tested over a wide range of concentrations, and the data shown represent responses obtained at optimal concentrations. Of the growth factors tested, only mGM-CSF was as potent as mIL-3 in stimulating growth of BeSUtA^ cells. This factor was therefore utilized in subsequent studies (see below) to delineate protein phosphorylation events common to both growth factors from those specific to the action of mIL-3. Granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), erythropoietin, and interleukins-1. -2, -5, and -6 had no effect while insulin, insulin-like growth factor-I (IGF-I) and interleukin-4 were moderately stimulatory. Of a number of low molecular weight agents 116 TABLE 3 - Ability of Various Agents to Substitute for mIl-3 in Stimulating 3H-Thymidine Incorporation into BeSUtAn Cells Agent 3H-Thymidine Incorporation (cpm) Control 458 ± 34 mIL-3 (10 ng/ml) 12,792 ± 1,086 mGM-CSF (10 ng/ml) 13,020 ± 350 hGM-CSF (1 ug/ml) 470 ± 26 Insulin (40 pg/ml) 5,280 ± 60 IGF-I (2 pg/ml) 8,542 ± 210 hIL-ip (170 U/ml) 422 ± 43 hIL-2 (100 U/ml) 450 ± 36 mIL-4 (400 U/ml) 1,065 ± 127 mIL-5 (25 pg/ml) 487 ± 50 hIL-6 (1000 U/ml) 532 ± 8 hEp (200 U/ml) 472 ± 26 Sodium orthovanadate (20 pM) 5,322 ± 186 TPA (100 ng/ml) 2,021 ± 278 OAG (100 pM) 461 ± 58 Calcium Chloride (1.2 mM) 434 ± 16 A-23187* (0.5 pg/ml) 471 ± 34 TPA (100 ng/ml)+A-23187 (0.5 pg/ml) 2.181 ± 312 Bt 2cAMP (10 pM) 734 ± 55 Monensin* (12.5 ng/ml) 812 ± 39 BeSUtAj cell 24 h proliferation assays were performed as described i n Materials and Methods. A l l agents were tested over a wide range of concentrations. Concentrations given are those that produced the maximal response. Numbers represent the mean of 3 determinations ± SEM. A b b r e v i a t i o n s i n c l u d e m ( m u r i n e ) , h ( h u m a n ) , IL ( i n t e r l e u k i n ) , G M - C S F (granulocyte/macrophage-colony stimulating factor), IGF-I (insulin-like growth factor-I). Ep (erythropoietin), TPA (12-0-tetradecanoylphorbol-13-acetate), OAG (l-oleoyl-2-acetyl-sn-glycerol). * A-23187 and monensin are calcium and sodium ionophores, respectively. 117 known to stimulate or augment mitogenesis in a variety of other cells, dibutyryl cyclic GMP, CaCl2 and the calcium ionophore A23187 had no effect, while dibutyryl cyclic AMP and the s o d i u m ionophore m o n e n s i n were only s l i g h t l y s t i m u l a t o r y . However, s o d i u m orthovanadate was found to have sig n i f i c a n t s t i m u l a t o r y activity. Since sodium orthovanadate is a potent inhibitor of phosphotyrosine phosphatase activity (3,8), this finding suggested that tyrosine phosphorylation may be involved i n the mechanism of action of mIL-3. We also tested the growth-stimulating activities of TPA and 1-oleoyl-2-acetyl-sn-glycerol (OAG), i n view of recent evidence for the involvement of PKC in the response of target cells to mIL-3 (6,9). While OAG had no effect, TPA-treated cells demonstrated a small dose-dependent increase in thymidine incorporation over control cells (Table 3). The response was dose-dependent and maximal at 100 ng/ml TPA, which typically represented 10-15% of the maximal 3H-thymidine incorporation observed with mIL-3 or mGM-CSF. However, when 100 ng/ml TPA was added to cells growing in the presence of a range of mIL-3 concentrations, marked inhib i t i o n of mIL-3-stimulated thymidine incorporation was observed (see Figure 10a). As indicated, there was no effect when the inactive phorbol ester, 4-a-phorbol-12,13-didecanoate, was substituted for TPA. A similar effect was observed when cells were co-incubated with mGM-CSF and 100 ng/ml TPA (see Figure 10b). The inhibitory effect of TPA was dose-dependent, with the maximal effect seen at 100 ng/ml TPA. To test whether the response of these cells to co-incubation with TPA was limited to an inhibition of DNA synthesis, cells were seeded at 2x10^ cells/ml and grown i n the presence of various agents, as stipulated i n the legend to Figure 11. Viable cell counts were then determined at 24 h (day 1) and at 48 h (day 2). Figure 11 indicates that co-incubation with 100 ng/ml TPA inhibits the growth-promoting activity of high (11 ng/ml) and low (0.23 ng/ml) initial concentrations of mIL-3, and of high (9 ng/ml) and low (0.18 ng/ml) initial concentrations of mGM-CSF. In fact, in the presence of TPA, viable cell counts were decreased by 18+13% and 94±17%, respectively, after 24 h and 48 h in culture with an initial mIL-3 concentration of 11 ng/ml, and by 24±23% and 94±4%, 1 1 8 0.01 0.02 0.05 0.09 0.18 0.36 0.72 1.43 2.85 5.70 11.40 mIL-3 (ng/ml) Figure 10A. Figure legend on following page. 119 16 0 0 01 0 02 0 04 0 08 0.15 0 29 0.57 113 2 25 4 50 9.00 G M - C S F (ng /ml) Figure 10B. Effects of mIL-3, GM-CSF and TPA on the proliferation of B 6 S U t A j cells. Washed BeSUtAj c e l l s were grown for 24 h i n RPMI 1640 + 2 0 % FCS containing: (A) increasing concentrations of mIL-3, mIL-3 + 100 ng/ml TPA, or mIL-3 + 100 ng/ml 4-a-phorbol 12.13-didecanoate (4-ct-phorbol). or; (B) increasing concentrations of mGM-CSF, mGM-CSF + 100 ng/ml TPA, or mGM-CSF + 100 ng/ml 4-a-phorbol. After 22 h. 3H-thymidine was added and, 2 h later, the cells were harvested and radioactivity was counted. 120 respectively, after 24 h and 48 h in culture with an initial mGM-CSF concentration of 9 ng/ml. A simple toxic effect of TPA on these cells is unlikely in view of the following data: (i) incubation with 100 ng/ml TPA alone typically resulted in the maintenance of 30-50% cell viability compared with 0 % for unstimulated control cells over 24 h in culture (see Figure 11); (ii) co-incubation with the inactive phorbol ester 4-oc-phorbol had no effect on mIL-3 or mGM-CSF stimulated proliferation (see Figure 11) and; (iii) cell viabilities remained at 8 0 % after 24 h i n co-cultures of 11 ng/ml mIL-3 + 100 ng/ml TPA or 9 ng/ml GM-CSF + 100 ng/ml TPA. To investigate whether the inhibitory effect of TPA was due to down regulation of PKC, as has been observed following long term (24-72 h) treatment with TPA (10,11,12), a time course study was performed. B6SUtA2 cells were grown in the presence of mIL-3 at 2x10^ cells/ml for 24 h and TPA was added at different times during this period. The medium was then pulsed with 3H-thymidine for the final 2 h of incubation, prior to harvesting the cells as before. As indicated in Figure 12, the inhibitory effect is observable within 2 h of adding TPA, strongly suggesting that the effect is not due to exhaustion of PKC. Therefore, it appears that while TPA is stimulatory alone, it inhibits growth factor-stimulated proliferation of BGSUtA^ cells. Based on the above results, we concluded that an examination of growth factor-stimulated phosphorylation events might provide important insights into the mechanism of action of mIL-3. Specifically, we compared the phosphorylation of cytosolic and membrane proteins in intact B e S U t A T cells exposed to mIL-3, mGM-CSF or activators of PKC. 2. Phosphorylation of Cytosolic Proteins in Intact Factor-stimulated Cells B6SUtAi cells were depleted of growth factors as described in Chapter II, labeled w i t h 3 2P-orthophosphate in phosphate-free medium for 1 h at 37°C and then incubated w i t h either control buffer, mIL-3, mGM-CSF, OAG or TPA for various time periods. Cells w e r e then subfractionated into cytosolic and membrane fractions for electrophoresis. To facilitate the rapid isolation of cytosolic phosphoproteins, thus minimizing protein dephosphorylation and proteolysis, the method of Janski and Cornell (13,14) was utilized. 13 -• • • day 0 i i iuii indny 1 • l a i a d o y 2 -* T P A 4-<.-Ph T P A 4-«-Ph Figure 11. Effects of TPA on growth and viability of BeSUtAj^ cells treated with mIL-3 or GM-CSF. BSSUtAj cells were seeded at 2 x 10 5 cells/ml in RPMI 1640 + 2 0 % FCS containing the indicated agents, and counted after 24 h (day 1) and 48 h (day 2) in culture. Results shown are viable cell counts, as determined by trypan blue dye exclusion, and each point represents the mean of duplicates ± SEM. C = control. 4-a-phorbol; TPA = 100 ng/ml; low mIL-3 = 0.23 ng/ml; high mIL-3 = 11 ng/ml; low mGM-CSF = 0.18 ng/ml; high GM-CSF = 9 ng/ml. 122 Figure 12. Time course study of TPA-induced i n h i b i t i o n of proliferation. B 6 S U t A j cultures were grown for 24 h i n RPMI 1640 + 2 0 % FCS containing 5 ng/ml mIL-3. At time points corresponding to 2, 4, 6, 8, 18, and 24 h prior to harvesting, one set of cultures (mIL-3 + TPA) was adjusted to 100 ng/ml TPA, while the other set (mIL-3) was adjusted with an equal volume of RPMI 1640. Cultures were pulsed for the final 2'h with 3H-thymidine and then harvested to determine the extent of thymidine incorporation. Each data point represents the mean of duplicates ± SEM. 123 With this procedure (see Chapter II), cytoplasmic samples can be boiled in SDS sample buffer within 1 min of cell lysis in preparation for electrophoretic analysis. No differences in phosphorylation patterns were observed with one-dimensional SDS-PAGE. However, O'Farrell two-dimensional gel electrophoresis of cytosolic proteins revealed an obvious increase over controls (Figure 13A) in the phosphorylation of a cytosolic 68 kDa protein (cp68) when cells were.treated for 15 min at 37°C with mIL-3 (Figure 13B) or mGM-CSF (Figure 13C). Isoelectric point determinations for cp68 revealed a pi of 5.1-5.2. However, there was no increased phosphorylation of the 68. kDa protein in cells treated with 100 ng/ml TPA (Figure 13D), or a range of other TPA concentrations. Furthermore, there was no effect of OAG. Our findings, therefore, indicate that the growth-promoting agents, mIL-3 and mGM-CSF, stimulate the phosphorylation of a 68 kDa cytosolic protein in BeSUtA.! cells, while the PKC activators TPA and OAG do not elicit this response. These results could not be demonstrated with mIL-3 non-responsive P815 murine mastocytoma cells (15). These studies were also undertaken using more conventional methods for isolating cytosolic proteins (see Chapter II), with similar results. Certain other differences in cytosolic protein phosphorylation between control and treated cells were occasionally observed in the two-dimensional gel patterns, but increased phosphorylation of cp68 was the only consistent observation over the course of 12 experiments. 3. Time Course and Concentration Dependence of cp68 Phosphorylation The kinetics of cp68 phosphorylation were examined by incubating B6SUtA! cells with mIL-3 or mGM-CSF for varying times prior to processing of the cells for two-dimensional gel electrophoresis. The relative extent of incorporation of 3 2 P into cp68 was determined by laser densitometric scanning of gel auto-radiograms. A typical result is shown in Figure 14; the phosphorylation of cp68 in cells treated with mIL-3 or mGM-CSF was transient: incorporation of label was detectable within 5 min, with maximal levels attained within 15 min. This was followed by a slow decay towards basal levels. A) Control basic a c i d i c B) m IL - 3 basic acidic 14.3-Figures 13A and B. Figure legend on following page. Figure 13C and D. O G M - C S F basic 200- | 92.5- •** 69-46-30-D)TPA acidic basic acidic 200 92.5 30-14.3-14.3-Effects of mIL-3, GM-CSF and TPA on the phosphorylation of cytosolic p r o t e i n s i n B 6 S U t A i cells. 3 2 P -labeled B6SULAJ cells were incubated with factors for ten minutes at 37°C. Cytosolic proteins were th e n isolated by digiton i n fractionation as described i n Chapter II. Proteins were separated by two-dimensional gel e l e c t r o p h o r e s i s u s i n g 8 % to 2 0 % S D S p o l y a c r y l a m i d e gels. A r r o w s i n d i c a t e p o s i t i o n of a 68-kDa cytosolic protein phosphorylated i n cells treated with 10" 8 mol/L mIL-3 (B) and 10" 8 mol/L mGM-CSF (C). hut not in control cells (A) or in cells treated with 100 ng/ml TPA (D). to 126 0 30 60 90 120 Time (min) Figure 14. Time course of cp68 p h o s p h o r y l a t i o n . For k i n e t i c a n a l y s i s of cp68 phosphorylation, 3 2P-labeled BeSUtA , cells were stimulated with 10~ 8 mol/L mIl-3, 10" 8 mol/L mGM-CSF. or 100 ng/ml TPA at 37°C for the Indicated times. C e l l s were then s u b f r a c t i o n a t e d into c y t o s o l i c f r a c t i o n s , and phosphoproteins were analyzed by two-dimensional gel electrophoresis for cp68. Relative levels of 3 2 P i n c o r p o r a t i o n were determined by laser densltometric scanning of phosphorylated cp68, u s i n g a c o r t i t u t l v e l y phosphorylated protein spot evident on autoradiograms to standardize 3 2 P incorporation between gels. Similar results were obtained i n two separate experiments. 127 Phosphorylation of cp68 was dependent on the concentration of mIL-3 or mGM-CSF added to the cells. E ^ S U t A ^ ce l l s were s t i m u l a t e d w i t h v a r i o u s c o n c e n t r a t i o n s of mIL-3 for 15 m i n at 37°C, a n d t h e n c y t o s o l i c p r o t e i n s were a n a l y z e d b y t w o - d i m e n s i o n a l gel electrophoresis. A s shown i n Figure 15, phosphorylation of cp68 was typi c a l l y observed in a dose-dependent manner over a concentration range of 10" 1 *-10"^ M for both mIL-3 and mGM-CSF, with half-maximal incorporation of 3 2 P at approximately 1 0 " 1 0 M i n each case. 4. Phosphoamino A c i d Analysis of 3 2 P - l a b e l e d cp68 The p h o s p h o a m i n o a c i d c o m p o s i t i o n of cp68 was determined after s t i m u l a t i o n of 3 2 P - l a b e l e d B G S U t A j cells for 15 m i n with mIL-3 or mGM-CSF. Spots c o r r e s p o n d i n g to l a b e l e d c p 6 8 were l o c a l i z e d by autorad i o g r a p h y a n d excised f r o m t w o - d i m e n s i o n a l gels. P r o t e i n was electroeluted from gel slic e s , a n d t h e n subjected to a c i d h y d r o l y s i s a n d t h i n l a y e r c h r o m a t o g r a p h y by m e t h o d s d e s c r i b e d i n C h a p t e r II. A s s h o w n i n F i g u r e 16, phosphate was incorporated predominantly, if not exclusively, into serine residues i n cp68 when cells were stimulated with these agents. 5. Phosphorylation of Membrane Proteins i n Intact Factor-Stimulated Cells To investigate the phosphorylation of membrane proteins, 3 2 P - l a b e l e d B 6 S U t A ^ cells were i n c u b a t e d i n the presence a n d absence of mIL-3, mGM-CSF, T P A or O A G at 37°C. prior to isolation of membranes by differential centrifugation. One-dimensional SDS-PAGE of m e m b rane s a m p l e s revealed a n i n c r e a s e d p h o s p h o r y l a t i o n of a 67 k D a membrane protein (mp67) when intact cells were treated for 15 m i n at 37°C with either mIL-3, mGM-C S F or TPA, but not with control buffer (see Figure 17). T h i s was also not detected when cells were exposed to OAG, possibly because th i s reagent is more labile t h a n TPA (16). In addition, membranes from TPA treated cells displayed an enhanced phosphorylation of a 12 k D a p r o t e i n (Figure 17). S i m i l a r r e s u l t s were obtained when membrane samp l e s were enriched for plasma membranes by the method of Belsham et al (17), suggesting that mp67 may be associated with plasma membranes. Membrane phosphoproteins were further 128 Growth Factor Concentration (M> Figure 15. C o n c e n t r a t i o n dependence of cp68 p h o s p h o r y l a t i o n , ^ - l a b e l e d B S S U t A , c e l l s w e r e s t i m u l a t e d f o r 15 m i n u t e s at 37°C w i t h t h e i n d i c a t e d concentrations of mIl-3 or GM-CSF. Cytosolic fractions were then analyzed by two-dimensional gel electrophoresis as i n Figures 13 and 14. Relative levels of 3 2 P I n c o r p o r a t i o n Into c p 6 8 was d e t e r m i n e d by l a s e r d e n s i t o m e t r y of autoradiograms. 129 P - S E R ^ - O P-THR* - O o o P-TYR^- O mIL-3 G M Figure 16. Phosphoamino acid analysis of cp68. 3 2P-labeled B6SUtA1 cells were incubated with 10"8 mol/L mIL-3 (mIL-3) or 10 - 8 mol/L mGM-CSF (GM) for ten minutes at 37°C, and then processed into cytosolic fractions f o r electrophoresis. Autoradiographically identified cp68 was electroeluted from gel slices and subjected to phosphoamino acid analysis using a 1:10:189 pyridine/acetic acid/water solvent system and thin-layer electrophoresis. Phosphoamino acid standards were identified by ninhydrin staining: P-Ser. phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine. S i m i l a r results were obtained in two separate experiments. 130 C I T O G Figure 17. Analysis of membrane phosphoproteins by one-dimensional SDS-PAGK Intact, 3 2 P - l a b e l e d B6SUtA 1 cells were incubated for ten minutes at 3 7 ( ' with control buffer (C), 10" 8 mol/L mIL-3 (I), 100 ng/ml TPA (T), 100 umol/l. OAG (O) or 10" 8 mol/L mGM-CSF (G). Membranes were then isolated and electrophoresed as described i n Chapter II us i n g an 8% to 2 0 % S D S polyacrylamide gel. Arrow indicates position of a 67-kDa membrane protein phosphorylated with mIL-3, mGM-CSF, or TPA added. 131 analyzed by O'Farrell two-dimensional gel electrophoresis. C o m p a r i s o n of the phosphoprotein constellations (Figure 18) confirmed the results obtained with one-dimensional SDS-PAGE and suggest that the increased phosphorylation observed with mlL-3, mGM-CSF and TPA is due to the same 67 kDa protein with a pi of approximately 5.7-5.9. As a control, similar experiments were carried out with P815 murine mastocytoma cells. Murine 11-3 did not induce specific phosphorylation events in the membranes of these cells as assessed by two-dimensional gel electrophoresis. As was the case with the cytosolic phosphoprotein patterns, increased and decreased phosphorylations of other proteins were occasionally observed when membranes from mIL-3-, mGM-CSF- and TPA-treated cells were compared to controls. However, over the course of 6 experiments, only increases in the phosphorylation of mp67 were consistently observed. 6. Time Course and Concentration Dependence of mp67 Phosphorylation The time course of mp67 phosphorylation for a typical experiment is shown in Figure 19. The phosphorylation of this protein appears to occur slightly more rapidly in response to mIL-3 than in cells treated with mGM-CSF or TPA, with peak levels attained within 5 min versus 15 min. In all cases phosphorylation was transient, with a slow decay to basal levels within 2 h. The phosphorylation of mp67 occurred in a dose-dependent manner when increasing concentrations of mIL-3 or mGM-CSF were added to B-eSUtAj cells (typical results shown i n Figure 20). For both growth factors, mp67 p h o s p h o r y l a t i o n was detected al concentrations of 10" 1 ^-lO'^M, with half-maximal incorporation of 3 2 P at growth factor concentrations of approximately 10" ^ M. Similar studies with TPA revealed an optimal concentration of 100 ng/ml for TPA-induced phosphorylation of mp67. A) Control B)mlL-3 Figures 18A and B. Figure legend on following page. C) GM-CSF D)TPA basic acidic basic acidic Figure 18C and D. Analysis of membrane phosphoproteins by two-dimensional gel electrophoresis. ^ P - l a b e l e d BeSUtAj cells were incubated for ten minutes at 37°C with control buffer (A), 1 0 ~ 8 mol/L mIL-3 (B), 1 0 " 8 mol/L mGM-C S F (C), or 100 ng/mL TPA (D). Membrane phosphoproteins isolated from factor-stimulated cells were then analyzed by two-dimensional gel electrophoresis using 8 % to 2 0 % SDS polyacrylamide gels. Arrows indicate presence of a 67-kDa membrane protein. 500 H 0 30 60 90 120 Time (min) Figure 19A. Figure legend on following page. CO 0 5 15 30 60 120 Figure 19B. Time course of mp67 phosphorylation. J 2P-labeled B6SUIA! cells were stimulated with 10"° mol/L mIL-3, 10" 8 mol/L mGM-CSF, or 100 ng/mL TPA at 37°C for the indicated times (A). Membrane fractions were then isolated and phosphoproteins were analyzed by one-dimensional SDS-PAGE for mp67. Relative levels Q f 32p incorporation were determined by liquid scintillation counting of gel slices containing 3 2P-labeled mp67. Similar results were obtained in two separate experiments. The autoradiogram in B demonstrates co the phosphorylation of mp67 after stimulation of cells with mIL-3 (10" 8 mol/L) for the indicated times. 0 1 136 7. Phosphoamino A c i d A n a l y s i s of 3 2 P - l a b e l e d mp67 P h o s p h o a m i n o a c i d a n a l y s i s of m p67 was performed f o l l o w i n g a d d i t i o n of mIL-3, mGM-CSF, a n d T P A to c e l l s for 15 m i n at 37°C. In c e l l s treated w i t h mGM-CSF or TPA (Figure 21C), phosphorylation of mp67 occurred only on serine residues. R e s u l t s obtained w i t h mIL-3-treated c e l l s , however, i n d i c a t e t h a t w h i l e the m a j o r i t y of p h o s p h a t e was i n c o r p o r a t e d i n t o s e r i n e r e s i d u e s , a s i g n i f i c a n t a m o u n t w a s a l s o p r e s e n t as phosphotyrosine; t h i s was observed u s i n g both a formic a c i d / a c e t i c a c i d / w a t e r solvent s y s t e m ( F i g u r e 2 1A) a n d a p y r i d i n e / a c e t i c a c i d / w a t e r s o l v e n t s y s t e m ( F i g u r e 2 IB). Therefore, of the agents tested, only mIL-3 appears to stimulate tyrosine phosphorylation of mp67. 8. Treatment of Gels with A l k a l i after SDS-PAGE to E n r i c h for Phosphotyrosine A l k a l i treatment of SDS-polyacrylamide gels c o n t a i n i n g 3 2 P - l a b e l e d proteins results i n the p r e f e r e n t i a l h y d r o l y s i s of b a s e - l a b i l e p h o s p h o s e r i n e a n d p h o s p h o t h r e o n i n e phosphate ester linkages, with preservation of the relatively base-stable phosphate ester l i n k a g e s of p h o s p h o t y r o s i n e (18). T h i s technique c a n therefore be u t i l i z e d to e n r i c h for labeled phosphotyrosine-containing proteins i n gels. 3 2 P - l a b e l e d BeSUtAT cells were incubated i n the presence and absence of mIL-3 for the i n d i c a t e d times at 37°C (see Figure 22). F r o m these c e l l s , membrane samp l e s were p r e p a r e d as above a n d e l e c t r o p h o r e s e d o n o n e - d i m e n s i o n a l 8 - 2 0 % g r a d i e n t SDS-polyacrylamide gels. Gels were then treated with 1 M NaOH for 1 h at 55°C. Th i s treatment re v e a l e d the presence of a 67 k D a base-stable membrane p h o s p h o p r o t e i n , s t i m u l a t e d w i t h i n 15 m i n of a d d i t i o n of mIL-3, that was absent f r o m c o n t r o l c e l l s . T h i s f i n d i n g is c o n s i s t e n t w i t h m I L - 3 - i n d u c e d t y r o s i n e p h o s p h o r y l a t i o n of a 67 k D a s p e c i e s i n the membranes of BeSUtAj cells. 137 C. DISCUSSION As a first step in investigating the mechanism of action of mIL-3, we screened various agents for their ability to substitute for mIL-3 in stimulating BSSUtA]^ cells to proliferate. Of the growth factors tested, only mGM-CSF was as potent as mIL-3. Of a panel of low molecular weight mitogens tested, both sodium orthovanadate and TPA were shown to induce DNA synthesis i n these cells. Sodium orthovanadate is known to be a potent inhibitor of phosphotyrosine phosphatase (3,8), thus raising the possibility that tyrosine phosphorylation is involved in the signal transduction pathways of mIL-3 and mGM-CSF. Furthermore, the stimulatory activity of TPA i n these assays suggested a role for the serine/threonine kinase activity of PKC. However, when TPA was added to BGSUtAj cells growing i n the presence of mIL-3 or mGM-CSF, it markedly inhibited both mIL-3- and mGM-CSF-induced proliferation of these cells. This inhibition did not appear to be due to PKC exhaustion since the effect was observable within 2 h of exposure to TPA. In this regard, it should be noted that TPA has also recently been demonstrated to inhibit the proliferation of vascular smooth muscle cells in response to a-thrombin (19). Our findings are significant in view of previous reports implicating PKC in the mechanism of action of mIL-3. Specifically, it has been demonstrated that incubation of mIL-3-dependent cell lines with mIL-3, TPA or OAG leads to the rapid translocation of PKC from the cytosol to the plasma membrane (9). Moreover, it has also been reported that TPA potentiates the growth stimulating effects of suboptimal levels of mIL-3 on FDC-Mix 1 cells (6). Given that TPA appears to stimulate the growth of some mIL-3-dependent cell lines while inhibiting others, the activation of PKC may have diverse functions i n the growth regulation of mIL-3 responsive cells. Interestingly, a recent report has demonstrated the ability of TGF-(3 to inhibit the growth-promoting effect of mIL-3 on B6SUtA cells (20). Whether the inhibitory effect of TGF-p is operating through the PKC pathway i n these cells remains to be determined. 138 Growth Factor Concentration (*o Figure 20. Concentration dependence of mp67 phosphorylation. ^P-labeled cells were stimulated for 10 minutes at 37°C with the indicated concentrations of mIl-3 or GM-CSF. Membrane fractions were then analyzed by one-dimensional SDS-PAGE as in Figures 17 and 19. Relative levels of 3 2 P incorporation into mp67 was determined by liquid scintillation counting of gel slices representing mp67. C) C I C I T G Figure 21. Phosphoamino acid analysis of mp67. J ZP-labeled mp67 from cells incubated with mIL-3 (I), TPA (T), and mGM-CSF (G), was identified by autoradiography and elector-eluted from gel slices. Gel slices from areas of similar electrophoretic mobility from unstimulated control cells (C) were identically processed. Phosphoamino acid analysis of these samples was carried out as described in Chapter II using either a formic acid/acetic acid/water solvent system (A) or a pyridine/acetic acid/water solvent (B and C) for thin layer electrophoresis. Phosphoamino acid standards were identified by nin h y d r i n staining. P-Ser. phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine. Similar levels of radioactivity were added to the mIL-3, mGM-CSF and TPA lanes in B & C, but autoradiographic exposure time was 3 times longer for C. 140 CONTROL mIL-3 15 60 I20 15 60 120 mjns mins Figure 22. Alkali treatment of SDS-PAGE gels to enrich for phosphotyrosine. d^P-labeled B6SUtA 1 cells were treated with (mIL-3) or without (control) 1 0 - 8 M mIL-3 for the indicated times at 37°C. Membrane samples were then isolated and analyzed by SDS-PAGE on an 8-20% SDS polyacrylamide gel. The gel was then t r e a t e d w i t h 1 M NaOH for 1 h at 55°C. f o l l o w e d by f i x a t i o n and autoradiography. The arrow indicates the presence of a 67 kDa alkali-resistant band in mIL-3-treated cells. 141 G i v e n the above f i n d i n g s , we examined the p h o s p h o r y l a t i o n of c e l l u l a r p r o t e i n s i n cells stimulated with mIL-3, mGM-CSF, TPA and OAG. Studies u s i n g 3 2 P - l a b e l e d BSSUtAi^ c e l l s r e v e a l e d t h a t b o t h mIL-3 a n d m G M - C S F s t i m u l a t e the r a p i d , time- a n d dose-dependent p h o s p h o r y l a t i o n of a 68 k D a cytosolic p r o t e i n (cp68) and a 67 k D a membrane protein (mp67) i n these cells. Half-maximal incorporation of 3 2 P into cp68 and mp67 was observed at growth factor concentrations of 10" ^ M for both mIL-3 a n d mGM-CSF. T h i s s t r o n g l y s u g g e s t s t h a t t h e above p h o s p h o r y l a t i o n events o c c u r u n d e r p h y s i o l o g i c a l c o n d i t i o n s when BeSUtA^ c e l l s are exposed to either of these growth factors, since both mIL-3 a n d m G M - C S F s t i m u l a t e h a l f - m a x i m a l p r o l i f e r a t i o n of these c e l l s at p i c o m o l a r concentrations (see Figure 10). Phosphoamino acid analysis revealed that cp68 and mp67 were both phosphorylated on serine residues i n response to mIL-3 and mGM-CSF, and, i n a d d i t i o n , t h a t mIL-3 s t i m u l a t e d t y r o s i n e p h o s p h o r y l a t i o n of m p67 (see below). Serine phosphorylation of cp68 was not observed when cells were treated w i t h T P A or OAG over a wide range of c o n c e n t r a t i o n s . T h i s p h o s p h o r y l a t i o n event i s t h e r e f o r e u n l i k e l y to be m e d i a t e d by P K C , a l t h o u g h t h i s p o s s i b i l i t y c a n not, as yet, be r u l e d out. S e r i n e p h o s p h o r y l a t i o n of m p 6 7 w a s d e t e c t e d i n r e s p o n s e to T PA, a n d t h e r e f o r e t h i s p h o s p h o r y l a t i o n event may be m e d i a t e d t h r o u g h the a c t i o n of PKC. In f a c t , m p 6 7 p h o s p h o r y l a t i o n m a y be i n v o l v e d i n the m e c h a n i s m by w h i c h T P A s t i m u l a t e s D N A synthesis i n BeSUtA} cells. The curves shown i n Figs. 14,15,19A and 20 are a l l plotted without error bars, due in large part to the d i f f i c u l t y i n o b t a i n i n g c o n s i s t e n t levels of i n c o r p o r a t i o n of 3 2 P into the bands or spots of interest. However, the trends observed were consistent over the course of m u l t i p l e e x p e r i m e n t s , a n d the p l o t s s h o w n r e p r e s e n t t y p i c a l e x a m p l e s of the g i v e n experiments. Because of the s i m i l a r i t y i n apparent molecular masses, it i s possible that cp68 and mp67 are identical proteins. Against this, however, i n addition to their different s u bcellular l o c a t i o n s , are t h e i r d i f f e r e n t p h o s p h o a m i n o a c i d p r o f i l e s i n mIL-3-treated c e l l s , t h e i r 142 different i s o e l e c t r i c points, and, f i n a l l y , the demonstration that TPA s t i m u l a t e s phosphorylation of the membrane phosphoprotein but not the cytosolic phosphoprotein. - The 68 kDa cytosolic phosphoprotein may be identical to a protein of s i m i l a r molecular mass that has very recently been reported to be phosphorylated in certain factor dependent c e l l l i n e s when st i m u l a t e d w i t h mIL-3, G -CSF or IL-2 (21,22). These investigators found, however, that TPA, and not mGM-CSF, stimulated phosphorylation of this protein and that phosphorylation occurred only on threonine residues. These discrepancies may reflect differences in the cell types used in the two studies. A possible functional role for the 68 kDa cytosolic protein is suggested by the recent finding that a 65 kDa cytosolic S6 kinase is activated in chick embryo fibroblasts in response to various mitogenic agents (23). The activated protein then autophosphorylates on serine residues and phosphorylates the ribosomal protein S6. Since phosphorylation of S6 is a common event in many mitogenically stimulated cells (24), cp68 may function as an S6 kinase. We have previously shown that, in addition to a 140 kDa protein, mIL-3 binds to a 67 kDa receptor on BeSUtA^ cells (reference 25 and see Chapter III), in close agreement w i t h others using different cell lines (26-28). It is therefore possible that the 67 kDa membrane phosphoprotein reported here is the 67 kDa mIL-3 receptor, although this relationship obviously requires further investigation. If this is indeed the case, then one can a l s o postulate that serine phosphorylation of this protein, possibly by PKC, is a regulatory e v e n t , as has been suggested in recent reports where phosphorylation of occupied growth factor receptors by PKC results in subsequent desensitization of target cells to the respective growth factor (reviewed in 29). Therefore, mIL-3 may be regulating its own receptor by-activating PKC, which i n turn phosphorylates the receptor on serine residues. TPA m a y mimic this effect by directly activating PKC, thus providing a possible basis for the ability of TPA to inhibit the proliferative activity of mIL-3 and mGM-CSF on B6SUtAi cells. In fact , the moderate stimulatory activity of TPA in proliferation assays may be a consequence o f mIL-3 receptor internalization or alteration, induced by PKC phosphorylation of t h e receptor. With respect to a role for mGM-CSF in such a scheme, there is recent evidence 143 for the cross-modulation of hemopoietic growth factor receptors by heterologous growth factors within the hemopoietic system (30), and it is possible that receptor phosphorylation may be involved in this form of cross-modulation. Of the factors tested, only mIL-3 induced the tyrosine-specific phosphorylation of mp67, as evidenced by phosphoamino acid analysis and a l k a l i treatment of SDS-polyacrylamide gels. The fact that sodium orthovanadate can stimulate DNA synthesis in BSSUtA^ cells is also consistent with a role for tyrosine phosphorylation in the mechanism of action of mIL-3. These studies therefore provide preliminary evidence that mIL-3 binding a c t i v a t e s p r o t e i n t y r o s i n e k i n a s e s i n BSSUtA^ c e l l s . Whether the presence of p h o s p h o t y r o s i n e i n p h o s p h o r y l a t e d mp67 represents l i g a n d - i n d u c e d t y r o s i n e phosphoryation of the 67 kDa mIL-3 receptor is the subject of a later chapter. A similar role for tyrosine phosphorylation in the mechanism of action of mGM-CSF can not be ruled out from these studies. We have d e m o n s t r a t e d that mIL-3 and mGM-CSF both s t i m u l a t e s e r i n e phosphorylation of 68 kDa cytosolic and 67 kDa membrane proteins in BeSUtA^ cells. Furthermore, mIL-3 was shown to induce tyrosine phosphorylation of the membrane p r o t e i n . S i n c e n e i t h e r p h o s p h o r y l a t i o n of the c y t o s o l i c p r o t e i n n or t y r o s i n e phosphorylation of the membrane protein could be reproduced i n cells treated with activators of PKC, it appears that, in addition to PKC, other phosphorylation systems a r e activated when mIL-3 and mGM-CSF interact with BeSUtAj cells. It remains to b e determined how each of the phosphoproteins described is functionally involved in the signal transduction pathways activated by these growth factors. 144 D. REFERENCES 1. Rozengurt E. Early signals in the mitogenic response. Science 234: 161, 1986. 2. Chambard JC, Paris S. L'Allemain F, Pouyssegur J . Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin. Nature 326: 800, 1987. 3. Hunter T, Cooper JA. Protein-tyrosine kinases. Ann Rev Biochem: 897, 1985. 4. Nishizuka Y. Studies and perspectives of protein kinase C. Science 233: 305, 1986. 5. Rozengurt E. Cyclic AMP, a growth-promoting signal for mouse 3T3 cells. Adv Cycl Nucl Res 14: 429, 1981 6. Whetton AD, Heyworth CM, Dexter TM. Phorbol esters activate protein kinase C and glucose transport and can replace the requirement for growth factor in interleukin-3-dependent multi-potent stem cells. J Cell Sci 84: 93. 1986. 7. Metcalf D. The granulocyte-macrophage colony-stimulating factors. Science 229: 16, 1985. 8. Swarup G, Cohen S. Garbers DL. Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate. Biochem Biophys Commun 107: 1104, 1982. 9. F a r r a r WL, Thomas TP, A n d e r s o n WB. A l t e r e d cytosol/membrane enzyme distribution on interleukin-3 activation of protein kinase. Nature 315: 235, 1985. 10. Solanki V, Slaga TJ. Specific binding of phorbol ester tumour promoters to intact primary epidermal cells from Senear mice. Proc Natl Acad Sci 78: 2549, 1981. 11. Collins M, Rozengurt E. Stimulation of DNA synthesis i n murine fibroblasts by the tumour promoter teleocidin: relationship to phorbol esters and vasopressin. Biochem Biophys Res Commun 104: 1159, 1982. 12. Rodriguez-Pena A, Rozengurt E. Disappearance of C a 2 + - s e n s i t i v e , phospholipid -dependent protein kinase activity in phorbol ester-treated 3T3 cells. Biochem Biophys Res Commun 120: 1053, 1984. 13. J a n s k i AM, Cornell NW. Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes. Biochem J 186: 423, 1980. 14. Garrison JC. Measurement of hormone-stimulated protein phosphorylation in intact cells. Methods Enzymol 99F: 20, 1983. 15. P a l a s z y n s k i EW, Ihle JN. Evidence for specific receptors for i n t e r l e u k i n 3 on lymphokine-dependent cell lines established from long-term bone marrow cultures. J Immunol 132: 1872, 1984. 16. Krishnamurthi S, Joseph J , Kakkar W. 1,2-Dioctanoylglycerol but not 1-oleoyl-2-acetylglycerol inhibits agonist-induced platelet responses. Dependence of effects on extent of 45-kDa protein phosphorylation and agonist type. Eur J Biochem 167: 585. 1987. 145 17. Belsham GJ, Dentom RM, Tanner MJA. Use of a novel rapid preparation of fat-cell plasma membranes employing Percoll to investigate the effects of i n s u l i n and adrenaline on membrane protein phosphorylation in intact cells. Biochem J 192: 457, 1981. 18. Buss J E , S t u l l JT. Measurement of chemical phosphate i n proteins. Methods Enzymol 99: 5, 1983. 19. Huang C-L, Ives HE. Growth inhibition by protein kinase C late i n mitogenesis. Nature 329: 849, 1987. 20. Ohta M, Greenberger JS, Anklesaria P et al. Two forms of transforming growth factor-P distinguished by multipotential haemopoietic progenitor cells. Nature 329: 539, 1987. 21. Evans SW, Rennick D, Fa r r a r WL. Multilineage hematopoietic growth factor interleukin 3 and direct activators of protein kinase C stimulate phosphorylation of common substrates. Blood 68: 906, 1986. 22. Evans SW, Rennick D, Farrar WL. Identification of a signal-transduction pathway shared by haematopoietic growth factors with diverse biological specificity. Biochem J 244: 683, 1987. 23. Blenis J , Kuo CJ, E r i k s o n RL. Identification of a ribosomal protein S6 kinase regulated by transformation and growth-promoting stimuli. J Biol Chem 262: 14373, 1987. 24. Blenis J , Erikson RL. Regulation of a ribosomal protein S6 kinase activity by the Rous sarcoma virus transforming protein, serum, or phorbol ester. Proc Natl Acad Sci USA 82: 7621, 1985. 25. Sorensen P, Farber NM, Krystal G. Identification of the interleukin-3 receptor using an iodinatable, cleavage, photoreactive cross-linking agent. J Biol Chem 261: 9094, 1986. 26. Park LS, Friend D, Gillis S, Urdal DL. Characterization of the cell surface receptor for a multi-lineage colony-stimulating factor (CSF-2a). J Biol Chem 261: 205, 1986. 27. May WS, Ihle JN. Affinity isolation of the interleukin-2 surface receptor. Biochem Biophys Res Commun 135: 870, 1986. 28. Nicola NA, Peterson L. Identification of distinct receptors for two hemopoietic growth factors (granulocyte colony-stimulating factor and multipotential colony-stimulating factor) by chemical cross-linking. J Biol Chem 261: 12384, 1986. 29. Sibley DR, Benovic JL. Caron MG, Lefkowitz RJ. Regulation of transmembrane signaling by receptor phosphorylation. Cell 48: 913, 1987. 30. Walker F, Nicola NA, Metcalf D. Burgess AW. Hierarchical down-modulation of hemopoietic growth factor receptors. Cell 43: 269, 1985. 146 C H A P T E R V INTERLEUKIN-3 STIMULATES TYROSINE-SPECIFIC PROTEIN PHOSPHORYLATION IN FACTOR-DEPENDENT E^SUtA, CELLS A. INTRODUCTION The a c t i v a t i o n of tyrosine-specific protein kinases has been implicated as an intermediate step i n the mitogenic response of cells to several polypeptide growth factors, including EGF, insulin, PDGF and M-CSF (1). The surface receptors for these molecules are relatively large membrane proteins containing intracellular protein tyrosine kinase (PTK) domains. The kinase moieties become activated by ligand binding and subsequently catalyze receptor autophosphorylation on tyrosine residues (2), as well as tyrosine phosphorylation of as yet poorly characterized cellular substrate proteins. A role for tyrosine phosphorylation in mIL-3 mediated signal transduction was suggested by the finding that mIL-3 stimulates tyrosine phosphorylation of a 67 kDa membrane protein in B6SUtA cells, and by the ability of sodium orthovanadate to stimulate proliferation of these cells. Although the 67 kDa mlL-3 receptor is likely too small to harbour an intracellular tyrosine kinase domain (3), this does not rule out that mIL-3 also activates protein tyrosine kinases in the signal transduction pathways of mIL-3-dependent cells. In fact, the larger 140 kDa binding protein described in Chapter III, is theoretically large enough to be a receptor protein tyrosine kinase. We have, therefore, examined more rigorously the ability of mIL-3 as well as mGM-CSF and TPA to stimulate tyrosine phosphorylation in B6SULAT cells. Specifically, we have used polyclonal antiphosphotyrosine antibodies to identify phosphotyrosine-containing proteins in factor-stimulated cells. Antiphosphotyrosine antibodies (aPTyrAbs) have been used extensively in recent years to investigate tyrosine phosphorylation in a variety of growth factor systems (4-8). These ant i b o d i e s were o r i g i n a l l y derived by i m m u n i z i n g r a b b i t s with the phosphotyrosine analogue, p-azobenzyl phosphonate (ABP), derivatized to keyhole limpet 147 h emocyanin (9). M onoclonal antibodies to phosphotyrosine have also been produced using A B P a s a h a p t e n (10). T h i s c h a p t e r d e s c r i b e s the use of r a b b i t p o l y c l o n a l a P T y r A b s , previously s h own to interact w ith phosphorylated tyrosine residues of P D G F receptors (11), to identify a n u m b e r of phosphotyrosine-containing proteins i n mIL-3-stimulated B SSUtA^ cells. B. R E S U L T S 1. W e s t e r n B l o t A n a l y s i s of C y t o s o l i c a n d M e m b r a n e P h o s p h o p r o t e i n s u s i n g  Antiphosphotyrosine Antibodies To examine phosphotyrosine-containing proteins i n BeSUtAj cells, mIL-3, mGM-CSF, or TPA was added to cells for 10 m i n at 37°C. Cells were then subfractionated into cytosolic a n d membrane fr a c t i o n s as before, and analyzed by Western b l o t t i n g u s i n g either affinity p u r i f i e d r a b b i t p o l y c l o n a l a P T y r A b s or c o n t r o l p r o t e i n A-sepharose p u r i f i e d r a b b i t IgG. C y t o s o l i c s a m p l e s are s h own i n F i g u r e 23A. A s i n d i c a t e d i n l a n e s 1-4, t y r o s i n e - s p e c i f i c p h o s p h o r y l a t i o n of a 68 k D a c y t o s o l i c p r o t e i n i n f a c t o r s t i m u l a t e d c e l l s w a s not demonstrated, i n agreement with previous phosphoamino acid analysis r e s u l t s (see Chapter IV). However, a number of differences i n labeling patterns were consistently observed using t h i s t e c h n i q u e . The most p r o m i n e n t f i n d i n g was the appearance of a 90 k D a tyros i n e -specific phosphoprotein i n cells stimulated with the growth-promoting agents mIL-3 (lane 2) and mGM-CSF (lane 3), but not i n control cells (lane 1) nor i n cells treated with TPA (lane 4). T h i s was not observed when pu r i f i e d rabbit IgG was used (lanes 6 a n d 7). Moreover, only mIL-3 stimulated the tyrosine-specific phosphorylation of a 55 k D a cytosolic protein (lane 2), while T P A unique l y induced the tyrosine-specific phosphorylation of a 40 k D a protein (lane 4). These findings were not observed i n conventional 3 2 P T a b e l i n g studies, perhaps reflecting the low relative a b u ndance of these phosphoproteins or the m a s k i n g of these p r o t e i n s in SDS-polyacrylamide gels by other phosphoproteins with s i m i l a r mobilities. Western blot a n a l y s i s of membrane phosphotyrosine-containing proteins is shown in F i g 23B. A s i n d i c a t e d i n l a n e 2, mIL-3, b u t not m G M - C S F (lane 3), n o r T P A (lane 4). s t i m u l a t e d the t y r o s i n e - s p e c i f i c p h o s p h o r y l a t i o n of 68 a n d 140 k D a membrane proteins. 148 These two b a n d s were not seen when c o n t r o l r a b b i t IgG was u s e d (lane 6). The 68 k D a protein li k e l y represents the m67 species observed i n the 3 2 P - l a b e l i n g studies described i n C hapter IV, c o n f i r m i n g that tyrosine p h o s p h o r y l a t i o n of a 67-68 k D a membrane protein o c c u r s i n mIL-3-treated cells. The absence of a 68 k D a b a n d i n the mGM-CSF and TP A lanes c o n f i r m s the r e s u l t s of the phosphoamino a c i d analyses w h i c h suggested that these two agents s t i m u l a t e only serine-specific p h o s p h o r y l a t i o n of t h i s p r o t e i n (see Chapter IV). P h o s p h o r y l a t i o n of the 140 k D a membrane p r o t e i n on tyrosine r e s i d u e s i n mIL-3 treated c e l l s was n o t d e t e c t e d i n 3 2 P - l a b e l i n g s t u d i e s , p e r h a p s a g a i n r e f l e c t i n g low r e l a t i v e abundance or m a s k i n g by other phosphoproteins. Figure 23B also provides evidence for the tyrosine-specific phosphorylation of an 80 k D a membrane protein i n TPA-treated cells (lane 4) w h i c h was not s e e n i n c o n t r o l , mIL-3 or m G M - C S F - t r e a t e d c e l l s (lanes 1, 2 a n d 3, respectively). 2. I m m u n o p r e c i p i t a t i o n of P h o s p h o t y r o s i n e - c o n t a i n i n g P r o t e i n s u s i n g  Antiphosphotyrosine Antibodies To further investigate mIL-3-induced tyrosine phosphorylation i n B 6 S U t A j cells, 32p_ labeled cells were incubated with control buffer or mIL-3 for 10 m i n at 37°C. Cells were then s o l u b i l i z e d u s i n g HEPES-buffered 1 % NP40, and the r e s u l t i n g c e l l lysates dialyzed against the lysi s buffer at 4°C. Lysates were dialyzed i n order to remove cytosolic nucleotides, which have been reported to interfere with the interaction between aPTyrAbs and phosphotyrosine residues. Phosphotyrosine-containing proteins were then immunoprecipitated from dialyzed lysates (containing equal 3 2 P cpm) w i t h these antibodies as described i n C h a p t e r II. One d i m e n s i o n a l S D S - P A G E r e v e a l e d t h a t a d d i t i o n of mIL-3 l e a d s to i n c r e a s e d t y r o s i n e p h o s p h o r y l a t i o n of a number of proteins, as i n d i c a t e d i n Figure 24, lane 3. These include proteins with apparent molecular masses of 140, 90, 68, 55, and 40 kDa. The presence of t h e s e p h o s p h o p r o t e i n s i n m I L - 3 - s t i m u l a t e d c e l l s w a s f u r t h e r d e m o n s t r a t e d by two-dimensional gel electrophoresis of immunoprecipitates from untreated (Figure 25A) and mlL-3-treated (Figure 25B) cells. T h i s study therefore coriflrms the results of the Western blot 150 Figure 23B. W e s t e r n b l o t a n a l y s i s of p h o s p h o p r o t e i n s f r o m B G S U t A ^ c e l l s u s i n g antiphosphotyrosine antibodies. Cytosolic proteins (A) or membrane proteins (B) from control cells (lanes 1 and 5) and cells treated for ten minutes at 37 C with 10" 8 mol/L mIL-3 (lanes 2 and 6), 10" 8 mol/L mGM-CSF (lanes 3 and 7). a n d 100 n g / m l T P A ( l a n e s 4 a n d 8) were s u b j e c t e d to S D S - P A G E a n d e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d onto n i t r o c e l l u l o s e f i l t e r s as d e s c r i b e d in Chapter II. F i l t e r s were then incubated with 3.5 ug/ml rabbit affinity-purified a n t i p h o s p h o t y r o s i n e a n t i b o d i e s (lanes 1 t h r o u g h 4) or 4 ug/ml P r o t e i n A-pu r i f i e d non-immune rabbit IgG (lanes 5 through 8) and v i s u a l i z e d with 1 2 5 i -labeled Staphylococcus aureus protein A. Autoradiographic exposure time was 24 h o u r s for lanes 1 through 4 and four days for lanes 5 t h r o u g h 8 for both panels A and B. S i m i l a r results were obtained i n four separate experiments. 151 Figure 24. Immunoprecipitation of BSSUtA! phosphoproteins with antiphosphotyrosine antibodies. Cells were labeled with o n [oz,P|orthophosphate and incubated for ten minutes at 37°C with (lanes 1 and 3) or without (lanes 2 and 4) 10"°" mol/L mIL-3. Cell lysates were then prepared as described in Chapter II and dialyzed against the solubilization buffer at 4°C. SDS-PAGE of aliquots from the cell lysates are shown in lanes 1 and 2. Phosphotyrosine-containing proteins were immunoprecipitated from dialyzed lysates with antiphosphotyrosine antibodies and analyzed by SDS-PAGE (lanes 3 and 4). Arrows indicate phosphoproteins discussed in the text. Similar results were obtained in four separate experiments. A) Control B) mll_-3 Basic Acidic Basic Acidic Figure 25. Two-dimensional gel analysis of mIL-3-induced tyrosine phosphorylation in B6SUIAJ cells. ^P-labeled B6SUtA^ cells were stimulated with (B) or without (A) mIL-3 for 5 min at 37°C. Cell lysates were then prepared and phosphotyrosine-containing proteins isolated by immunoprecipitation with aPTyrAbs as described in Materials and Methods. Labeled proteins were subsequently analyzed by O'Farrell two-dimensional gel electrophoresis. Arrows indicate the position of mIL-3 induced phosphoproteins. 153 analyses (Figure 23), where mIL-3 was shown to s t i m u l a t e the tyrosine p h o s p h o r y l a t i o n of 140, 90, 68, a n d 55 k D a p r o t e i n s , a n d p r o v i d e s e v i d e n c e f o r the a d d i t i o n a l t y r o s i n e phosphorylation of a 40 k D a protein. 3. Time Course Study of mII-3-induced Tyrosine Phosphorylation To determine the k i n e t i c s of mIL-3-stimulated tyrosine phosphorylation, 3 2 P - l a b e l e d B6SU1A.! cells were incubated at 37°C with mIL-3 for va r i o u s time periods. After reactions were stopped, phosphotyrosine-containing proteins were immunoprecipitated from whole-cell extracts as described above an d subjected to SDS-PAGE. T y p i c a l r e s u l t s are i n d i c a t e d i n Figure 26, showing that phosphorylation of the 140, 90, 68, 55, and 40 k D a proteins was in a l l c a s es detectable w i t h i n 2 min. F o r the 140 a n d 68 k D a p h o s p h o p r o t e i n s , m a x i m a l phosphorylation was reached w i t h i n 2 min, while m a ximal levels were reached w i t h i n 5 min for the 90 k D a p r o t e i n a n d w i t h i n 15 m i n for the 55 a n d 40 k D a p r o t e i n s . W h e t h e r the different time intervals to reach maximal incorporation of 3 2 P reflect the chronological order of phosphorylation of these proteins cannot be ascertained from the present studies. A slow decay towards basal levels was observed i n a l l cases after maximal levels were attained. 4. Concentration Dependence of mIl-3-induced Tyrosine Phosphorylation Tyrosine p h o s p h o r y l a t i o n was dependent on the concentration of mIL-3 added to the c e l l s . A dose response of the p h o s p h o r y l a t i o n of the above p r o t e i n s was d e t e r m i n e d by s t i m u l a t i n g BeSt l rA^ c e l l s w i th v a r i o u s c o n c e n t r a t i o n s of mIL-3 for 10 m i n at 37°C, and then analyzing immunoprecipitated phosphotyrosine-containing proteins by SDS-PAGE. As s h o w n i n F i g u r e 27, tyrosine p h o s p h o r y l a t i o n of each of the 140, 90, 68, 55, a n d 40 kDa proteins was typically detectable at 10 pM mIL-3. Concentrations of mIL-3 required for half-m a x i m a l s t i m u l a t i o n of p h o s p h o r y l a t i o n r a n g e d f r o m 3 0 to 8 0 p M f o r t h e f i v e p h o s p h o p r o t e i n s . S i n c e h a l f - m a x i m a l p r o l i f e r a t i o n of BeSUtA^ c e l l s i n 3 H - t h y m i d i n e incorporation assays occurs at approximately 50 pM mIL-3 (see Figure 10), the present Figure 26A. Figure legend on following page. B) 0 15 30 45 60 Time (mia) Figure 26B. Time course of mIl-3-induced tyrosine phosphorylation. 3 2P-labeled BGSUtA, cells were incubated with 10-8 mol/L mIl-3 at 37°C for various times. Phosphotyrosine-containing proteins were then Immunoprecipitated with antiphosphotyrosine antibodies as described in the legend to Fig. 24. (A) SDS-PAGE of Immunoprecipitated phosphoproteins after stimulation of cells with mIl-3 for the indicated times. (B) Graphic representation of data in A. Relative levels of 3 2 P incorporation were determined by laser densitometric scanning. Similar results were obtained ln two separate experiments. cn Figure 27. Dose-dependence of mIl-3-induced tyrosine phosphorylation. Immunoprecipitation of phosphotyrosine-containing proteins with antiphosphotyrosine antibodies was performed after incubation of 3 2P-labeled B6SUT-A! cells with the indicated concentrations of mIl-3 at 37°C. Laser densitometric scanning of phosphoprotein bands was used to determine relative levels of 3 2 P incorporation. Similar results were obtained in two separate experiments. » U l 157 f i n d i n g s s t r o n g l y suggest t h a t the above t y r o s i n e p h o s p h o r y l a t i o n s are i n d u c e d u n d e r physiological conditions. 5. Phosphoamino A c i d A n a l y s i s of Immunoprecipitated Phosphoproteins To c o n f i r m the presence of phosphotyrosine i n the immunoprecipitated, 3 2 P - l a b e l e d 40-140 k D a proteins, the phosphoamino acid content of each of these species was analyzed. 3 2 P - l a b e l e d B G S U t A j cells were stimulated with mIL-3 for 5 m i n at 37°C as described above. A f t e r i m m u n o p r e c i p i t a t i o n w i t h a P T y r A b s a n d a n a l y s i s of i m m u n o p r e c i p i t a t e s b y SDS-P A G E , p h o s p h o a m i n o a c i d a n a l y s i s of the 40, 55, 68, 9 0 a n d 140 k D a p r o t e i n s was performed. Figure 28 clearly demonstrates the presence of phosphotyrosine, i n addition to phosphoserine, i n a l l of these proteins after stimulation of cells with mIL-3. C. DISCUSSION The r e s u l t s presented i n t h i s chapter provide strong evidence that mIL-3 st i m u l a t e s t y r o s i n e - s p e c i f i c p r o t e i n p h o s p h o r y l a t i o n i n mIL-3-dependent BeSUtA^ c e l l s . T y r o s i n e p h o s p h o r y l a t i o n i n t h e s e c e l l s w as e x a m i n e d u s i n g a f f i n i t y p u r i f i e d a n t i b o d i e s to phosphotyrosine. W e s t e r n blot a n a l y s i s u s i n g these antibodies demonstrated that mIL-3 induces tyrosine phosphorylation of 68 and 140 k D a membrane proteins. In addition, mIL-3 s t i m u l a t e s t y r o s i n e p h o s p h o r y l a t i o n of 55 and. 90 k D a c y t o s o l i c p r o t e i n s . T y r o s i n e p h o s p h o r y l a t i o n of the 90 k D a prot e i n was also detected i n mGM-CSF-treated ce l l s , while the 55, 68, a n d 140 k D a s p e c i e s were ob s e r v e d o n l y w i t h mIL-3. T h e r e f o r e , t y r o s i n e p h o s p h o r y l a t i o n of a 90 k D a c y t o s o l i c p r o t e i n may be a c o m m o n c o m p o n e n t of s i g n a l t r a n s d u c t i o n i n response to both growth factors i n B6SUtAT cells. The i n a b i l i t y of TPA to stimulate s i m i l a r tyrosine phosphorylations suggests that these phosphorylation events are not indir e c t l y mediated through the action of PKC. Proteins immunoprecipitated from 32p_ labeled B 6 S U t A ^ cells w i th antiphosphotyrosine antibodies after short term i n c u b a t i o n with mIL-3 confirmed that this growth factor stimulates tyrosine phosphorylation of 140, 90, 68. p40 p55 p90 p68 p140 Figure 28. Phosphoamino acid analysis of the mIL-3-induced phosphotyrosine-containing proteins. The five ^ P - l a b e l e d proteins identified in Fig. 24 and Fig. 25 were electroeluted from gel slices and subjected to phosphoamino acid a n a l y s i s u s i n g a 1:10:189 p y r i d i n e / a c e t i c acid/water solvent system and t h i n - l a y e r electrophoresis. Phosphoamino a c i d s t a n d a r d s were i d e n t i f i e d by n i n h y d r i n staining: P-Ser, phosphoserine; P-Thr, phosphothreonine; P-tyr, phosphotyrosine. 159 and 55 kDa proteins, and provided evidence for an additional phosphotyrosine-containing protein of 40 kDa i n these cells. The c o n c e n t r a t i o n of mIL-3 r e q u i r e d to s t i m u l a t e h a l f - m a x i m a l t y r o s i n e phosphorylation typically ranged from 30 to 80 pM for the five phosphotyrosine-containing proteins described above. Since the concentration of mIL-3 required to half-maximally stimulate proliferation of BeSUtAj cells is approximately 50 pM, the above data strongly suggest that these phosphorylation events are induced under physiological conditions. Kinetic analysis of tyrosine phosphorylation revealed that mIL-3-stimulated tyrosine phosphorylation of 68 and 140 kDa proteins were the earliest phosphorylation events detected, in both cases reaching maximal levels within 2 min. The other phosphorylation events noted reached maximal levels within 5-15 min of stimulation. It will be of interest to determine if these data reflect the physiological order of protein phosphorylation in growth factor-treated BeSUtAj cells. It should be noted that in other growth factor systems such as those involving epidermal growth factor, insulin, and platelet-derived growth factor, tyrosine phosphorylation also occurs rapidly and transiently after growth factor binding (1,2). The results presented in this chapter demonstrate that an increase in tyrosine-specific protein phosphorylation is an early event in the stimulation of BSSUtA! cells by mIL-3 and also by mGM-CSF. Therefore these growth factors can be placed In a category of polypeptide mitogens, along with EGF, insulin, PDGF, M-CSF and others, whose signal transduction mechanisms appear to involve the activation of protein tyrosine kinases i n target cells Elucidating the nature of the tyrosine kinases activated in mIL-3-stimulated cells, as well as the functional roles of the tyrosine phosphorylated substrates, will likely be fundamental to the^understanding of how mIL-3 and mGM-CSF stimulate the growth of hemopoietic cells. A number of recent reports provide some suggestions as to the nature of the phosphoproteins described in this study. One candidate for the identity of the 40 kDa phosphoprolein described above is one or more members of the lipocortin family (calpactins), which are 34-39 kDa tyrosine phosphorylated proteins, found in EGF stimulated cells (12), that inhibit phospholipase A 2 (see Chapter I). Another candidate is the protein known as p42, a 42 kDa 160 molecule that is rapidly and transiently tyrosine phosphorylated in fibroblasts in response to a number of mitogens including EGF and PDGF (13). Recent studies suggest that p42 is related to or id e n t i c a l to a cytosolic serine/threonine kinase which phosphorylates microtubule associated protein-2 (MAP-2) as well as ribosomal protein S6 kinase in mitogen-activated cells (14). The protein has therefore been designated as MAP-2 kinase (14), and its kinase activity appears to be activated by tyrosine phosphorylation. With regard to the 55 kDa phosphoprotein, two 56-59 kDa members of the src family of non-receptor protein tyrosine kinases have recently been identified In hemopoietic cells. The product of the c-lck gene, expressed predominantly i n T lymphocytes (15,16), is a 56 kDa PTK (pp56^c^) which is highly phosphorylated in vivo on tyrosine residues 394 and 505 (17). Tyrosine-394 is the autophosphorylation site of p p 5 6 ^ and leads to increased PTK activity (15). Since mutation of tyrosine-505 to phenylalanine increases the PTK activity and evokes the transforming potential of this protein (15), phosphorylation of tyrosine-505 may represent negative regulation of the kinase activity of pp56^c^. It has also been found that CD45, or leukocyte common antigen (LCA), is responsible for the dephosphorylation of tyrosine-505 i n activated T cells (18). Recently, it was demonstrated that pp56^ c^ is p h y s i c a l l y a s s o c i a t e d w i t h the CD4 a n d C D 8 m o l e c u l e s on T h e l p e r a n d T cytotoxic/suppressor cells, respectively. Therefore one possible physiological scenario in antigen-binding T cells is that CD4 or CD8-associated pp56^ c^ is first brought into contact with CD45, and dephosphorylated on tyrosine-505 with activation of the PTK activity. The pp56^c^- molecule is subsequently brought into contact with the T cell receptor-CD3 complex, resulting i n tyrosine phosphorylation of the 5 ch a i n of CD3 (19) and signal t r a n s d u c t i o n across the T cell membrane. It also appears that pp56^ c^ is serine phosphorylated on amino-terminal residues by both PKC and another C a 2 + dependent serine/threonine protein kinase (20). Another src-related protein found i n cells of hemopoietic origin is p p 5 9 ^ c ^ , a 59 kDa membrane-associated PTK that is found predominantly i n mature hemopoietic cells of low i n vivo mitotic potential including granulocytes and monocytes (21,22). Activated macrophages show large increases in 161 expression of c-hck (23). As with pp56^ c k, there appears to be a carboxy terminal tyrosine whose phosphorylation results i n negative regulation of the PTK activity and mutation of which to a phenylalonine results in transforming ability of the protein (24). The c-hck gene has been localized to chromosome 20 at bands q l l - 1 2 , a region that is affected by interstitial deletions i n some acute myeloid leukemias and myeloproliferative disorders (22). Whether mIL-3 is involved in the activation of src-related genes remains to be determined. Tyrosine phosphorylation of the 67 kDa mIL-3 receptor could in theory explain the presence of a 68 kDa phosphotyrosine-containing protein i n mIL-3-stimulated B6SU+A 1 cells. However, data to be presented in Chapter VI makes such a possibility highly unlikely. Another candidate for the 68 kDa phosphoprotein is the proto-oncogene product Raf-1. This 68 kDa serine/threonine kinase has been implicated i n signal transduction mediated by membrane-bound oncogene products and growth factor receptors (25). Recent evidence suggests that this protein associated with and is tyrosine phosphorylated by ligand-activated PDGF receptor (26), with a resultant increase in the kinase activity of the Raf-1 protein (26). An identity for the 90 kDa phosphotyrosine-containing protein observed in mIL-3- (and MGM-CSF) s t i m u l a t e d cells is suggested by recent reports of an 85 k D a tyrosine phosphorylated protein that associated with p p 6 0 v " s r c polyoma middle T / p p 6 0 c " s r c complexes, and ligand-activated PDGF and M-CSF receptors (27,28,29). The 85 kDa protein is i n fact of p h o s p h a t i d y l i n o s i t o l (PI) kinase, termed PI-3 kinase as it catalyzes phosphorylation of the D-3 position of the inositol ring (not involved in the generation of IP3) (27,28). The kinase activity, increased by tyrosine phosphorylation of PI-3 kinase, appears to be important in signal transduction as mutant PDGF receptors unable to associate with and phosphorylate PI-3 kinase are unable to signal DNA synthesis when cells bind PDGF (30). Two other proteins that have recently been shown to be tyrosine phosphorylated in response to growth factors are the y isozyme of the phospholipase C family (PLC-y) and ras-associated GTPase-activating protein (GAP). PLC-y, one of the PLC isozymes involved in P I P 2 metabolism to generate IP3 and DAG, is a 145 kDa membrane-associated protein that is reportedly tyrosine phosphorylated (and subsequently activated) by ligand-activated EGF 162 and PDGF receptors (31-34). Interestingly. PLC-y is known to contain src homology (SH) region domains (SH2 and SH3) (33), though to be involved in the association of proteins bearing these regions with protein tyrosine kinase domains (35). Tyrosine phosphorylation of PLC-y was not observed in response to bombesin and bradykinin (33), both of which are known to induce P I P 2 metabolism. Therefore there are likely two ways to activate PLC, one involving tyrosine phosphorylation of PLC-y, and the other, as described i n Chapter I, involving the activation of a G protein. GAP is a 120 kDa cytosolic protein known to interact with p 2 1 r a s proteins at a site previously identified as the effector site of p 2 1 r a s (36). GAP has therefore been implicated as the biological target for regulation by ras proteins. Upon association of ras proteins GAP, the GTPase activity of the normal p 2 1 r a s is dramatically increased while that of oncogenic forms of ras protein is not (20). It is hypothesized that interaction of p2 l r a s with GAP draws the latter to the membrane where it can interact with other signal transduction elements, with activation of the GTPase activity of p 2 1 r a s resulting i n disassociation from GAP and dampening of the response (20). It has recently been reported that GAP, which contains two SH2 regions (20,35), is tyrosine phosphorylated i n cells transformed by cytoplasmic and receptor-like PTKs, and in EGF-stimulated fibroblasts (37). These results, therefore, suggest a mechanism, involving GAP, by which tyrosine kinases might modify p 2 1 r a s function. Either PLC-y or GAP, i n view of their respective apparent molecular masses, are theoretically candidates for the 140 kDa phosphotyrosine-containing protein observed in the mIL-3 stimulated cells. However, as it is now well established that the receptors for several polypeptide growth factors contain intracellular tyrosine kinase domains which become tyrosine autophosphorylated upon ligand binding (1), another strong possibility is that the 140 kDa phosphoprotein represents the 140 kDa mIL-3 receptor. It was therefore of interest to determine if mIL-3 stimulates tyrosine phosphorylation of its surface receptor proteins, and this is the subject of the following chapter. 163 D. REFERENCES 1. Hunter T, Cooper JA. Proteln-tyrosine kinases. Ann Rev Biochem: 897. 1985. 2. Carpenter G. Receptors for epidermal growth factor and other polypeptide mitogens. Ann Rev Biochem: 881, 1987. 3. May WS, Ihle JN. Affinity isolation of the interleukin 3 surface receptor. Biochem Biophys Res Commun 135: 870, 1986. 4. Frackelton J r AR, Tremble PAA, Williams LT. Evidence for the platelet-derived growth factor-stimulated tyrosine phosphorylation of the platelet-derived growth factor receptor i n vivo. J Biol Chem 259: 7909, 1984. 5. Morla AO, Wang JYJ. Protein tyrosine phosphorylation in the cell cycle of BALB/c 3T3 fibroblasts. Proc Natl Acad Sci USA 83: 8191, 1986. 6. Haring HU, White MF, Machicao F et al. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins In intact fat cells. Proc Natl Acad Sci USA 94: 113, 1987. 7. Kadowaki T, Koyasu S, Nishida E et al. Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell. J Biol Chem 262: 7342, 1987. 8. Saltzman EM, Thorn RR, Casnellie JE. Activation of a tyrosine protein kinase is an early event in the stimulation of T lymphocytes by interleukin-2. J Biol Chem 263: 6956, 1988. 9. Ross AH, Baltimore D, Eisen HN. Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature 294: 654, 1981. 10. Frackelton J r . AR, Ross AH, Eisen HN. Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells. Mol Cell Biol 3: 1343, 1983. 11. Kawahara RS, Kennedy BB, Deuel TF. Monoclonal antibody C3.1 is a platelet-derived growth factor (PDGF) antagonist. Biochem Biophys Res Comm 147: 839, 1987. 12. Carpenter G. Receptors for epidermal growth factor and other polypeptide mitogens. Ann Rev Biochem: 881, 1987. 13. Hunter T, Alexander CB, Cooper JA. Protein phosphorylation and growth control. Ciba Foundation Symposia 116: 188, 1985. 14. Rossomando AJ, Payne DM, Weber MJ, Sturgill TW. Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. Proc Natl Acad Sci USA 86: 6940, 1989. 15. Marth JD, Cooper JA, King CS et al. Neoplastic transformation induced by an activated lymphocyte-specific protein kinase (pp56* c k). Mol Cell Biol 8: 540, 1988. 164 16. Veillette A. Ross FM. Sausville EA et al. Expression of the lck tyrosine kinase gene in human colon carcinoma and other non-lymphoid human tumour cell lines. Oncogene Res. 1: 357. 1987. 17. Veillette A. Horak ID, Horak EM et al. Alternations of the lymphocyte-specific protein tyrosine kinase (p56 l c k) during T-cell activation. Mol Cell Biol 8: 4353, 1988. 18. Mustelin, T, Coggeshall KM, Altman A. Rapid activation of the T-cell tyrosine protein kinase pp56* c k by the CD45 phosphotyrosine phosphatase. Proc Natl Acad Sci USA 86: 6302. 1989. 19. Mustelin T and Altman A. Do CD4 and CD8 control T-cell activation via a specific tyrosine protein kinase? Immunology Today 10: 189, 1989. 20. Storms RW and Bose HR Jr. Oncogenes, protooncogenes, and signal transduction: toward a unified theory? Adv in Vir Res 37: 1, 1989. 21. Ziegler SF, Marth JD, Lewish DB, Perlmutter RM. Novel protein-tyrosine kinase gene (hck) preferentially expressed i n cells of hematopoietic origin. Mol and Cel Biol 7:2276, 1987. 22. Quintrell N, Lebo R, Varmus H et al. Identification of a human gene (HCK) that encodes a protein-tyrosine kinase and is expressed in hemopoietic cells. Mol and Cel Biol 7: 2267, 1987. 23. Zeigler SF, Wilson CB, Perlmutter R. Augmented expression of a myeloid-specific protein tyrosine kinase gene (hck) after macrophage activation. J Exp Med 168: 1801. 1988. 24. Ziegler SF, Levin SD Perlmutter RM. Transformation of NIH 3T3 fibroblasts by an activated form of p59" c k. Mol Cel Biol 7: 2724, 1989. 25. Morrison DK, Kaplan DK, Rap U, Roberts TM. Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity. Proc Natl Acad Sci USA 85: 8855. 1988. 26 Morrison DK. Kaplan DR, Escobedo JA et al. Direct activation of the serine/threonine kinase activity of RAF-1 through tyrosine phosphorylation by the PDGF (3-receptor Cell 58: 649, 1989. 27. Whitman M, Kaplan D, Schauffhausen BS et al. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature 315: 239, 1985. 28. Kaplan DR, Whitman M, Schauffhausen B et al. Common elements in growth factor s t i m u l a t i o n a n d onco g e n i c t r a n s f o r m a t i o n : 85 k d p h o s p h o p r o t e i n and phosphatidylinositol kinase activity. Cell 50: 1021, 1987. 29. Varticovski L, Druker B, Morrison D et al. The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3 kinase. Nature 342: 699, 1989. 30. Coughlin SR, Escopedo JA, Williams L. Rose of phosphatidylinosital kinase in PDGF receptor signal transduction. Science 243: 1191, 1989. 165 31. N i s h i b e S. W a h l M, Rhee SG. C a r p e n t e r G. T y r o s i n e p h o s p h o r y l a t i o n of phospholipase C-II i n vitro by epidermal growth factor receptor. J Biol Chem 264: 10335. 1989. 32. Margolis B, Rhee SG, Felder S et al. E GF induces tyrosine phosphorylation of phospholipse C-II: a potential mechanism for EGF receptor signaling. Cell 57: 1101, 1989. 33. Meisenhelder J. Suh PG, Rhee SG, Hunter T. Phospholipase C-y is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell 57: 1109, 1989. 34. Kumjian DA, Wahl MI, Rhee SG, Daniel TO. Platelet-derived growth factor (PDGF) binding promotes physical association of PDGF receptor with phospholipase C. Proc Natl acad Sci USA 86: 8232, 1989. 35. Koch CA, Moran M, Sadowski I, Pawson T. The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function. Mol Cell Biol 9: 4131. 1989. 36. Cales C, Hancock JF, Marshall CJ. Hall A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature 332: 548, 1989. 37. E l l i s C, Moran M, McCormick F, Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature 343: 377, 1990. 166 CHAPTER VI INTERLEUKIN-3 STIMULATES THE TYROSINE PHOSPHORYLATION OF THE 140 kDA INTERLEUKIN-3 RECEPTOR A. INTRODUCTION We have previously investigated protein phosphorylation events that occur rapidly following the binding of mIL-3 to mIL-3-dependent B e S U t A j cells. As described i n Chapter V, p h y s i o l o g i c a l c o n c e n t r a t i o n s of mIL-3 i n d u c e s the r a p i d , t r a n s i e n t t y r o s i n e phosphorylation of 40, 55, 68, 90 and 140 kDa proteins. Similar findings have also been reported by other investigators, although the phosphotyrosine-containing proteins detected appear to vary somewhat depending on the mIL-3 dependent cell line studied (1-4). Since tyrosine-specific protein phosphorylation has been implicated In the mechanism of action of a number of polypeptide growth factors (5,6), it is likely that this post-translational modification is also involved in the signal transduction pathway of mIL-3. We have also observed that 1 2^I-mIL-3 can be chemically crosslinked to 67 and 140 kDa surface proteins on B e S U t A j cells (Chapter III). It was therefore of interest to determine the relationship between these 67 and 140 kDa mIL-3-binding proteins and the 68 and 140 kDa proteins which are transiently tyrosine-phosphorylated in mIL-3-stimulated B6SUtAj c e l l s , i.e. to investigate the p o s s i b i l i t y that mIL-3 b i n d i n g s t i m u l a t e s t y r o s i n e phosphorylation of mIL-3 receptor proteins. In this section, we demonstrate, using a variety of techniques, that a 140 kDa but not a 67 kDa mIL-3-binding protein becomes phosphorylated on tyrosine residues when B6SUtAi cells bind mIL-3. 167 B. RESULTS 1. Crosslinking of 1 2 5I-mIl-3 to BeSUtAj Cells and Immunoprecipitation with  Antiphosphotyrosine Antibodies In preliminary experiments to investigate whether ligand binding stimulates tyrosine phosphorylation of mIL-3 receptor proteins, BeSUtA^ cells were exposed to 1 2^I-labeled recombinant mIL-3 for 5 min at 37°C as described in Chapter II. Cells were then washed, and 1 2 5I-mIL-3 was crosslinked to surface receptors using either DSS or DSP at 4°C for 30 min. Cells were then solubilized and processed for immunoprecipitation with aPTyrAbs as in Chapter V. SDS-PAGE of immunoprecipitates from DSS-crosslinked samples revealed the presence of radiolabeled bands corresponding to 85 and 155 kDa cr o s s l i n k e d complexes (Figure 29A, lane 3). These bands were not detected in the presence of a 20-fold excess of unlabeled mIL-3 (lane 1), in the absence of DSS (lane 2), nor when cell lysates were incubated i n the presence of purified rabbit IgG instead of antiphosphotyrosine antibodies (lane 4). Similar results were obtained when DSP was used as the crosslinking agent (Figure 29B, lane 2), except that the intensity of the lower molecular mass band was substantially greater than that of the 155 kDa band. Again, no bands were detected when cells were incubated in the presence of both ^ 2^I-mIL-3 and a 20-fold excess of unlabeled mIL-3 (Figure 29B, lane 1). These results indicate that the crosslinked 67 and 140 kDa proteins could be immunoprecipitated with aPTyrAbs. To demonstrate that the above findings reflect a specific interaction between tyrosine phosphorylated mIL-3 binding proteins and aPTyrAbs, the effect of phosphorylated amino acids on immunoprecipitation from cell lysates was tested. Cells to which ^ 2^I-mIL-3 had been DSS-crosslinked were solubilized as above. However, prior to immunoprecipitation with antiphosphotyrosine antibodies, dialyzed cell lysates were brought to a fina l concentration of 2 mM with phosphoserine, phosphothreonine or phosphotyrosine. 168 169 B)DSP 2 0 0 -9 2 . 5 -6 9 -4 6 -3 0 -Figure 29. (A) A n t i p h o s p h o t y r o s i n e p r e c i p i t a t i o n of 1 2 & I - m I L - 3 c r o s s l i n k e d to B6SULA j c e l l s . C e l l s were i n c u b a t e d w i t h 1 2 5 I - m I L - 3 f o r 5 m i n at 37°C a n d t h e n c r o s s l i n k e d w i t h D S S as d e s c r i b e d i n C h a p t e r II, l y s e d , s u b j e c t e d to antiphosphotyrosine immunoprecipitation and analyzed by SDS-PAGE (lane 2). This was also carried out i n the presence of a 20-fold excess of unlabeled mIL-3 (lane 1), no DSS (lane 3) and with p u r i f i e d non-immune rabbit IgG instead of antiphosphotyrosine antibodies (lane 4). (B) A s i n (A), except that DSP substituted for DSS. Lane 1: experiment carried out i n presence of 20-fold excess of unlabeled mIL-3. 170 W h i l e p h o s p h o s e r l n e or p h o s p h o t h r e o n i n e h a d l i t t l e o r no e f f e c t on the immunoprecipitation of the 85 and 155 kDa protein complexes, phosphotyrosine markedly reduced the intensity of these bands (Figure 30, lane 3). These findings strongly suggest that the results of Figure 29 are a consequence of a reaction between antiphosphotyrosine antibodies and phosphotyrosine residues on phosphotyrosine -containing proteins. 2. E f f e c t of S D S D e n a t u r a t i o n of C e l l L y s a t e s on A n t i p h o s p h o t y r o s i n e  Immunoprecipitation While the above results suggest that the 67 and 140 kDa mIL-3 binding proteins may be tyrosine phosphorylated when cells bind mIL-3, an alternative explanation is that the mIL-3 receptor exists as a complex of proteins and that, if this complex remains intact during aPTyrAbs immunoprecipitation procedures, the tyrosine phosphorylation of any component i n the complex would produce the pattern observed i n Figs. 29A and 29B, i.e., the results shown i n Fig. 29 do not prove that the proteins that become tyroslne-phosphorylated are mIL-3 b i n d i n g proteins. To d i s c r i m i n a t e between these two possibilities, crosslinking experiments similar to those shown in Fig. 29A were carried out except that, prior to addition of aPTyrAbs, cell lysates were supplemented with 2 % SDS and 5 % p-ME and heated for 1 min at 100°C. This would be expected to disrupt non-covalent protein-protein interactions. SDS was then removed from cell lysates by incubation at 4°C for 1 h with 40 mM KCI (7). After microfuging at top speed for 15 min, the supernatants were diluted 4-fold with lysis buffer and dialyzed against the same buffer overnight at 4°C. aPTyrAbs were then added and the samples processed as usual. Preliminary studies demonstrated that similar SDS treatment of 3 2 P - l a b e l e d lysates did not alter the relative intensities of phosphotyrosine-containing proteins isolated from mIL-3-treated B6SUtA 1 cells (ie. results similar to Fig. 25 were obtained), although the absolute intensities were diminished (data not shown). Results of two experiments are shown in Fig. 31, and 171 Figure 30. Antiphosphotyrosine precipitation of 1^ DI-mIL-3 crosslinked to BeSUtAj^ cells in the presence of various phosphoamino acids. Lysates from cells to which 125l-mIL-3 had been DSS-crosslinked were prepared as for Fig. 29A, lane 2 and then brought to a final concentration of 2 mM with either phosphothreonine (P-Thr), phosphoserine (P-Ser), or phosphotyrosine (P-Tyr). Immunoprecipitation with antiphosphotyrosine antibodies was then performed as for Fig 29. 172 1 2 3 92.5 69 — 46 30 Figure 31. Antiphosphotyrosine precipitation of SDS treated DSS-crosslinked i Z O I - m I L -S-BSSUtA! cell protein complexes. Cells to which 1 2 5I-mIL-3 had been DSS-crosslinked were lysed by heating at for 1 min 100°C with 2% SDS, 5% B -ME. Lysates were then made 40 mM with KC1 and incubated for 60 min at 4°C (39). After mlcrofuging for 15 min at 16,000 g, the 600 ul supernatants were diluted to 2 ml with lysis buffer containing 0.5% NP40 and dialyzed against this buffer overnight at 4°C. The samples were then immunoprecipitated with ccPTyrAbs and eluates analyzed by SDS-PAGE. Lane 3 is a control lane in which an aliquot of SDS lysed cells was electrophoresed directly without immunoprecipitation. Lanes 1 and 2 represent the experimental lanes of two separate experiments. The bottom most arrow points to what is most likely crosslinked 12^I-mIL-3 since it is observed irregularly and occurs in the absence of cells. m 173 demonstrate that only a 140 kDa mIL-3 binding protein is immunoprecipitated with ofPTyrAbs under these conditions. These results, therefore, strongly suggest that the 140 kDa, but not the 70 kDa mIL-3 receptor protein, is tyrosine phosphorylated i n mlL-3-stimulated BeSUtAj^ cells. Of course, these results do not rule out the possibility that other non-mIL-3 binding phosphotyrosine-containing proteins are present i n the mIL-3 receptor complex i n B6SUtAi cells. 3. Affinity Precipitation of Phosphorylated mIL-3 Binding Proteins To confirm that the 140 kDa mIL-3 binding protein is tyrosine phosphorylated in mIL-3-treated BSSUtA^ cells, and to determine if other phosphorylated proteins are indeed present i n the mIL-3 receptor complex, a different experimental approach was utilized. Cells were labeled with [ 3 2P]-orthophosphate and incubated i n the presence or absence of either fluorescein- or biotin-labeled mIL-3 (Fl-mIL-3 and Bi-mIL-3, respectively). These derivatives were prepared as described i n Chapter II and were shown to retain >90% bioactivity after derivatization, as assessed by 3H-thymidine incorporation assays. That derivatized molecules actually possessed bioactivity was demonstrated by showing that the bioactivity could be adsorbed from solutions of Fl-mIL-3 or Bi-mIL-3 by passage through a column containing immobilized anti-FITC antibodies or streptavidin, respectively (8). After stimulation with these mIL-3 derivatives for 5 min at 37°C, cell lysates were prepared. Lysates from Fl-mIL-3-treated cells were pre-cleared with blocked Affi-Gel 10 beads, incubated with mouse anti-FITC antibodies and then immobilized by addition of goat anti-mouse antibodies coupled to Affi-Gel 10 beads as described i n Chapter II. SDS-PAGE of eluted proteins revealed a marked enrichment of a 140 kDa protein (Fig. 32). This band was not detected i n the absence of Fl-mIL-3, when purified non-immune mouse IgG was substituted for anti-FITC antibodies, nor when cells were co-incubated with a 20-fold excess of underivatized mIL-3. Fl-mIL-3 Bi-mIL-3 1 1 i 1  1 2 3 4 1 2 3 4 Figure 32. D e t e c t i o n of p h o s p h o r y l a t e d mIL-3 b i n d i n g p r o t e i n s u s i n g fluoresceinated and b i o t i n y l a t e d mIL-3. 3 2 P - l a b e l e d B 6 S L H A J ce l l s were i n c u b a t e d w i t h Fl-mIL-3 or Bi-mIL-3 for 5 m i n at 37°C. C e l l lysates were prepared a n d pre-cleared by a d d i t i o n of block e d A f f i - G e l 10 beads. Lysates from Fl-mIL-3-treated cells were t h e n i n c u b a t e d w i t h anti-FITC a n t i b o d i e s a n d t h e n exposed to a n t i i m m u n o g l o b u l i n antibodies b o u n d to A f f i - G e l 10 beads. B o u n d proteins were eluted by addition of a pH 3.5 citrate buffer and analyzed by SDS-PAGE (lane 2). Lane 1, no Fl-mIL-3 added; lane 3, non-immune IgG instead of anti-FITC antibodies; lane 4, 20-fold excess of underivatized mIL-3 added with Fl-mIL-3. Lysates from Bi-mIL-3-treated cells were in c u b a l e d with streptavidin-agarose beads and bound proteins eluted with a p H 3.5 ci t r a t e b u f f e r a n d analyzed by SDS-PAGE (lane 2). Lane 1. no Bi-mIL-3 added; lane 3. 20-fold excess of u n d e r i v a t i z e d mIL-3 a d d e d w i t h B i - m l L - 3 ; l a n e 4, 100-fold e xcess of free b i o t i n added p r i o r to a d d i t i o n of s t r e p t a v i d in a g a r o s e b e a d s . 175 Similar results were obtained when cell lysates from Bi-mIL-3-treated cells were processed as described in Chapter II: Again, a major 140 kDa phosphoprotein was detected in the streptavidln-agarose eluted fraction (Fig. 32) which was not present in the absence of Bi-mIL-3, when cells were co-incubated with a 20-fold excess of underivatized mIL-3, nor when a 100-fold excess of free biotin was added prior to addition of streptavidin-agarose beads. No other specific phosphoprotein bands were detected suggesting again that the 67 kDa mIL-3 binding protein is not phosphorylated and that there are likely no other phosphorylated proteins in the mIL-3 receptor complex (Fig. 32). Phosphoamino acid analysis of the 140 kDa protein detected in Fig. 32, was carried out and the results were similar for Fl-mIL-3- and Bi-mIL-3-treated cells (Fig. 33). Both showed a phosphotyrosine.phosphoserine ratio of at least 1:1, thus confirming the presence of phosphotyrosine in the 140 kDa phosphoprotein. C. DISCUSSION These two independent studies provide compelling evidence that the 140 kDa mIL-3 binding protein in B6SULA, cells is tyrosine phosphorylated in mIL-3-stimulated B e S U t A ^ cells. Moreover, when samples prepared as for Fig. 32, lane 2, were subjected to two-dimensional gel electrophoresis, the 32P-labeled 140 kDa protein was found to migrate identically with the 32P-labeled 140 kDa phosphotyrosine-containing protein identified in Fig. 25 (8). This strongly suggests that the 140 kDa protein identified in Fig. 32 is the same protein as the 140 kDa molecule that becomes tyrosine phosphorylated in response to mlL-3 In Fig. 25, i.e. that mIL-3 stimulates tyrosine phosphorylation of the 140 kDa receptor. Interestingly, when B e S U t A ^ cells were labeled with ^S-methionine a n ( j then processed as in the legend to Fig. 32, lane 2 (i.e., treated with Bi-mIL-3, solubilized, and incubated with streptavidin-agarose beads), two 3^S-labeled proteins with apparent molecular masses of 67-70 and 140 kDa were specifically enriched in the eluate (8). In view of these data and those from the present study, two models can be put forth regarding the nature of the Figure 33. Phosphoamino acid analysis of pi40. The 140 kDa phosphoproteins identified in Fig. 32 were electroeluted from gel slices and subjected to phosphoamino acid analysis using thin layer electrophoresis with a 1:10:189 pyridine/acetic acid/water solvent system. Phosphoamino acid standards were identified by ninhydrin staining: P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine. >— 177 mIL-3 receptor. There may be two distinct mIL-3 receptor molecules, with only the larger 140 kDa species being tyrosine-phosphorylated when cells bind mIL-3. Alternatively, as has been suggested previously (3,4), there may be only one mIL-3 binding protein, pl40, which becomes cleaved, after mIL-3 binding, to a 67 kDa protein that retains the ligand binding domain. Consistent with this second model is the finding that most if not all mlL-3-dependent cell lines tested to date show only a single class of receptors by Scatchard analysis (Chapter III, ref. 9 and 10). The data from Figs. 31 and 32 could then be explained on the basis of the 67 kDa binding fragment containing no phosphotyrosine residues. The fact that an 85 kDa 12E*I-labeled complex is also Immunoprecipitated by aPTyrAbs in Fig. 29 would then suggest that the cleavage products remain associated during the cell processing procedures of Fig. 29. Of the above two models, we favor the latter, single receptor model first put forward by Isfort et al (3), especially in view of recent kinetic studies from our laboratory suggesting the evolution of the 67 kDa mIL-3 binding molecule from the 140 kDa protein (11). Interestingly, a lesser exposure of the autoradiogram in Fig. 32 suggests that the 140 kDa band may actually be a doublet (with phosphotyrosine present in both bands). The lower molecular weight species of this doublet may represent an initial cleavage step or a less phosphorylated receptor species. Studies are currently in progress to determine whether the 67 kDa protein is indeed a cleavage product of the 140 kDa mIL-3 binding protein. In view of the above findings, the identity of the 68 kDa phosphotyrosine-containing protein observed in Fig. 25 remains to be determined. As discussed in Chapter V, the phosphorylated Raf-1 product is a candidate for the identity of this protein. Another possibility is that it represents the tyrosine phosphorylated non-mIL-3 binding cleavage product of the 140 kDa receptor. These resul ts also imply that the 67 kDa phosphotyrosine-containing protein (mp67) described in Chapter IV is unlikely to be the 67 kDa mIL-3 receptor, as a 3 2 P-labeled 67 kDa mIL-3 binding protein was not observed in Fig. 32. It is probable that mp67 is identical to the tyrosine phosphorylated protein of 178 similar apparent molecular mass detected i n Fig. 25, and therefore may represent the non-binding cleavage product of the 140 kDa receptor. The reason that it was observed i n membrane samples from B6SULA! cells may be that, either physiologically or non-physiologically, the protein was membrane-associated in these experiments. If mp67 is in fact the non-binding cleavage product of the 140 kDa receptor, then it will be interesting to determine the significance of the serine phosphorylation of this protein in response to GM-CSF and TPA. The initial step by which a number of polypeptide growth factors stimulate target cell growth involves the binding to and activation of cell surface receptors that possess an intrinsic, ligand-responslve tyrosine kinase activity (5,6). In all cases studied to date (6), receptor tyrosine kinases are relatively large binding proteins (>100 kDa) and ligand-bindlng stimulates a rapid and transient tyrosine autophosphorylation of the receptor molecules. It is therefore tempting to speculate that the 140 kDa mIL-3 receptor also possesses tyrosine kinase a c t i v i t y , and that mIL-3 b i n d i n g s t i m u l a t e s tyrosine autophosphorylation of the receptor. However, cloning of an mIL-3 receptor gene h a s recently been reported, and a consensus sequence for a tyrosine kinase domain was not identified (12). The predicted size of the mature protein is 94,723 daltons, with two potential N-Iinked glycosylation sites (12). Although a tyrosine kinase domain is apparently not present in the cytosolic portion of this protein, an analysis of the extracellular domains of the protein reveals two conserved motifs which are also found in the external receptors for the receptors for IL-2 (p-subunit), IL-4, IL-6, and erythropoietin (12). The first conserved motif in the IL-3 receptor is composed of approximately 60 amino acids and contains four conserved cysteine residues. The second motif, separated from the first by a sequence of 90-100 unique amino acids, is located close the the transmembrane region and consists of approximately 30 amino acids. Similar motifs have already recently been reported in the external domains of the human and murine IL-7 receptors as well as growth hormone and prolactin receptors (13), and the suggestion has been made that the receptors for IL-2 (p-179 subunit), IL-3, IL-4, IL-6, IL-7, erythropoietin, growth hormone and prolactin are members of a receptor superfamily (13). Interestingly, the cytoplasmic domains of the mIL-3 receptor have been found to be both proline- and serine-rich ( 1 7 % and 1 2 % of cytoplasmic amino acids, respectively), also recognized in the cytosolic portions of the IL-2 receptor fj-chain, the IL-4 receptor, and the erythropoietin receptor (12). A l l of the cytokine receptors may therefore belong to a new gene family and may have evolved from a common ancestor. These studies suggest that the mIL-3 receptor and the tyrosine kinase involved in mIL-3-induced tyrosine phosphorylation are distinct molecules. As described in Chapter V, two members of the src family of non-receptor PTKs have been identified i n hemopoietic cells, pp56 c"^ c k and p p 5 9 c- ^ c k , and therefore a src-related protein may act as a signal transducing PTK in mIL-3-stimulated cells. Interestingly, a novel tyrosine kinase encoding gene, termed ltk (leukocyte tyrosine kinase), has recently been identified in leukocytes and a number of murine hemopoietic cell lines, including an mIL-3-dependent cell line (14). The predicted apparent molecular mass is 52.2 kDa and the protein has a PTK domain. It appears to be a transmembrane protein devoid of an extracellular domain (14). The ltk protein therefore could be a signal transduction subunit associated with one or more of the hemopoietic growth factor receptors. In conclusion, we provide evidence that one of the substrates for mIL-3-stimulated tyrosine phosphorylation is the 140 kDa mIL-3 surface receptor. Other recent reports indicate that activation of the protein kinase C pathway is important i n mIL-3-induced signal transduction (15-18) and so it w i l l be interesting to determine the interaction between the tyrosine kinase and protein kinase C pathways during the stimulation of mIL-3 dependent cells by mIL-3. 180 D. REFERENCES 1. Koyasu S, Tojo A, Miyajima A et al. Interleukin 3-specific tyrosine phosphorylation of a membrane glycoprotein of Mbr 150,000 in multi-factor-dependent myeloid cell lines. EMBO J 6: 3979, 1987. 2. Morla AO, Schreurs J, Miyajima A, Wang JYJ. Hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in interleukin-3-dependent murine cell lines. Mol Cell Biol 8: 2214, 1988. 3. Isfort R, Huhn RD, Frackelton AR Jr., Ihle JN. Stimulation of factor-dependent myeloid cell lines with Interleukin-3 induces tyrosine phosphorylation of several cellular substrates.. J Biol Chem 263: 19203, 1988. 4. Isfort RJ, Stevens D, May WS, Ihle JN. Interleukin-3 binds to a 140 kDa phosphotyrosine-containing cell surface protein. Proc Natl Acad Sci USA 85: 7982, 1988. 5. Hunter T, Cooper JA. Protein-tyrosine kinases. Ann Rev Biochem: 897, 1985. 6. Yarden Y, Ullrich A. Growth factor tyrosine kinases. Ann Rev Biochem 57: 443, 1988. 7. Suzuki H, Tereda T. Removal of dodecyl sulphate from protein solution. Anal Biochem 172: 259, 1988. 8. Mui AL-F, Sorensen PHB, Kay RJ, Krystal G. Purification and characterization of the murine interleukin-3 receptor. In: Redding C (ed). Hematopoiesis, Vol. 120. New York: Alan R. Liss Inc. (in press) 9. Palaszynski EW, Ihle JN. Evidence for specific receptors for interleukin 3 on lymphokine dependent cell lines established from long-term bone marrow cultures. J Immunol 132: 1972, 1984. 10. Park LS, Friend D, Gillis S, Urdal DL. Characterization of the cell surface receptor for a multi-lineage colony-stimulating factor (CSF-2a). J Biol Chem 261: 205, 1986. 11. Murthy SC, Mui AL-F, Krystal G. Characterization of the interleukin-3 receptor. Exp Hematol 18: 11. 1990. 12. Itoh N, Yonehara S, Schreurs J et al. Cloning of an Interleukin-3 receptor gene: a member of a distinct receptor gene family. Science 247: 324, 1990. 13. Goodwin RG, Friend D, Zeigler SF et al. Cloning of the human and murine Interleukin-7 receptors: demonstration of a soluble form and homology to a new receptor superfamily. Cell 60: 941, 1990. 14. Ben-Neriah Y and Bauskin AR. Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain. Nature 333: 672, 1988. 15. Farrar WL, Thomas TP, Anderson WB. Altered cytosol/membrane enzyme distribution on interleukin-3 activation of protein kinase. Nature 315: 235, 1985. 181 16. Evans SW, Rennick D, F a r r a r WL. Multilineage hematopoietic growth factor interleukin-3 and direct activators of protein kinase C stimulate phosphorylation of common substrates. Blood 68: 906, 1986. 17. Whetton AD, Heyworth CM, Dexter TM. Phorbol esters activate protein kinase C and glucose transport and can replace the requirement for growth factor in interleukin-3-dependent multi-potent stem cells. J Cell Sci 84: 83, 1986. 18. Whetton AD, Monk PN, Consalvey SD et al. Interleukin 3 stimulates proliferation via protein kinase C activation without increasing inositol lipid turnover. Proc Natl Acad Sci USA 85: 3284, 1988. 1 8 2 C H A P T E R VII S U M M A R Y A N D C O N C L U S I O N S H e m o p o i e s i s , t h e p r o c e s s o f b l o o d c e l l f o r m a t i o n , a p p e a r s t o b e r e g u l a t e d , at l e a s t i n p a r t , b y a s e r i e s o f s o l u b l e p o l y p e p t i d e s k n o w n a s c o l o n y - s t i m u l a t i n g f a c t o r s (1). T h e s e f a c t o r s r e p r e s e n t a n a b s o l u t e r e q u i r e m e n t fo r t h e s u r v i v a l , p r o l i f e r a t i o n a n d d e v e l o p m e n t o f s p e c i f i c t a r g e t c e l l p o p u l a t i o n s w h e n g r o w n i n v i t r o i n t h e a b s e n c e o f s u p p o r t i n g s t r o m a l c e l l s (2). A m o n g t h e f a c t o r s t h a t h a v e b e e n i m p l i c a t e d i n t h e c o n t r o l o f h e m o p o i e s i s i n t h e m o u s e i s m u r i n e i n t e r l e u k i n - 3 ( m I L - 3 ) , w h i c h i s t h o u g h t to p l a y a p a r t i c u l a r l y i m p o r t a n t r e g u l a t o r y r o l e a s i t n o t o n l y s t i m u l a t e s a v a r i e t y o f m y e l o i d (3-5) a n d l y m p h o i d p r o g e n i t o r s (6), b u t a l s o p r o m o t e s t h e s e l f - r e n e w a l a n d d e v e l o p m e n t o f p l u r i p o t e n t h e m o p o i e t i c s t e m c e l l s (7 -9 ) . H o w e v e r , v e r y l i t t l e i s k n o w n r e g a r d i n g t h e m e c h a n i s m b y w h i c h m I L - 3 e x e r t s i t s e f f ec t s o n t a r g e t c e l l s . I n c r e a s i n g o u r k n o w l e d g e o f t h e n o r m a l b i o c h e m i c a l p a t h w a y s i n v o l v e d w h e n t a r g e t c e l l s r e s p o n d t o m I L - 3 m a y l e a d n o t o n l y to p h a r m a c o l o g i c a l s t r a t e g i e s f o r m i m i c k i n g t h e b i o l o g i c a l e f f e c t s o f t h i s g r o w t h f a c t o r , b u t a l s o t o a g r e a t e r u n d e r s t a n d i n g o f t h e l o s s o f g r o w t h c o n t r o l o b s e r v e d i n h e m a t o l o g i c a l m a l i g n a n c i e s . We h a v e t h e r e f o r e i n v e s t i g a t e d t h e m e c h a n i s m o f a c t i o n o f m I L - 3 . A . I D E N T I F I C A T I O N O F T H E m I L - 3 R E C E P T O R W e f i r s t e x a m i n e d a n u m b e r o f m I L - 3 - d e p e n d e n t c e l l l i n e s f o r t h e i r a b i l i t y to b i n d b i o a c t i v e , 1 2 ^ I - l a b e l e d r e c o m b i n a n t m I L - 3 . O f t h e c e l l s t e s t e d , t h e B 6 S U t A c e l l l i n e was f o u n d to b i n d s u b s t a n t i a l l y h i g h e r l e v e l s o f 1 2 ^ I - m I L - 3 t h a n o t h e r w e l l c h a r a c t e r i z e d l i n e s s u c h a s F D C - P 1 a n d 3 2 D c l o n e 2 3 . I n f ac t , S c a t c h a r d a n a l y s i s d e m o n s t r a t e d t h a t B 6 S U t A c e l l s e x p r e s s e d u n u s u a l l y h i g h l e v e l s o f a s i n g l e c l a s s o f m I L - 3 s u r f a c e r e c e p t o r s , i.e.. a p p r o x i m a t e l y 1 1 5 , 0 0 0 r e c e p t o r s / c e l l w i t h a K D o f 3 . 1 n M . T h e s e c e l l s w e r e t h e r e f o r e 183 utilized in subsequent studies to identify the mIL-3 receptor and to investigate the mechanism of action of mIL-3. A novel heterobifunctional, cleavable, iodlnatable cross-linking agent, SASD, was used to identify a monomeric 67 kDa mIL-3 binding protein. This result was confirmed using glutaraldehyde as a cross-linking agent. In addition, the 67 kDa receptor was shown to have a pi of approximately 6.2 and appeared to contain intramolecular disulphide bridges. Crosslinking studies using DSS and intact BeSUtA} cells led to the identification of a second mIL-3 binding protein, with an apparent molecular mass of 140 kDa. Identification of the mIL-3 surface receptors allowed us to test for growth factor-induced post-translational modification of these proteins, such as by protein phosphorylation, in later studies. B. MURINE IL-3. mGM-CSF AND TPA INDUCE DISTINCT PHOSPHORYLATION EVENTS  IN B6SUtA1 CELLS As a first step in investigating the mechanism of action of mIL-3, we surveyed a number of known mitogens and polypeptide growth factors for their capacity to stimulate proliferation of the B6SUtA clone, B6SUtA!. Murine GM-CSF, sodium orthovanadate, and TPA were all found to stimulate DNA synthesis in these cells, with mGM-CSF being as potent as mIL-3 in this regard. The proliferative activity of sodium orthovanadate and TPA suggested that tyrosine and serine/threonine protein phosphorylations respectively, may be involved in the signal transduction pathways of mIL-3 and mGM-CSF. Moreover, it was found that TPA markedly inhibited the proliferative activity of either mIL-3 or mGM-CSF in cell cultures. This effect did not appear to be due to PKC exhaustion. Both mIL-3 and mGM-CSF induced the rapid, transient, and concentration-dependent phosphorylation of a 68 kDa cytosolic protein, cp68, on serine residues. This phosphorylation event likely occurs under physiological conditions as it was observed at 184 TABLE 4 - Apparent Molecular Masses of Receptors for Murine CSFs Receptor Apparent Molecular Mass (kDa) Reference mIL-3 GM-CSF G-CSF M-CSF Erythropoietin IL-5 50, 65-70, 140 51, 130 150 165 85-90, 100-105 Chapter III, 10-14 15,16 13 17 18,19 185 g r o w t h factor concen t r a t i ons tha t s t i m u l a t e d h a l f - m a x i m a l p ro l i fe ra t ion of B G S U t A j ce l ls . P K C d i d not appear to mediate the phosphory la t ion of cp68 . These growth factors also s t imu la t ed the r ap id , t rans ient serine p h o s p h o r y l a t i o n of a 6 7 k D a m e m b r a n e p r o t e i n , m p 6 7 , a g a i n at p h y s i o l o g i c a l l y r e l e v a n t c o n c e n t r a t i o n s of g r o w t h factor. B e c a u s e T P A also i n d u c e d the ser ine p h o s p h o r y l a t i o n of m p 6 7 , P K C m a y m e d i a t e t h i s p h o s p h o r y l a t i o n e v e n t . K i n e t i c a n a l y s i s i n d i c a t e d t h a t m p 6 7 w a s p h o s p h o r y l a t e d more r a p i d l y i n response to m I L - 3 t h a n i n response to m G M - C S F or T P A . Moreover , p h o s p h o a m i n o ac id ana lys i s of 3 2 P - l a b e l e d m p 6 7 demons t ra ted that of the three agents , on ly m I L - 3 i n d u c e d tyros ine-speci f ic as we l l as ser ine-speci f ic p h o s p h o r y l a t i o n of m p 6 7 . C . T Y R O S I N E P H O S P H O R Y L A T I O N IS A N E A R L Y E V E N T I N T H E S T I M U L A T I O N O F  BeSUtA]^ C E L L S B Y mIL-3 A n t i p h o s p h o t y r o s i n e an t ibod ies (aPTyrAbs) were u s e d to e x a m i n e ty ros ine-spec i f ic p r o t e i n p h o s p h o r y l a t i o n i n BeSUtA^ ce l l s . W e s t e r n b lo t a n a l y s i s u s i n g these an t ibod ie s r e v e a l e d t ha t m I L - 3 i n d u c e d t y r o s i n e p h o s p h o r y l a t i o n of 68 a n d 140 k D a m e m b r a n e p ro te ins as w e l l as 55 a n d 90 k D a cy toso l ic pro te ins . O f these p ro te ins , on ly the 90 k D a s p e c i e s w a s t y r o s i n e p h o s p h o r y l a t e d i n r e sponse to m G M - C S F . It i s u n l i k e l y tha t P K C ind i rec t ly media tes these phosphory la t ion events i n view of the inab i l i ty of T P A to s t imula te s i m i l a r t y r o s i n e p h o s p h o r y l a t i o n s . I m m u n o p r e c i p i t a t i o n w i t h a n t i b o d i e s to p h o s p h o t y r o s i n e c o n f i r m e d the p resence of 55 ;, 6 8 , 9 0 a n d 140 k D a p h o s p h o t y r o s i n e -c o n t a i n i n g p ro te ins i n m I L - 3 - s t i m u l a t e d ce l ls , a n d provided evidence for a n add i t i ona l 40 k D a t y r o s i n e p h o s p h o r y l a t e d p r o t e i n i n these ce l l s . T h e 68 a n d 140 k D a p r o t e i n s were t y r o s i n e p h o s p h o r y l a t e d w i t h i n 2 m i n a f t e r a d d i t i o n of m I L - 3 , w h i l e t h e o t h e r phosphopro t e in s were a l l detectable w i t h i n 15 m i n of exposure to the g rowth factor. Half-m a x i m a l incorpora t ion of 3 2 P into each of the proteins descr ibed was demons t ra ted at mIL-186 3 concentrations of 30-80 pM, strongly suggesting that these phosphorylation events occur under physiological conditions. D. MURINE IL-3 STIMULATES TYROSINE PHOSPHORYLATION OF T H E 140 kDA  mIL-3 RECEPTOR Using aPTyrAbs, 1 2 5 I - m I L - 3 crosslinked to 68 and 140 k D a proteins could be specifically Immunoprecipitated from BeSUtAj cells. If cell lysates were treated with SDS and p-mercaptoethanol prior to addition of aPTyrAbs, 1 2 ^I-mIL-3 crosslinked to only the 140 kDa protein was detected in immunoprecipitates. To confirm this finding, 3 2 P-labeled B 6 S U t A i cells were treated with biotinylated or fluoresceinated mIL-3. Addit ion of immobilized streptavidin or antifluorescein antibodies, respectively, to cell lysates from these cells resulted in the enrichment of only a 140 kDa tyrosine phosphorylated protein. Taken together, these results strongly suggest that only the 140 kDa receptor protein is tyrosine phosphorylated upon mIL-3 binding. E . G E N E R A L COMMENTS Three aspects of the present study are of special significance to the field of hemopoietic growth factor research. First, the identification of the mIL-3 receptor means that receptor sizes have now been determined for all the currently recognized murine CSFs, except for 11-5, as s u m m a r i z e d in Table 4. F u r t h e r m o r e , p r i m a r y b iochemica l characterization of the mIL-3 receptor proteins coupled with the isolation of a cell line bearing high receptor levels should facilitate efforts to purify the mIL-3 receptors and eventually clone the genes for these proteins. The second significant outcome of the present work is the demonstration that tyrosine-specific protein phosphorylation is an early event in the stimulation of target cells 187 by mIL-3. Murine IL-3 therefore joins an expanding group of polypeptide growth factors which are known to activate tyrosine kinases in responsive cell populations. It remains to be conclusively shown that tyrosine phosphorylation is essential for the proliferative activity of mIL-3. However, the observation that transfection of mIL-3-dependent cells with v-src or v-abl oncogenes, which both encode tyrosine kinases, renders the cells independent of mlL-3 for growth (see Chapter I) provides compelling evidence that tyrosine phosphorylation is involved in the proliferative response of target cells to mIL-3, as does the demonstration that the phosphotyrosine phosphatase inhibitor, sodium orthovanadate, can induce DNA synthesis in BeSUtAT cells. Tyrosine phosphorylation was also observed in B e S U t A ! cells stimulated with mGM-CSF, although no phosphotyrosine-containing proteins unique to mGM-CSF-treated cells were detected. A d d i t i o n a l studies are required to further characterize tyrosine phosphorylation in response to this growth factor. In a number of growth factor systems where tyrosine phosphorylation is known to be involved i n signal transduction, the protein tyrosine kinases involved are c e l l u l a r homologues of transforming vir a l oncogene products (see Chapter I). Therefore, the evidence presented here that mIL-3 stimulates tyrosine phosphorylation may prove to be of considerable importance. It remains to be determined whether the protein tyrosine kinases activated by mIL-3 binding are proto-oncogene products whose biochemical alteration leads to transformation within the hemopoietic system. The third significant aspect of the present study is the demonstration that while mlL-3 and mGM-CSF do induce phosphorylation of common proteins, a dis t i n c t set of phosphotyrosine-containing proteins was detected only i n response to mlL-3, as summarized in Table 5. This suggests not only that mIL-3 activates protein kinases that are additional to or distinct from those activated by mGM-CSF, but also that certain common phosphorylation events may be obligatory for growth factor-promoted proliferation of BeSUtAT cells. Furthermore, since the majority of the growth factor-stimulated phosphorylations could not be reproduced in cells treated with activators of PKC, it is 188 TABLE 5 - Murine IL-3-induced Protein Phosphorylation in B6SUtA Cells Apparent Molecular Mass of Phosphoprotein (kDa) Phosphoamino Acid Profile Agents Inducing Similar Response 40 Tyr/Ser — 55 Tyr/Ser — 68 (cp68) Ser mGM-CSF 67-68 (mp67) Tyr Ser mGm-CSF TPA 90 Tyr/Ser mGM-CSF 140 Tyr/Ser — This Table summarizes data described in detail in Chapters IV-VI. Abbreviations: Tyr, tyrosine; Ser, serine; TPA, 12-0-tetradecanoyl-phorbol-13-acetate. 189 unlikely that these events are mediated by the PKC system. The findings, however, do not rule out a role for PKC in the mitogenic response of mIL-3 target cells. Although mIL-3 does not appear to stimulate PIP2 breakdown (20), many studies have demonstrated the activation of PKC in cells stimulated to divide by mIL-3 (21-23). Interestingly, mIL-3 has recently been shown to induce phosphatidylcholine hydrolysis i n mIL-3 dependent cells (24), a potentially novel pathway for the activation of PKC not involving PIP 2 metabolism. It is possible, therefore, that PKC activation, by this or another pathway, functions in a negative feedback loop by catalyzing serine phosphorylation of the mIL-3 receptor or other proteins i n mIL-3-stimulated target cells. Clearly, much work is required to more fully u n d e r s t a n d which p h o s p h o r y l a t i o n events are c r u c i a l to the pathways of signal transduction in cells stimulated by mIL-3. It is hoped that the data presented in this thesis will help to open new avenues for research into the mechanism of action of this interesting hemopoietic growth factor. 190 F. REFERENCES 1. Sieff. CA. Hematopoietic growth factors. J Clin Invest 79: 1549, 1987. 2. Whetton AC, Dexter TM. Hemopoietic growth factors. Trends Biochem Sci 11: 207, 1986. 3. Ihle JN, Keller J , Greenberger J S et al. Phenotypic characteristics of cell lines requiring interleukin 3 for growth. J Immunol 129: 1377, 1982. 4. Ihle JN, Rebar L, Keller J , Lee JC, Hapel A. Interleukin 3: possible roles in the regulation of lymphocyte differentiation and growth. Immunol Rev 63: 5, 1982. 5. Iscove NW, Roitsch CA, Williams N, Guilbert LJ. Molecules stimulating early red cell, granulocyte, macrophage, and mega-karyocyte precursors in culture: similarity in size, hydrophobicity, and charge. J Cell Physiol 1: 65, 1982. 6. Palacios J. Cloned lines of interleukin 2 producer human T lymphocytes. J Immunol 129: 2586, 1982. 7. Broxmeyer HE, Wi l l i a m s DE, Cooper S et a l . Comparative effects i n vivo of recombinant murine interleukin 3, natural murine colony stimulating factor-1, and recombinant murine granulocyte-macrophage c o l o n y - s t i m u l a t i n g factor on myelopoiesis in mice. Blood 69: 913, 1987. 8. Lord B, Molineux G, Testa NG et al. The kinetic response of haemopoietic precursor cells, in vivo, to highly purified, recombinant interleukin-3. Lymphokine Res 5: 97, 1985. 9. Metcalf D, Begley CG, Johnson GR et al. Effects of purified bacterially synthesized murine multi-CSF (IL-3) on hematopoiesis in normal adult mice. Blood 68: 46, 1986. 10. Palacios R, Neri T, Brockhaus M. Monoclonal antibodies specific for interleukin-3-sensitive murine cells. J Exp Med 163: 6652, 1986. 11. May WS, Ihle JN. Affinity isolation of the interleukin 3 surface receptor. Biochem Biophys Res Commun 135: 870, 1986. 12. Park LS, Friend D, Gillis S, Urdal DL. Characterization of the cell surface receptor for a multi-lineage colony-stimulating factor (CSF-2cc). J Biol Chem 261: 205, 1986. 13. Nicola NA, Peterson L. Identification of distinct receptors for two hemopoietic growth factors (granulocyte colony-stimulating factor and multipotential colony-stimulating factor) by chemical cross-linking. J Biol Chem 261: 12384, 1986. 14. Isfort R J . Stevens D, May WS, Ihle JN. I n t e r l e u k i n - 3 b i n d s to a 140 kDa phosphotyrosine-containing cell surface protein. Proc Natl Acad Sci USA 85: 7982, 1988. 15. Walker F, Burgess AW. Specific binding of radioiodinated granulocyte-macrophage colony-stimulating factor to hemopoietic cells. EMBO 4:933, 1985. 16. Park LS, Friend D, Gillis S, Urdal DL. Characterization of the cell surface receptor for granulocyte-macrophage colony-stimulating factor. J Biol Chem 261: 4177, 1986. 191 17. Morgan CJ. Stanley ER. Chemical crosslinking of the mononuclear phagocyte specific growth factor CSF-1 to its receptor at the cell surface. Biochim Biophys Res Commun 119: 35. 1984. 18. Sawyer ST, Krantz SB, Luna J. Identification of the receptor for erythropoietin by cross-linking to Friend virus-infected erythroid cells. Proc Natl Acad Sci USA 84: 3690, 1987. 19. Broudy VC, Lin N, Egrie J et al. Identification of the receptor for erythropoietin on human and murine erythroleukemia cells and modulation by phorbol ester and dimethyl sulfoxide. Proc Natl Acad Sci USA 85: 6513, 1988. 20. Whetton AD, Monk PN, Consalvey SD et al. Interleukin 3 stimulates proliferation via kinase C activation without increasing inositol lipid turnover. Proc Natl Acad Sci USA 85: 3284, 1988. 21. F a r r a r WL, Thomas TP, A n d e r s o n WB. A l t e r e d cytosol/membrane enzyme distribution on interleukin-3 activation of protein kinase. Nature 315: 235, 1985. 22. Evans SW, Rennick D, Fa r r a r WL. Multilineage hematopoietic growth factor interleukin-3 and direct activators of protein kinase C stimulate phosphorylation of common substrates! Blood 68: 906, 1986. 23. Whetton AD, Heyworth CM, Dexter TM. Phorbol esters activate protein kinase C and glucose transport and can replace the requirement for growth factor in interleukin-3-dependent multi-potent stem cells. J Cell Sci 84: 83, 1986. 24. Duronio V, Nip L, Pelech SL. Interleukin-3 stimulates phosphatidylcholine turnover in a mast/megakaryocyte cell line. Biochem Biophys Res Commun 164: 804, 1989. 

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