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Regulation of the stress-activated protein kinase pathways in hematopoietic cells Foltz, Ian Nevin 1999

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REGULATION OF THE STRESS-ACTIVATED PROTEIN KINASE PATHWAYS IN HEMATOPOIETIC CELLS Ian Nevin Foltz B . S c , The University of Guelph, 1994  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Experimental Medicine Program  We accept this thesis as conforming Jp^fteoecjuired standard  THE UNIVERSITY OF BRITISH C O L U M B I A March 1999 © Ian Nevin Foltz, 1999  In  presenting  degree freely  at  this  the  available  copying  of  in  partial  fulfilment  University  of  British  Columbia,  for  this  department publication  thesis  thesis  or of  reference  by this  for  his thesis  and  scholarly  or for  her  of  rA^T-Cx^e  The University of British Vancouver, Canada  Date  DE-6  (2/88)  ftf  ;l  [<t  )  Columbia  1 further  purposes  gain  the  requirements  I agree  shall  that  agree  may  representatives.  financial  permission.  Department  study.  of  It not  be is  that  the  Library  an  granted  by  allowed  advanced  shall  permission  understood be  for  the that  without  for  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT The regulation of the p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways in hematopoietic cells is largely uncharacterized. We demonstrated the tyrosine phosphorylation and activation of p38 MAPK by interleukin (IL) -3, granulocytemacrophage colony-stimulating factor (GM-CSF) and Steel locus factor (SLF). We also showed the activation of p38 MAPK is required for the activation of MAPKAP kinase-2 by these cytokines. The activation of JNK often parallels the activation of p38 MAPK. We found that these cytokines also activated the 46 and 55 kDa splice variants of JNK1 and JNK2, and induced threonine phosphorylation of MAPK kinase 4 (MKK4), an upstream activator of JNK. Together, these results demonstrated that p38 MAPK and JNK are activated not only by environmental stresses, but also by hematopoietic growth factors. In contrast, a related cytokine IL-4 failed to activate p38 MAPK, MAPKAP kinase-2, JNK1, JNK2 or MKK4. Therefore, IL-4 was unique in failing to activate any MAPK pathways in hematopoietic cells. Both SLF and GM-CSF activated JNK comparably with similar kinetics, however SLF induced greater phosphorylation of MKK4 than GM-CSF, suggesting the existence of other JNK kinases. A cDNA with homology to MKK4 was identified in the Expressed Sequence Tag database, and the full-length cDNA was cloned using Rapid-Amplification of Cohesive Ends (RACE) PCR. This cDNA encoded a 419 amino acid protein, hereafter termed MKK7, that contained a putative kinase domain. We identified several splice variants of MKK7, and transcripts encoding MKK7 were expressed ubiquitously. Co-expression of MKK7 with either JNK1 or p38a2 MAPK led to the activation of JNK1, but not p38a2 MAPK. Epitope-tagged MKK7 was activated by IL-3, TNFa, UV light, hyperosmolarity, heat shock and anisomycin, but not by IL-4 or EGF. We also showed that endogenous MKK4 and MKK7 were activated by IL-3, TNFa or the Fc receptor for IgG. MKK7 was also activated by constitutively-active Rac and Cdc42 when expressed in HeLa or Ba/F3 cells. In contrast, constitutively-active Ras activated MKK7 in HeLa cells, but not in Ba/F3 cells, demonstrating that signalling from Ras to MKK7  ii  varies between cell types. In conclusion,  M K K 7  is regulated by diverse stresses and physiological  stimuli in hematopoietic cells.  iii  Table of Contents  Title  i  Abstract  ii  Table of Contents  iv  List of Figures Chapter 1  viii  Chapter 3  viii  Chapter 4  ix  Chapter 5  ix  List of Tables Chapter 5  xi  Preface  xii  Abbreviations  xiv  Acknowledgments  xvi  CHAPTER 1 1.1  General Overview  1  1.2  Signal Transduction by Cytokines and Growth Factors  3  1.3  Phosphorylation in Signal Transduction  1.3.1  Protein Kinases  8  1.3.2  Mechanism of Phosphotransfer by Kinase Domains  10  1.3.3  Phosphatases  1.4 1.4.1  1.5 1.5.1  14  The Mitogen-Activated Protein Kinase Cascades The M A P K Cascades  15  The Stress-Activated Protein Kinases: JNK and p38 MAPK Overview of the genes encoding J N K  i  v  24  1.5.2  Activators and Substrates of JNK  24  1.5.3  Overview of different p3 8 genes  28  1.5.4  A p38 M A P K inhibitor SB 203580  28  1.5.5  Activators and Substrates of p38 M A P K  30  1.5.6  Stimuli upstream of p38 M A P K and JNK  35  1.6  Biological Roles of the Stress-Activated Protein Kinases  1.6.1  Role of Stress-Activated Protein Kinases in Yeast.  39  1.6.2  Role of Stress-Activated Protein Kinases in Drosophila.  40  1.6.3  Role of Stress-Activated Protein Kinases in Embryogenesis  42  and Hematopoiesis. 1.6.4  Role of Stress-Activated Protein Kinases in Cytokine  44  Production and Inflammation. 1.6.5  Role of Stress-Activated Protein Kinases in Proliferation  47  and Tumorigenesis. 1.6.6  Role of Stress-Activated Protein Kinases in Apoptosis.  CHAPTER 2 - Materials and Methods  50 56  CHAPTER 3 - Activation of p38 MAPK in Hematopoietic Cells. 3.1  Introduction  3.2  Results  76  3.2.1  Identification of p38 M A P K - an unknown phosphoprotein.  78  3.2.2  Activation of p38 M A P K by IL-3, G M - C S F or SLF but not  78  IL-4 or IL-13. 3.2.3  Activation of p38 M A P K by CSF-1 in FDMACII cells.  81  3.2.4  Activation of p38 M A P K by cross-linking of the Fc Receptor  84  3.2.5  for Immunoglobulin G. Hematopoietic growth factors activate M A P K A P kinase-2.  84  v  3.2.6  GM-CSF or SLF activate M A P K A P kinase-2 via p38 M A P K .  87  3.2.7  p38a is sufficient for activation of M A P K A P kinase-2.  92  3.2.8  Role of p38 M A P K in D N A synthesis.  92  3.2  Discussion  96  CHAPTER 4 - Activation of JNK in Hematopoietic Cells. 4.1  Introduction  4.2  Results  4.2.1  102  Activation of JNK 1 and JNK2 by hematopoietic growth  103  factors with the exception of IL-4 or IL-13. 4.2.2  Threonine Phosphorylation of M K K 4 is induced by G M - C S F  107  or S L F . 4.3  Discussion  107  CHAPTER 5 - MKK7 - A Specific Activator of JNK. 5.1  Introduction  5.2  Results  113  5.2.1  Cloning strategy to discover unknown M K K .  114  5.2.2  Expression of M K K 7 in human and murine cells.  122  5.2.3  M K K 7 specifically activates JNK, but not p3 8 M A P K .  125  5.2.4  M K K 7 is activated by TNF or multiple stress stimuli.  125  5.2.5  Activation of M K K 7 by IL-3, but not IL-4, in Ba/F3 cells.  129  5.2.6  Activation of endogenous M K K 4 and M K K 7 by IL-3, but  129  notIL-4. 5.2.7  Activation of endogenous M K K 4 and M K K 7 by TNF.  132  5.2.8  Activation of endogenous M K K 4 and M K K 7 by  132  cross-linking the Fc receptor for IgG. vi  5.2.9 5.3 CHAPTER  Constitutively active GTPases activate M K K 4 and M K K 7 . Discussion  132 137  6 - CONCLUSION  144  Bibliography  148  vii  LIST O F FIGURES:  Chapter 1  Fig. 1.1  Comparison of Signalling Pathways Initiated by Stimulation  6  of Cells with IL-3, GM-CSF, SLF or IL-4. Fig. 1.2  Crystal structure of c A M P dependent protein kinase.  11  Fig. 1.3  Model of the active site of c A M P dependent protein kinase.  13  Fig. 1.4  The M A P K Cascade.  16  Fig. 1.5  Alignment of the Isoforms of JNK.  25  Fig. 1.6  Activators and Substrates of JNK.  26  Fig. 1.7  Alignment of the Isoforms of p3 8 M A P K .  29  Fig. 1.8  Structure of SB 203580 - The p38 M A P K inhibitor.  31  Fig. 1.9  Crystal Structure of p38 M A P K bound to A T P or SB 203580.  32  Fig. 1.10  Activators and Substrates of p38 M A P K .  33  Analysis of phosphoproteins induced by IL3 and SLF using 2D  77  Chapter 3  Fig. 3.1  SDS-PAGE. Fig. 3.2  IL-3 or SLF, but not IL-4, induce tyrosine phosphorylation of  79  p38 M A P K in bone marrow-derived mast cells. Fig. 3.3  Activation of p38 M A P K by hematopoietic growth factors in  80  MC/9 mast cells. Fig. 3.4  Kinetics of tyrosine phosphorylation of p38 M A P K by G M - C S F  82  or SLF. Fig. 3.5  IL-13 fails to activate p38 M A P K in FD5/IL13R cells.  viii  83  Fig. 3.6 - Colony stimulating factor (CSF)-l induces the tyrosine  85  phosphorylation of p38 M A P K in FDMACII cells. Fig. 3.7 - Cross-linking of the FcR for IgG induces tyrosine phosphorylation  86  of p38 M A P K in MC/9 mast cells. Fig. 3.8 - Activation of M A P K A P kinase-2 by hematopoietic growth factors  88  Fig. 3.9 - IL-3 activates M A P K A P kinase-2 via p38 M A P K .  89  Fig. 3.10 - G M - C S F activates M A P K A P kinase-2 via p38 M A P K .  90  Fig. 3.11 - SLF activates M A P K A P kinase-2 via p38 M A P K .  91  Fig. 3.12 - p38a M A P K is sufficient for activation of M A P K A P kinase-2.  93  Fig. 3.13 - p38 M A P K activity is required for D N A synthesis in MC/9 cells.  94  Fig. 3.14 - p38cc M A P K fails to rescue the inhibition of D N A synthesis in  95  Ba/F3 cells.  Chapter 4  Fig. 4.1 - Activation of JNK1 by hematopoietic growth factors with the  104  exception of IL-4 in MC/9 cells. Fig. 4.2 -IL-13 fails to activate JNK1 in FD5/IL13R cells.  105  Fig. 4.3 -Activation of JNK2 by G M - C S F and SLF, but not IL-4, in MC/9 cells.  106  Fig. 4.4 -Kinetics of JNK activation by GM-CSF or SLF in MC/9 cells.  108  Fig. 4.5 -Threonine phosphorylation of M K K 4 by SLF or GM-CSF, but not IL-4.  109  Chapter 5  Fig. 5.1  - Designing a probe to screen the EST database.  115  Fig. 5.2  - Strategy for cloning full length M K K 7 .  116  Fig. 5.3  - Amplification of fragments of M K K 7 using 5'-RACE PCR.  117  ix  Fig. 5.4 - Nucleotide and primary amino acid sequences of human M K K 7 .  118  Fig. 5.5 - Alignment of primary structures of human and murine isoforms  119  of M K K 7 . Fig. 5.6 - Alignment of human M K K 7 with its orthologs in D.  C. elegans  and  120  melanogaster.  Fig. 5.7 - Alignment of the catalytic domains of known human M K K .  121  Fig. 5.8 - Dendrogram of known human M K K and the orthologs of M K K 7 .  123  Fig. 5.9 -  Expression of M K K 7 in Ba/F3 and MC/9 cells.  124  Fig. 5.10 - Tissue Distribution of mRNA encoding M K K 7 .  126  Fig. 5.11 - Co-expression of M K K 7 with JNK1 activates JNK1.  127  Fig. 5.12- Co-expression of M K K 7 with p38a M A P K fails to activate  128  p38a M A P K . Fig. 5.13 - Activation of M K K 7 by various stimuli in HeLa cells.  130  Fig. 5.14 - Activation of M K K 7 by IL-3, but not IL-4, in Ba/F3 cells.  131  Fig. 5.15 - Specificity of M K K 4 and M K K 7 antibodies.  133  Fig. 5.16- Activation of endogenous M K K 4 and M K K 7 by IL-3, but not IL-4.  134  Fig. 5.17- TNFa activates endogenous M K K 4 and M K K 7 in HeLa cells.  135  Fig. 5.18 - Cross-linking of the Fc receptor for IgG activates M K K 4 and  136  M K K 7 in MC/9 mast cells. Fig. 5.19- Activation of M K K 7 or M K K 4 by the small GTPases Ras,  138  Fig. 5.20 - Activation of M K K 7 by the small GTPases Rac and Cdc42.  139  x  LIST  OF  TABLES:  Chapter 5  Table 5 . 1 - The Genetic Code.  115  Table 5.2 - Analysis of Clones derived from the EST database.  116  Table 5.3 - Expression mRNA encoding M K K 7 in various cell lines.  126  xi  PREFACE  My thesis is divided into six chapters and organized into the following format. Chapter one is a general overview of protein kinases, the mitogen-activated protein kinase cascades and a current review of the stress-activated protein kinase pathways. Chapter two is a summary of the techniques used during my thesis. Chapters three to five describe my findings in the field of the stress-activated protein kinase pathways.  Due to the competitive nature of the field, I have  included a brief introduction for these chapters describing our knowledge at the time the work was being conducted. Similarly, the conclusions of these chapters represent those drawn when the work was done. Chapter six gives a summary of the data and their implications for the field in general. It is important to state that I have used the term "stress-activated protein kinase" to refer to both J N K and p38 M A P K .  I have also used the human nomenclature for J N K and M K K 4  (instead of S A P K and SEK1) throughout my thesis. These changes were made simply to reduce confusion for those who are reading the thesis and not intimately versed in the field.  Publications Arising F r o m The W o r k O f This Thesis  Foltz, I . N . , Lee, J. C , Young, P. R. and Schrader, J. W. (1997) Hemopoietic Growth Factors with the Exception of Interleukin 4 Activate the p38 Mitogen-Activated Protein Kinase Pathway. J. Biol. Chem. 272, 3296-3301.  Foltz, I . N . and Schrader, J. W. (1997) Activation of the Stress-Activated Protein Kinases by Multiple Hematopoietic Growth Factors with the Exception of Interleukin-4. Blood 89, 30923096.  xii  Salmon, R. A . , Foltz, I. N . , Young, P. R. and Schrader, J. W. (1997) The p38 mitogenactivated protein kinase is activated by ligation of the T or B lymphocyte receptors, Fas or CD40, but suppression of kinase activity does not inhibit apoptosis induced by antigen receptors. J. Immunol.  159, 5309-5317.  Foltz, I. N . , Gerl, R. E., Wieler, J., Luckach, M . , Salmon, R. A . and Schrader, J. W. (1998) Human Mitogen-Activated Protein Kinase Kinase 7 (MKK7) is a Highly Conserved c-Jun N terminal  Kinase  (JNK)/Stress-Activated  Protein  Environmental Stresses and Physiological Stimuli.  Kinase  J. Biol.  (SAPK)  Chem.  Kinase  Activated by  273, 9344-9351.  Scheid, M . P., Foltz, I. N . , Young, P. R., Schrader, J. W. and Duronio, V . (1999) Ceramide and Cyclic Adenosine Monophosphate (cAMP) Induce cAMP Response Element Binding Protein Phosphorylation via Distinct Signaling Pathways While Having Opposite Effects on Myeloid Cell Survival.  Blood  93, 217-225.  xiii  ABBREVIATIONS  ATF-2  Activating Transcription Factor 2  ATP  Adenosine triphosphate  BCR  B Cell Antigen Receptor  BSA  Bovine Serum Albumin  CSF-1  Colony Stimulating Factor - 1  DNA  Deoxyribonucleic acid  FCS  Fetal Calf Serum  FcR  Receptor for the Fc Fragment of Immunoglobulin G  GM-CSF  Granulocyte/Macrophage Colony Stimulating Factor  GTPase  Guanosine Triphosphatase  HEPES  N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]  Hogl  High Osmolality Glycerol  Hsp  Heat Shock Protein  Ig  Immunoglobulin  IL  Interleukin  JNK  c-Jun N-Terminal Kinase  LPS  Lipopolysaccharide  MAPK  Mitogen-Activated Protein Kinase  MAPKAPK  Mitogen-Activated Protein Kinase Activated Protein Kinase  MBP  Myelin Basic Protein  MEK  Mitogen-Activated Protein Kinase Kinase  MKK  Mitogen-Activated Protein Kinase Kinase  M K K 7  Mitogen-Activated Protein Kinase Kinase 7  MMLV  Murine Maloney Leukemia Virus  OVA  Ovalbumin  PBS  Phosphate Buffered Saline  PCR  Polymerase Chain Reaction  PTK  Protein Tyrosine Kinase  PTPase  Protein Tyrosine Phosphatase  RNA  Ribonucleic Acid  RT  Reverse Transcriptase  SAPK  Stress-Activated Protein Kinase  SDS-PAGE  Sodium Dodecyl Sulphate - Polyacylamide Gel Electrophoresis  SEK1  Stress-Activated Protein Kinase/Extracellular Regulated Kinase Kinase 1  SLF  Steel  TNF-a  Tumour Necrosis Factor alpha  locus factor  xv  ACKNOWLEDGMENTS  I am indebted to several people for their support and guidance during my time at the Biomedical Research Centre.  I would like to thank my supervisor John Schrader for his  enthusiastic "kid in a candy store" approach to science, and for giving me the resources and the freedom to pursue my own research interests. I would also like to extend my appreciation and thanks to the entire staff of the B R C , who have not only shared their scientific expertise, but also made coming to the B R C a pleasure.  In particular, I have to thank James Wieler for our  enthusiastic discussions of science and teaching me the importance of rounds down range, Melanie Welham for teaching me the art of protein biochemistry, Sam Abraham for our lengthy coffee breaks, Kevin Leslie for making every Christmas a Doozie, and Robert Gerl for his assistance on the M K K 7 project. I am also thankful to several other members of the JWS lab including Megan Levings, Frances Lee, Goetz Ehrhardt, John Babcook, Joshua Schafer, Ruth Salmon and Michael Luckach for their invaluable assistance, their patience and their friendship. I have also been fortunate to have the support of many people outside of the B R C . My family has provided a great deal of support and love during my studies at both the University of Guelph and the University of British Columbia. I have to especially thank my girlfriend Lynda Duncan for her understanding, her overwhelming support and her endless patience during my five minute hours. I would also like to thank Mike Scheid for teaching me how to lose gracefully at pool, and for our long scientific discussions over a pint. Several collaborators have also provided invaluable reagents to me, and without their assistance most of this work would not have been possible. In that light, I would like to thank Drs. Peter Young and John Lee at Smith-Kline Beecham, Dr. Leonard Zon at Harvard University Medical School, Dr. Richard Cerione at Cornell University, Dr. Frank McCormick at Onyx Pharmaceuticals, and Dr. Rob Kay at the Terry Fox Research Laboratories.  xvi  CHAPTER 1  1.1  General Overview  Life as we know it likely began as a single-cellular organism similar to the bacteria of today.  This organism had to acquire nutrients for growth and survival, remove dangerous  metabolic byproducts, and replicate its genetic material for future generations.  Besides these  problems, this early life form had to respond to and overcome harsh environmental conditions such as osmolarity of its environment, temperature changes and ultraviolet irradiation. As life evolved from an anareobic to an aerobic state, these organisms had to overcome the problems inherent to aerobic life such as oxygen free-radicals. These early organisms needed to interpret and respond to their environment to overcome all these problems. The ability to sense and respond to the external environment was in essence the evolution of signal transduction. With the evolution of multicellular organisms, the requirement for cells to respond to their environment was as important as for the earliest forms of life.  However, these organisms had  many new problems to overcome. They needed to devise a means to provide nutrients and oxygen to all their cells, to remove waste from these cells, to communicate between cells, and to keep their cells associated. The cells became less individualized and more specialized during this process, and evolved to form specific organs and organ systems to overcome these problems. Vasculature systems were developed to supply nutrients and remove waste from cells in the organism when simple diffusion was no longer adequate to meet the organisms needs. Cell to cell communication is observed in single-cellular organisms such as yeast and multicellular organisms exploited similar systems to co-ordinate many aspects of life. Chemokines evolved to direct cells throughout the body, and growth factors, transforming growth factors, and cytokines evolved to control cellular growth, proliferation and differentiation. Multicellular organisms also evolved an extracellular matrix not only act to keep cells together, but also to prevent their death.  1  Besdes these problems, the requirement to control total cell number was possibly the biggest hurdle facing multicellular life. This dynamic process involves the controlled production and the ehmination of cells. A failure in this process leading to an over-production of cells is known as cancer, and involves a dysregulation of the same genes, known as proto-oncogenes, normally involved in cellular growth, proliferation and survival. The deregulated expression or mutation of proto-oncogenes, forming oncogenes, has been implicated in most forms of cancers. For example, the p21 Ras family of small GTPases are constitutively active in as many as 90% of human pancreatic cancers.  Other genes encoding tumor suppressor proteins such as p53 that  function to inhibit cellular proliferation, and are often deleted or non-functional in human cancers. Besides proliferation, total cell number is regulated by the elimination of cells, known as apoptosis. My thesis focuses on two families of stress-activated protein kinases that are activated by pro-inflammatory cytokines, hyperosmolarity, oxygen free-radicals, ultraviolet irradiation, or basically any cellular insult imaginable. When I began working in this field with the exception of the proinflammatory cytokines, no physiological stimuli were known to activate these kinases. I examined the regulation of the stress-activated protein kinases in hematopoietic cells by growth factors that regulate their growth, proliferation and survival. The fact that hematopoietic cells living inside a multicellular organism sense and respond to environmental stresses that they would never encounter was strange. I reasoned this phenomenon might reflect a remnant of an ancient signalling cascade leftover from single-cellular life as these enzymes do exist in yeast. As new pathways were evolved to co-ordinate the diverse functions of multicellular life, it seems logical for a cell to harness the pre-existing protein machinery instead of re-inventing the wheel.  We  hypothesized that the stress-activated protein kinases would play many physiological roles in cells of multicellular organisms.  Consistent with this notion, the stress-activated protein kinase  pathways are now known to be activated by an incredibly diverse range of stimuli. Clearly, these kinases are not only important as effectors of ancient signals regulating single cellular life, but also  2  have been adapted to regulate many aspects of multicellular life including apoptosis, proliferation, tumorigenesis, chemotaxis, organogenesis, embryogenesis, and the immune system.  1.2  Signal Transduction by Cytokines and Growth Factors  Hematopoietic growth factors including Steel locus factor (SLF), interleukin (IL) -3, IL-4, and granulocyte/macrophage colony-stimulating factor (GM-CSF), are members of a large structural family of polypeptide growth factors or cytokines that regulate the growth, survival, and differentiation of cells in many tissues, including the immune system. S L F acts on pluripotent hematopoietic stem cells, the progenitors of many hematopoietic lineages, and mature mast cells, and also has an important developmental role on cells of non-hematopoietic origin, including germ cells and cells derived from the neural crest (Fleischman, 1993). S L F is produced constitutively by stromal cells, and is essential for the development of the hematopoietic system. IL-3, G M - C S F and IL-4 are released by activated cells of the immune system and link the immune system to the regulation of the growth and function of cells of hematopoietic origin. IL-3 stimulates the growth, survival and differentiation of pluripotent hematopoietic stem cells, progenitors of all erythroid and myeloid cells, mature macrophages, eosinophils, megakaryocytes, mast cells, or basophils (Miyajima et al., 1993). G M - C S F is more restricted in its activity, and targets the progenitors and mature cells of the macrophage, neutrophil, and eosinophil lineages (Miyajima et al., 1993). Mice lacking G M - C S F have normal hematopoiesis, but surprisingly exhibit altered lung homeostatis and chronic pulmonary infections (Dranoff and Mulligan, 1994; Stanley et al., 1994). IL-4 is active on cells of both hematopoietic and non-hematopoietic origin, and is involved in the differentiation of specific classes of B - and T-lymphocytes and the downregulation of macrophage activity (Taniguchi, 1995).  Mice lacking IL-4 exhibit reduced  serum levels of IgGl and IgE due to a failure of immunoglobulin class switching in B cells, fail to differentiate CD4+ T cells to the Th2 subclass, and fail to produce germinal centers in Peyer's patches (Kopf et a l , 1993; Vajdy et al., 1995; von der Weid et al., 1994).  3  The receptor for SLF, c-Kit, is a homodimeric receptor tyrosine kinase, resembling the platelet-derived growth factor receptor (Fantl et al., 1993). The cytoplasmic tail of c-Kit contains an insert within its kinase domain, and the extracellular domain contains five immunoglobulin-like domains (Fantl et al., 1993). In contrast the receptors for IL-3, G M - C S F or IL-4 are distinctive heterodimers, the subunits of which lack enzymatic activity and belong to the hematopoietin receptor superfamily. The extracellular domains of these receptors contain four conserved cysteine residues, and the characteristic W S X W S motif that may be involved in protein folding or interaction with ligand (Bazan, 1990; Hilton et a l , 1995; Yoshimura et al., 1992). IL-3, IL-5 and GM-CSF share a common p-chain of their receptor complex known as KH97 (now referred to as (3-common) in humans or AIC2B in mice. Each cytokine also binds to a different low-affinity occhain, and the resulting complex interacts with the common p-chain to form the high-affinity receptor. The a-chains have very short cytoplasmic tails and require the p-common chain to transduce biological signals. In mice, IL-3 also signals through AIC2A, another p-chain arising from gene duplication of AIC2B.  However mice lacking AIC2A are phenotypically normal  (Nishinakamura et al., 1995). Mice lacking AIC2B show the same lung pathology as mice lacking GM-CSF, and have reduced numbers of eosinophils as seen in mice lacking IL-5 (Nishinakamura et al., 1995). Similarly, mice lacking both AIC2B and IL-3 are phenotypically identical to mice lacking AIC2B, and respond normally to immunological challenges such as infection with Listeria monocytogenes, raising the question of the physiological role of AIC2A (Nishinakamura et al., 1996). The IL-4 receptor consists of two distinct receptor complexes that use the IL-4 receptor achain (IL4Ra) as their low-affinity receptor. One high affinity receptor for IL-4 consists of the IL4Roc chain and the y-common chain of the IL-2R. The y-common chain is widely expressed and transduces IL-4 signals in most cells. The other high-affinity receptor for IL-4 consists of the IL4Ra chain and the a-chain of the IL-13 receptor (IL-13Ra). In fact, expression of the IL-13Roc lacking the cytoplasmic domain inhibits IL-4 signalling through the y-common chain in a dominant  4  negative fashion (Orchansky et al., 1997). IL-4 and IL-13 share many biological functions, most likely due to shared receptor subunits. Cytokine signal transduction is initiated by ligand-induced dimerization of their receptors and the subsequent activation of associated tyrosine kinases, including members of the J A K and src protein kinase families (Miyajima et al., 1993; Taniguchi, 1995). The J A K tyrosine kinases are constitutively associated with conserved domains in the cytoplasmic tails of these receptors known as boxl and box2. J A K activity is required for the tyrosine phosphorylation of specific residues in the cytoplasmic domains of these receptors that act as interaction sites for SH2 domains, a proteinprotein interaction domain found in proteins such as the signal transducers and activators of transcription (STAT).  IL-3 or G M - C S F induced signalling activates J A K 2 , a tyrosine kinase  required for the biological activity of these receptors (Parganas et al., 1998), and subsequently activates STAT5 (Fig. 1.1).  IL-4 signalling through the IL4Ra chain and y-common activates  JAK1 and J A K 3 , whereas signalling through IL4Ra and IL13Ra activates JAK1 and Tyk2. Thymocytes derived from mice lacking JAK1 are unresponsive to IL-4, demonstrating the importance of this tyrosine kinase for IL-4 signalling (Rodig et al., 1998). JAK3 is constitutively associated with y-common, and loss of either of these proteins results in X-linked severecombined immunodeficiency (SCID) syndrome (Leonard et al., 1995; Macchi et al., 1995; Nosaka et al., 1995; Russell et al., 1995b).  IL-4 induced signalling activates STAT6, a protein that  mediates many of the biological activities of IL-4 (Kaplan et al., 1996; Shimoda et al., 1996; Takeda etal., 1996). S L F induced signalling activates J A K 2 , and subsequently induces tyrosine phosphorylation of STAT1 (Deberry et al., 1997). Besides the JAK/STAT pathways, IL-3, G M - C S F , IL-4 or S L F activate PI-3 kinase through the relocalization of PI-3 kinase to its substrate at the plasma membrane (Gold et al., 1994).  PI-3 kinase phosphorylates phosphatidylinositol (PI), PI 4-phosphate, or PI 4,5-  bisphosphate on the 3'-position to generate PI 3-phosphate, PI 3,4-biphosphate or PI 3,4,5triphosphate (PIP3) respectively. PIP3 provides membrane docking sites for pleckstrin homology (PH) domains in proteins such as SOS, Akt, Pdkl and Pdk2. The recruitment of Pdkl and Pdk2  5  6  to the plasma membrane by PIP3 activates p 7 0 al., 1996).  The activation of p 7 0  phosphorylation of the S6 peptide.  S 6 K  S 6 K  and Akt (Burgering and Coffer, 1995; Scheid et  is required for efficient protein translation through  Akt is the mammalian homolog of v-Akt, a transforming  oncogene that acts by preventing apoptosis (Coffer and Woodgett, 1991; Staal, 1987).  IL-3,  SLF or IL-4 activates Akt (del Peso et al., 1997; Scheid and Duronio, 1998; Songyang et al., 1997), and over-expression of catalytically inactive Akt induces apoptosis even in the presence of IL-3 (Songyang et al., 1997). Furthermore, the inhibition of PI-3 kinase prevents the activation of Akt and induces apoptosis of hematopoietic cells, suggesting that Akt may mediate the antiapoptotic effects of hematopoietic growth factors (Scheid and Duronio, 1998). Cellular growth and proliferation are normally associated with the activation of the GTPase Ras and the E R K M A P K pathway. Accordingly, IL-3, G M - C S F or S L F activates both Ras and E R K M A P K (Duronio et al., 1992; Welham et al., 1992). Tyrosine kinase activity is required for the activation of Ras by cytokines (Duronio et al., 1992), possibly to provide docking sites for the SH2-containing proteins She or Grb2. SOS, an exchange factor for Ras and Rac/Cdc42, interacts with Grb2 through its SH3 domains, a protein-protein interaction domain that recognizes polyproline rich peptides (Chardin et al., 1993; Egan et al., 1993; L i et al., 1993). Grb2 recruits SOS by either directly binding the receptor, or indirectly binding receptor-associated She. SOS requires its P H domain to activate Rac or Cdc42 (Nimnual et al., 1998; Zheng et al., 1997), indicating that PI-3 kinase activity may regulate the activation of Rac or Cdc42 by IL-3, GM-CSF or SLF. These cytokines not only activate Ras and PI3K, but also lead to the activation of J N K and p38 M A P K (Foltz et al., 1997; Foltz and Schrader, 1997), two kinases proposed to be downstream of the Rac or Cdc42 (Bagrodia et al., 1995; Coso et al., 1995; Zhang et al., 1995). The activation of JNK by IL-3 requires activation of Ras (Rausch and Marshall, 1997; Terada et al., 1997), and may be prevented by inhibitors of PI-3 kinase (Ishizuka et al., 1997; Kawakami et al., 1998; Logan et al., 1997). While SOS is not necessarily upstream of JNK, these findings suggest that Ras and PI-3 kinase synergize to activate J N K by IL-3. IL-4 and IL-13 are unique among cytokines for their failure to activate Ras and the M A P K pathway (Duronio et al., 1992; Welham et al., 1992; Welham  7  et al., 1994). IL-4 also fails to activate JNK or p38 M A P K (Foltz et al., 1997; Foltz and Schrader, 1997; Salmon et al., 1997), suggesting that PI-3 kinase activity is not sufficient to activate Rac or Cdc42. Consistent with this hypothesis, a mutant form of SOS containing only the P H domain and the Dbl domain, which activates Rac, requires the expression of activated Ras to activate Rac or Cdc42 (Nimnual et al., 1998). Therefore, both PI-3 kinase and Ras activity appear to be required to activate Rac or Cdc42. SLF differs from IL-3, G M - C S F or IL-4 in its ability to regulate intracellular calcium channels by activating phospholipase (PL) C (Hallek et al., 1992).  P L C cleaves PI 4,5-  bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) (Bennett et al., 1988; Exton, 1994). IP3 releases intracellular calcium stores that subsequently allows the entry of extracellular calcium into the cell, effectively increasing the cellular concentration of free calcium. D A G and intracellular calcium activate several enzymes including conventional and novel P K C isoforms, calcium/calmodulin dependent protein kinases, the small GTPase Rapl, and the calcium sensitive phosphatase calcineurin (Crabtree and Clipstone, 1994; Huang and Huang, 1993; M'Rabet et al., 1998; Schonwasser et al., 1998). In summary, these highly related cytokines provide distinct biological functions that reflects the integrated signal provided through these receptors.  1.3  Phosphorylation in Signal Transduction  1.3.1. Protein Kinases - Kinases are enzymes that catalyze the transfer of the y-phosphate of adenosine triphosphate (ATP) to a hydroxyl group on either its protein or lipid substrate. A single phosphate group adds a large negative charge and drastically changes the appearance of the substrate. The first oncogene identified was the tyrosine kinase v-Src, the transforming agent of the Rous sarcoma virus. v-Src was later discovered to be a homolog of the mammalian protein cSrc. The Src family of proteins are membrane-associated tyrosine kinases, meaning these enzymes specifically phosphorylate proteins on the hydroxyl group of tyrosine residues.  8  Several other  cytosolic tyrosine kinases have been identified including the Janus kinases (JAK), the Tec kinase family,  and c-Abl. The other class of tyrosine kinases are the receptor tyrosine kinases that  include the receptors for EGF, PDGF, F G F and SLF.  These receptors are activated by ligand-  induced dimerization or oligomerization, and become trans or autophosphorylated.  This  phosphorylation creates binding sites for SH2 domains of adapter proteins or other kinases. Tyrosine kinases act, for the most part, as membrane proximal signal transducers and represent less than 1% of total cellular phosphorylation. Most phosphorylation occurs on serine, and to a lesser extent on threonine residues, although phosphorylation may occur on histidine, aspartate, and lysine residues. As with tyrosine kinases, these enzymes are classified as cytosolic and receptor serine/threonine kinases.  The  receptors for transforming growth factors are the only known receptor serine/threonine kinases, and as such the majority of serine/threonine kinases are cytosolic proteins.  The mammalian  homologs of S t e l l and Ste20 including Raf, M E K K 1 and P A K , as well as the broad-specificity E R K , J N K and p38 mitogen-activated protein kinases (MAPK) are all serine/threonine specific kinases. Besides the M A P K cascades, the protein kinase A , B and C families are specific for serine and threonine residues. Typically these enzymes are activated downstream of tyrosine kinases and provide a link from the plasma membrane of a cell to the nucleus. Most kinases are defined based on their substrate specificity, however a few dualspecificity kinases have been identified.  The best characterized dual-specificity kinases are the  mammalian homologs of Ste7. These enzymes phosphorylate M A P K proteins on tyrosine and threonine residues within their activation loops. Another family of nuclear dual-specificity kinases known as STY, or Clk have also been identified, and appear to regulate mRNA splicing by phosphorylating serine/arginine-rich proteins (Duncan et al., 1997; Lee et al., 1996). In contrast to many kinases, the dual-specificity kinases are very selective enzymes, and have a small number of highly-related physiological substrates.  9  1.3.2. Mechanism of Phosphotransfer by Kinase Domains -  The catalytic domain of  protein kinases has been highly conserved through evolution, and forms a highly globular structure consisting of 250 to 300 amino acids.  The first crystal structure of a kinase domain was  determined for cAMP-dependent protein kinase in 1991 (Figure 1.2), and has provided a structural model for other kinases (Knighton et al., 1991a,b). The catalytic domain consists of a p-stranded N-terminal lobe and an a-helical C-terminal lobe. The active site lies at the interface of the two lobes with the ATP-binding site being formed by residues on both lobes. The adenine moiety binds to a hydrophobic pocket, and the ribose group is stabilized by hydrogen bonds. A kinase catalyzes the transfer of the y-phosphate of ATP onto a hydroxyl group of its respective substrate. This reaction requires a magnesium ion as a co-factor to co-ordinate the p- and y-phosphates of ATP. Alignment of the primary structure of the catalytic domain of protein kinases reveals eleven conserved sub-domains (Hanks et al., 1988).  These sub-domains are important for catalytic  function and are involved either directly in the active site or indirectly in a structural role. The alignment of catalytic domains has also identified several invariant or nearly invariant amino acids that are critical for phosphotransferase activity. The alignment of the primary structures of catalytic domains has also defined peptides that predict the kinase specificity from their primary sequence. For example, serine/threonine kinases have (T/S)xx(Y/F)xAPE in sub-domain VIII, and DLKP(E/S)N in sub-domain VI, whereas tyrosine kinases have P(I7V)(K/R)W(T/M)APE in subdomain VIII, and D L R A ( A / R ) N in sub-domain V I (Hanks et al., 1988). Sub-domain I contains a GxGxxG motif, and the first two glycines of this motif are nearly invariant residues (Hanks et al., 1988; Johnson et al., 1996a).  This motif is present in many  nucleotide binding proteins including GTPases, and is involved in the proper co-ordination of ATP. The first glycine contacts the ribose group and the second glycine lies near the a-phosphate of ATP. Sub-domain I also contains a nearly invariant valine residue two amino acids C-terminal to the GxGxxG motif that is believed to properly position the glycines within the kinase domain .  10  Figure  1.2  - Crystal structure of c A M P dependent protein kinase.  The  structure of cAMP-dependent protein kinase (cAPK) in complex with an inhibitor peptide and ATP is shown. The N-terminal lobe of cAPK is predominantly P-stranded, whereas the C-terminal lobe is primarily a-helical in nature. The ATP molecule lies between the Nand C-terminal lobes. The serine residue that is the target of phosphorylation is depicted as PO. The activation segment is noted on the right hand side of the diagram. The catalytic base of cAPK is D166, and D184 interacts with the p- and y-phosphates of ATP. The critical lysine (K72) is positioned above the ATP molecule. This figure was adapted from Johnson et al., 1996.  1 1  Sub-domain II contains an invariant lysine residue that is directly involved in the coordination of the a- and P-phosphates of ATP (Johnson et al., 1996a). This lysine (K72, Fig. 1.3) is located within the active site and hydrogen bonds with an invariant glutamate residue (E91, Fig. 1.3) in sub-domain III. The importance of this residue in catalysis was initially discovered with the ATP analog, p-fluoro sulfonyl 5'-benzoyl adenosine, that reacts with the lysine residue and inhibits kinase activity. Site-directed mutagenesis supports a critical role for this lysine in catalysis as its mutation to any amino acid ablates kinase activity (Hanks et al., 1988). The central core of the catalytic domain lies in the C-terminal lobe, and is highly conserved between kinases. Sub-domain VI contains a highly conserved aspartate group found in an RD motif that is indicative of kinases regulated by phosphorylation (RD kinases), and site-directed mutagenesis of this aspartate group ablates phosphotransferase activity. (Johnson et al., 1996a). An oxygen group on the aspartate side chain (D166, Fig. 1.3) undergoes a base-catalyzed attack on the proton of the hydroxyl group on the substrate. This hydroxyl-group is highly reactive when deprotonated and attacks the y-phosphate of ATP mediating phosphotransfer.  The arginine group  (R165, Fig. 1.3) immediately N-terminal to the catalytic aspartate forms a salt bridge with the catalytic aspartate residue and keeps the kinase inactivated. Sub-domain VII is defined by the presence of the D F G motif that marks the N-terminus of the activation loop (Hanks et al., 1988; Johnson et al., 1996a). The aspartate (D184, Fig. 1.3) of the D F G motif is an invariant amino acid and is critical for kinase activity. The aspartyl side chain interacts with the magnesium ion that is complexed with ATP, and properly orients the p- and yphosphates of ATP for catalysis. Phosphotransferase activity is removed by mutation of this aspartate to any amino acid. Sub-domain VIII contains the A P E motif that defines the end of the activation loop (Hanks et al., 1988; Johnson et al., 1996a). The glutamate residue in this motif is invariant, and coordinates the invariant arginine in sub-domain X I (Johnson et al., 1996a). The activation loop undergoes dramatic structural changes upon kinase activation, and contains residues that require phosphorylation to activate RD kinases. The phosphorylation dramatically alters the charge of the  12  Figure 1.3 - Model of the active site of cAMP dependent protein kinase.  13  residue and creates new interactions with other positively charged residues.  A hydrogen bond  forms between the phosphorylated residue (T197, Fig. 1.3) and the arginine residue N-terminal of the catalytic aspartate residue removing its inhibition on catalysis, and allowing the aspartate group to attack the proton of the hydroxyl-group of its substrate. This phosphorylation may change the overall conformation of the kinase to correctly orient the catalytic base, may determine the conformation of residues in the activation loop involved in substrate recognition, and may convert the enzyme from an open to a closed conformation. A n open conformation allows ATP into the catalytic site, and allows the release of A D P after catalysis. A closed conformation brings residues into the correct conformation to promote catalysis (Johnson et al., 1996a).  1.3.3. Phosphatases - The advantages of phosphorylation as a post-translational modification he not only in the addition of a small highly negatively charge, but also in its reversibility. The removal of phosphate groups is catalyzed by a group of enzymes known as phosphatases. As with kinases, phosphatases can be specific for phosphotyrosine, phosphoserine and phosphothreonine or both. Protein tyrosine phosphatases exist as both transmembrane receptors and cytosolic proteins.  Receptor tyrosine phosphatases are fairly well characterized and include CD45 and  PTPa. These phosphatases generally contain two cytoplasmic phosphatase domains of which the N-terminal domain is more active.  In contrast to the receptor tyrosine kinases, the receptor  tyrosine phosphatases are inhibited by dimerization, and are active as monomers. These enzymes are very divergent in their extracellular domains, suggesting the possibility of many distinct ligands, however as yet no ligands that would dimerize and inhibit PTPase activity are known. The cytosolic protein tyrosine phosphatases and serine/threonine specific phosphatases generally contain a single phosphatase domain. The protein tyrosine phosphatases include the proteins SHP1, SHP2 and PTP1B, while the serine/threonine phosphatases include calcineurin and protein phosphatase 2A. These enzymes often contain domains involved in intracellular localization such as SH2 domains in the case of SHP1 and SHP2, or endoplasmic reticulum  14  retention sequences in the case of PTP1B.  SHP1 and SHP2 are recruited to receptors after  signalling and likely act to either attenuate or initiate signalling through dephosphorylation. The dual specificity phosphatases are characterized as either VHl-like or Cdc25-like phosphatases.  The Cdc25-like phosphatases regulate the activity of cyclin-dependent protein  kinases by removing inhibitory phosphate groups. The VHl-like phosphatases including M K P 1 , PAC1 and Pystl, are classified for their homology to the VH1 phosphatase from vaccinia virus, and function to dephosphorylate and inactivate M A P kinases. The expression of these enzymes is generally regulated at the transcriptional level and these PTPases differentially inactivate the E R K , JNK and p38 M A P K families of protein kinases.  1.4  The Mitogen-Activated Protein Kinase Cascades  1.4.1. The MAPK Cascades -  Yeast exist as haploid organisms that, in the presence of  peptide pheromones, fuse to become diploid organisms through a process known as the mating response. Mutations either preventing the mating response or inducing the mating response in the absence of pheromones led to the identification of protein kinases in the M A P K cascades. The ability of these different mutant proteins to enforce or prevent the mating response allowed the sequential determination of kinases in the pathway. In summary, this work defined Sterile (Ste) 20 gene as the kinase responsible for activating Stel 1. In turn, Stel 1 acts upstream of Ste'7, a kinase that phosphorylates the mitogen-activated protein kinase Fus3 (Fig. 1.4). This signalling cassette is held together by a scaffolding protein known as Ste5.  Proteins in the mammalian M A P K  cascades are placed into linear cascades based on their homology to these kinases. The canonical M A P K cascade in mammalian cells is the E R K M A P K pathway. I will describe this pathway in some detail as much that has been discovered has proven relevant for other M A P K cascades. Growth factors such as EGF or IL-3 activate this pathway downstream of Ras or P K C .  The Ras superfamily consists of 5 genes that encode N-Ras, H-Ras, 2 splice  variants of Ki-Ras, TC21 and M-Ras. Ras proteins are molecular switches that are inactive when  1 5  GTPase:  Rac/Cdc42  Ras  SOK  Ste20:  L O K Mst3  1 Stell:  1  Ste7:  Kssl/Fus3:  A-Raf  | Mos  Raft B - R a f |  MEK1  MEK2  ERK1/2  ERK3  MKK4  MKK7 |  JNK1/2/3  rsk  Mnkl  Elkl  Mnk2  Mskl  Sapl  Rsk2  Msk2  Myc  MAPKAPK5  p 9 0  GCK  MEKK1-5  Tpl2  |  PAK1-3  c-Jun ATF2 Bcl2 p53  MLK1-3  MKK3  p38a  ATF2 ATF1 CREB MEF2C CHOP  1 GLK  HPK1  D L K TAK1  MKK6  p38P  p38v/8  M A P K A P Kinase 2/3 PRAK1  MKK5  ERK5  MEF2C  I  Hsp25  Figure 1.4 - The M A P K Cascade. Summary of the signal transduction molecules presently identified as being involved or belonging to a M A P K cascade.  16  they bind GDP, and are active when they bind GTP. These highly related molecules contain two domains, switch 1 and switch 2, that undergo conformational changes upon binding GTP and are involved in binding to effectors.  Functional differences between the Ras proteins are poorly  characterized, however exchange factors such as Ras-GRF can selectively activate specific Ras proteins indicating that these proteins may be differentially regulated in vivo (Jones and Jackson, 1998). The greatest differences in the primary structure of the Ras proteins lies in their C-terminal tails. A l l Ras proteins are anchored to the membrane by prenylation within a CAAX-motif at their C-terminus. The C-termini of Ras proteins are also either modified by palmitoylation, or contain a poly-basic region likely involved in either membrane association or perhaps in recruiting GDP dissociation factors (Mizuno et al., 1991). The best described effectors of GTP-bound Ras are the serine/threonine protein kinases Raf-1, A-Raf and B-Raf. Raf proteins contain a Ras-interaction domain in their N-termini (Vojtek et al., 1993), and have approximately 1000-fold higher affinity for GTP-bound Ras than GDPbound Ras. Ras binding to Raf recruits Raf to the plasma membrane, however localization to the plasma membrane by Ras is only sufficient to activate B-Raf (Stanton et al., 1989; Yamamori et al., 1995). Experiments localizing Raf-1 to the plasma membrane suggest that Ras functions to recruit Raf to the plasma membrane (Stokoe et al., 1994), however these results have been contentious (Mineo et al., 1997; Tamada et al., 1997). Besides membrane recruitment, Ras may function to aggregate Raf. This hypothesis is supported by elegant experiments using drugs that artificially dimerize and activate Raf-1 (Farrar et al., 1996; Luo et al., 1996).  Raf also requires  phosphorylation for activation (Morrison et al., 1993), that may occur as a result of autophosphorylation, or phosphorylation by conventional and novel P K C isoforms (Schonwasser et al., 1998). Raf is phosphorylated within a highly conserved R S X S X P motif that provides an interaction domain for 14-3-3(3 and is required for activity in vitro and in vivo 1998).  (Thorson et al.,  The best characterized substrates for Raf are the dual-specificity kinases MEK1 and  M E K 2 . Rafl and B-Raf are capable of activating both M E K 1 and M E K 2 , while A-Raf appears to only activate M E K 1 (Wu et al., 1996). The Raf gene products have non-redundant functions in  17  vivo  as mice lacking A-Raf display strain specific intestinal defects and neurological abnormalities  (Pritchard et al., 1996). Mice lacking B-Raf die in utero at mid-gestation due to a premature apoptotic death of differentiated endothelial cells (Wojnowski et al., 1997).  Furthermore,  conditionally active mutants of A-Raf and B-Raf differ in their ability to promote the entry of cells into the cell cycle (Pritchard et al., 1995). M E K 1 and M E K 2 are dual-specificity kinases that are activated by phosphorylation on two serine residues within their activation loop by Raf. The recognition of M E K 1 or M E K 2 by Raf requires a large proline-rich insert in their C-terminus (Catling et al., 1995). Both MEK1 and M E K 2 activate ERK1 or E R K 2 by phosphorylating both threonine and tyrosine residues of the TEY motif in sub-domain VIII (Brott et al., 1993; Crews et al., 1992; Zheng and Guan, 1993). E R K is initially phosphorylated on tyrosine, and then on threonine (Burack and Sturgill, 1997; Ferrell and Bhatt, 1997). The phosphorylation of both residues is required for activation of E R K (Canagarajah et al., 1997). The activation of E R K by M E K is enhanced by a protein characterized as M E F , or MEK-enhancing factor (Scott et al., 1995), and cloned as MP-1 (Schaeffer et al., 1998). Interestingly, M E K 2 , but not M E K 1 , also activates E R K 3 , but the relevance of this observation in vivo is unclear (Robinson et al., 1996).  A n activator of ERK3 has been  characterized biochemically, but remains to be identified (Cheng et al., 1996b). The E R K M A P kinases are proline-directed serine/threonine kinases (Boulton et al., 1991; Charest et al., 1993; Clark-Lewis et al., 1991) and their activity may to regulate mitogenesis, differentiation and apoptosis.  The E R K M A P K pathway activates a broad spectrum of  downstream kinases including p90 k rs  (Blenis, 1993), M n k l (Fukunaga and Hunter, 1997;  Waskiewicz et al., 1997), Mnk2 (Waskiewicz et a l , 1997), Rsk2 (Xing et al., 1996) and M A P K A P kinase 5 (Ni et al., 1998). The functions of these kinases are largely unknown however M n k l phosphorylates eIF-4e possibly linking E R K activity with the regulation of translation (Waskiewicz et al., 1997). E R K M A P K also regulates the transcriptional activity of E l k l , Sapl and c-Myc, and in turn regulates the expression of many genes critical to mitogenesis such as cyclinDl (Greulich and Erikson, 1998; Lavoie et al., 1996). The E R K M A P K pathway is also  1 8  activated by non-rnitogenic stimuli including chemokines and pro-inflammatory cytokines indicating that E R K activity is not sufficient for mitosis. Mice lacking M E K 1 die in utero due to a failure in vascular endothelial cell migration within the placenta (Jean Charron, unpublished data). This finding suggests either non-redundant functions or insufficient gene dosages of MEK1 and M E K 2 as vascular endothelial cells normally express both enzymes. Upon activation, ERK1 and ERK2 translocate from the cytoplasm to the nucleus in an event that requires neither kinase activity nor phosphorylation on the activation loop (Chen et al., 1992; Lenormand et al., 1993), but may require homodimerization of E R K (Cobb, M . , unpublished data).  Unlike E R K 1 and E R K 2 ,  ERK3 is constitutively localized to the nucleus (Cheng et al., 1996a). Besides the Raf enzymes, MEK1 and M E K 2 are activated by the serine/threonine kinases c-Mos, Tpl-2 and M E K K 1 . c-Mos is expressed predominantly in germline cells and is required for the activation of M-phase promoting factor (MPF) (Haccard et al., 1993; Yew et al., 1992). Genetic disruption of c-Mos supports its role in early meiosis for the activation of M P F and M A P K (Araki et al., 1996). Tpl-2, or tumor progression locus-2, was discovered as a protooncogene in MMLV-derived T-lymphomas in rats, and shares 90 % identity with the human gene Cot (Patriotis et al., 1994). Tpl-2 directly phosphorylates and activates M E K 1 and M K K 4 in vitro (Salmeron et al., 1996). Supporting these observations, the over-expression of Tpl-2 activates both the E R K and J N K pathways, with little effect on p38 M A P K activity. The upstream regulators of c-Mos and Tpl-2 are poorly understood. M E K K 1 was identified based on its homology with the Stel 1 and Byr2 kinases from yeast (Lange-Carter et al., 1993; X u et al., 1996a). Since then five related M E K K enzymes have been identified (Blank et al., 1996; Deacon and Blank, 1997; Gerwins et al., 1997; Ichijo et al., 1997; Wang et al., 1996). M E K K 1 was initially characterized by an activator of M E K 1 (Lange-Carter et al., 1993), however a role for M E K K 1 upstream of M E K 1 and M E K 2 in vivo is contentious (Gardner et al., 1994; Minden et al., 1994a; X u et al., 1995). Like Raf, M E K K 1 is an effector for GTP-bound Ras (Russell et al., 1995a), and Ras function is required for M E K K 1 activation by E G F (Lange-Carter and Johnson, 1994). M E K K 1 and M E K K 4 bind and may be regulated by  19  Racl or Cdc42 (Fanger et al., 1997b; Gerwins et al., 1997). However, the regulation of M E K K 2 , M E K K 3 or A S K 1 / M E K K 5 by small GTPases of the Ras or Rho family has not been described. Over-expression of M E K K 1 potently activates the c-Jun N-terminal kinases (JNK) (Minden et al., 1994a; Yan et al., 1994) through the activation of M K K 4 (Lin et al., 1995; Yang et al., 1997b) and M K K 7 (Lu et al., 1997; X u and Cobb, 1997; Yao et al., 1997). Furthermore, mice lacking M E K K 1 fail to activate J N K and also E R K M A P K downstream of specific stimuli indicating that M E K K 1 functions to activate JNK in vivo (Yujiri et al., 1998). Enforced expression of M E K K 2 , M E K K 3 , or M E K K 4 activates the JNK pathway (Blank et al., 1996; Deacon and Blank, 1997; Gerwins et al., 1997; Wang et al., 1996). M E K K 5 / A S K 1 activates both J N K and p38 M A P K , but not E R K M A P K , by directly phosphorylating M K K 3 , M K K 4 and M K K 6 (Ichijo et al., 1997). Overexpression of M E K K 2 or M E K K 3 also activates the E R K pathway (Blank et al., 1996) and fails to activate p38 M A P K .  Interestingly, M E K K 3 activates M K K 3 , but not p38a M A P K ,  possibly demonstrating in vivo substrate specificity of M K K 3 for p38 isoforms (Deacon and Blank, 1997). The JNK family of M A P K are proline-directed serine-threonine kinases that phosphorylate the N-terminus of c-Jun (Derijard et al., 1994; Kyriakis et al., 1995). These enzymes are activated by dual phosphorylation on threonine and tyrosine residues in a TPY motif (Derijard et al., 1994) by the dual specificity kinases M K K 4 and M K K 7 (Derijard et al., 1995; Foltz et al., 1998; Holland et al., 1997; Lawler et al., 1997; L u et al., 1997; Moriguchi et al., 1997; Sanchez et al., 1994; Tournier et al., 1997; Wu et a l , 1997; Yang et al., 1998b; Yao et al., 1997). Mice lacking M K K 4 die in utero suggesting differential functions for M K K 4 and M K K 7 .  In support of this notion,  G C K and M E K K 5 preferentially activate M K K 7 in over-expression studies, however M E K K 1 and M E K K 2 activate both enzymes similarly (Wu et al., 1997). M L K 2 also preferentially activates M K K 7 over M K K 4 in vitro under conditions where M E K K 1 activated both enzymes comparably (Hirai et al., 1998). However, M E K K 1 preferentially activates M K K 4 in K B cells (Cuenda and Dorow, 1998), suggesting the activation of M K K 4 and M K K 7 is modulated by other molecules such as scaffolding proteins (Fanger et al., 1998).  20  Like J N K and E R K M A P K , the p38 M A P K family are proline-directed serine/threonine kinases (Goedertet al., 1997; Han et al., 1994; Han et al., 1995; Jiang et al., 1996; Jiang et al., 1997; Kumar et al., 1997; Lechner et al., 1996; Lee et al., 1994; L i et al., 1996; Mertens et al., 1996; Rouse et al., 1994; Stein et al., 1997; Wang et al., 1997b). These enzymes are activated by dual-phosphorylation on tyrosine and threonine residues within a T G Y motif on their activation loops (Doza et al., 1995; Raingeaud et al., 1995). Proteins of the p38 M A P K family are activated by two dual specificity kinases, M K K 3 and M K K 6 (Cuenda et al., 1996; Derijard et al., 1995; Han et al., 1996; Han et al., 1997; Lin et al., 1995; Moriguchi et al., 1996; Raingeaud et al., 1996). M K K 4 also activates p38 M A P K  in vitro,  however the relevance of this activation has yet  to be determined (Derijard et al., 1995; Lin et al., 1995). Several homologs of Ste20 and S t e l l activate the JNK and p38 M A P K pathways. mixed  lineage  kinases  M L K 1 , MLK2/MST,  MLK3/SPRK/PTK1  and  DLK/MUK  The are  serine/threonine kinases that are characterized by catalytic domains with similarity to both tyrosine and serine/threonine kinases (Creasy and Chernoff, 1995; Dorow et al., 1993; Ezoe et: al., 1994; Gallo et al., 1994; Hirai et al., 1996; Holzman et al., 1994; Ing et al., 1994; Katoh et al., 1995). The C-termini of these enzymes have leucine zippers that are likely involved in protein-protein interactions (Fanger et al., 1997b). Both M L K 3 and D L K contain Cdc42/Rac interaction domains (CRIB), however M L K 3 interacts weakly and D L K fails to interact with Rac or Cdc42 (Lamarche et al., 1996; Nagata et al., 1998; Teramoto et al., 1996). M L K 2 , M L K 3 and D L K activate M K K 4 directly, and over-expression of these enzymes increase J N K activity (Cuenda and Dorow, 1998; Hirai et al., 1996; Hirai et a l , 1997; Hirai et al., 1998; Nagata et al., 1998; Tibbies et al., 1996). M L K 2 co-localizes with activated JNK1 and JNK2 on microtubules, implying some biologically relevance for the activation of JNK by M L K 2 (Nagata et al., 1998).  Overexpression of M L K 3  also activates p38 M A P K through M K K 6 , but fails to activate E R K M A P K (Tibbies et al., 1996). Germinal center kinase (GCK), a mammalian homolog of Ste20, was identified as a protein kinase that is differentially expressed in germinal center B lymphocytes, but not in B cells from lymphoid follicles (Katz et al., 1994). Overexpression of G C K activates the J N K pathway, but  21  fails to activate p38 M A P K or E R K M A P K (Katz et al., 1994). The activation of JNK by G C K is inhibited by kinase dead M L K 3 , but not by kinase dead M E K K 1 (Tibbies et al., 1996), suggesting M L K 3 functions downstream of G C K in vivo.  Recently, a GCK-like protein kinase (GLK) was  identified that activates the J N K pathway without activating either p38 or E R K M A P K pathways (Diener et al., 1997). Unlike G C K , the activation of JNK by G L K is inhibited by kinase dead M E K K 1 suggesting these highly related enzymes use different S t e l l homologs (Diener et al., 1997). T A K 1 , or TGFp-activated kinase, was identified in a genetic screen to find mammalian proteins that rescue Stell defects in S. cerevisiae  (Yamaguchi et al., 1995). Overexpression of  TAK1 activates both the JNK and the p38 M A P K pathways (Moriguchi et al., 1996; Shirakabe et al., 1997), but fails to activate E R K M A P K . Yeast two-hybrid analysis identified TAB1, a TAK1 binding protein, that binds to TAK1 and regulates its enzymatic activity (Shibuya et al., 1996). While the function of these proteins has not been determined in mice, studies on homologs of TAK1 and TAB1 in X. laevis suggest a role for these proteins for expression of ventral mesoderm during embryogenesis (Shibuya et al., 1998). Hematopoietic protein kinase 1 (HPK1), a serine/threonine kinase with similarity to Ste20, is expressed predominantly in hematopoietic cells, including early progenitor cells (Hu et al., 1996; Kiefer et al., 1996). Overexpression of HPK1 activates the J N K pathway, and fails to activate p38 M A P K or E R K M A P K (Hu et al., 1996; Kiefer et al., 1996; Wang et al., 1997). H P K 1 directly phosphorylates and activates M E K K 1 providing a pathway for JNK activation (Hu et al., 1996). Catalytically inactive mutants of M L K 3 , M E K K 1 and TAK1 prevent the activation of J N K by HPK1 (Hu et al., 1996; Kiefer et al., 1996; Wang et al., 1997a), suggesting linear cascades from HPK1 to J N K involving three distinct Stell homologs. The ability of T A K 1 , a known activator of the p38 M A P K pathway, to inhibit HPK1 is confusing as HPK1 fails to activate p38 MAPK.  These differences may reflect cell-specific signal transduction pathways, or may  exemplify the problems associated with over-expression studies and the use of dominant negative proteins.  22  The p21-activated kinases; P A K 1 , PAK2 and P A K 3 , contain CRIB domains and are effectors for GTP-bound Cdc42 and Racl (Manser et al., 1995; Manser et al., 1994; Martin et al., 1995; Teo et al., 1995). These serine/threonine kinases are activated directly by binding to GTPbound Cdc42 or Rac. Over-expression of P A K 1 , P A K 2 or P A K 3 has been shown to activate both p38 M A P K and J N K pathways (Bagrodia et al., 1995; Brown et al., 1996; Frost et al., 1996; Zhang et al., 1995), the latter in a MEKKl-independent fashion (Fanger et al., 1997). Several Ste20-like serine/threonine kinases are known that fail to activate any M A P K pathways. Two highly related enzymes Krs-1 and Krs-2 are yet to be tested for their ability to activate p38, JNK or E R K M A P K (Taylor et al., 1996). However, these enzymes are activated by stress stimuli such as extreme heat shock or arsenite implying a role upstream of J N K or p38 M A P K (Taylor et al., 1996). Another enzyme known as SOK, or Ste20/oxidant stress response kinase-1, is activated specifically by oxidative stresses such as reactive oxygen intermediates (Pombo et al., 1996). SOK is related to lymphocyte-oriented kinase (LOK) and both enzymes are unable to activate E R K , J N K or p38 M A P K pathways (Kuramochi et al., 1997; Pombo et al., 1996) . Another Ste20 homolog that is highly related to S O K is Mst3 (Schinkmann and Blenis, 1997) . Mst3 is an unusual kinase as it uses either ATP or GTP and manganese as co-factors for the phosphotransferase activity. The over-expression of Mst3 fails to activate p38a, p38y, J N K , ERK1 or E R K 2 (Schinkmann and Blenis, 1997). The failure of these Ste20 homologs to activate known M A P K pathways suggests the existence of novel M A P K family members, or a need to analyze other isoforms of known M A P K as substrates. ERK5, or Big M A P K 1 (BMK1), is the least understood M A P K pathway (Lee et al., 1995; Zhou et al., 1995), and was identified in a yeast two-hybrid screen using MEK5 as bait (Zhou et al., 1995). ERK5 is activated by dual phosphorylation on tyrosine and threonine residues on a TEY motif within its activation loop by MEK5 (Kato et al., 1997).  ERK5 is activated by  oxidative stress possibly downstream of c-Src, and in some instances requires calcium release for activation (Abe et al., 1996, 1997). Serum and activated mutants of Ras, but not constitutively active mutants of Rafl, activate ERK5 (English et al., 1998).  23  Overexpression of constitutively  active M E K 5 activates ERK5 and leads to MEF2C phosphorylation in vivo (Kato et al., 1997). The phosphorylation of MEF2C is inhibited by a kinase dead form of ERK5 implying that MEF2C is an in vivo substrate of ERK5 (Kato et al., 1997).  1.5  The Stress-Activated Protein Kinases: JNK and p38 MAPK  1.5.1. Overview of the genes encoding JNK - The c-Jun N-terminal kinase (JNK) family of M A P kinases is encoded by three distinct genes: JNKl/SAPKy,  JNK2/SAPKa  and  JNK3/SAPK/3. The JNK1 and JNK2 genes are ubiquitously expressed. The JNK3 gene has a more restricted expression pattern and is found predominantly in the brain, heart and testis. These genes encode a total of 10 distinct JNK enzymes by differential splicing (Fig. 1.5) (Gupta et al., 1996). The JNK1 and JNK2 gene each encode 4 isoforms, whereas the JNK3 gene only encodes 2 isoforms. These genes encode both 46 and 55 kDa isoforms of J N K (Figure 1.4), that are formed by differential splicing at the 3'-end of the mRNA. The additional transcripts encoding a and p isoforms of JNK1 or JNK2 are generated by internal splicing within the kinase domain between sub-domains IX and X . The JNK3 gene does not appear to undergo this second splicing event.  1.5.2. Activators and Substrates of JNK -  The immediate upstream activators and some  downstream substrates of the JNK signalling pathway are fairly well characterized (Fig. 1.6). The JNK family of kinases is regulated by dual phosphorylation on both threonine and tyrosine residues within a TPY motif on their activation loop in sub-domain VIII.  This phosphorylation  event is regulated by two dual-specificity kinases known as M K K 4 and M K K 7 .  M K K 4 , also  known as S A P K / E R K Kinase 1 (SEK1), was initially identified as a component of the J N K signalling pathway based on its homology with X - M E K 2 , a M K K from X. laevis (Lin et al., 1995). M K K 4 can activate both p38 M A P K and JNK in vitro (Lin et al., 1995), whereas M K K 7  24  JNKl JNK2 JNK3  MSRSKRDNNFYSVEIGDSTFT . .D. .C.SQ. . . .OVA MSLHFLYYCSEPTLDVKIAFCQGFDKQVDVSYIAKHYN. .K. .V. .Q V I  II  III  JNKl JNK 2 JNK 3  WLKRYQNLKPIGSGAQGIVCAAYDAILERNVAIKKLSRPFQNQTHAKRAYRELVLMKC Q F.TV.GIS..V L. . V. D  JNKl JNK 2 JNK 3  VNHKNIIGLLNVFTPQKSLEEFQDVYIVMELMDANLCQVIQMELDHERMSYLLYQMLCGI S T L H S T L  JNKl JNK 2 JNK 3  KHLHSAGIIHRDLKPSNIWKSDCTLKILDFGLARTAGTSFMMTPYWTRYYRAPEVDILG C.N  IV  v  VI  VII  IX  JNKlalpha JNKlbeta JNK2alpha JNK2beta JNK 3  *  *  VIII  X  MGYKENVDLWSVGC1MGEMVCHKILFPGRDYIDQWNKVIEQLGTPCPEFMKKLQPTVRT I IKGGV....T.H I KGCVI.Q.T.H SA.I A. . . L . . V SA N I R P N XI  JNKl JNK2 JNK3  YVENRPKYAGYSFEKLFPDVLFPADSEHNKLKASQARDLLSKMLVIDASKRISVDEAL P . I K . . E . . . . WI . . SE . . RD. I . T PD LT . P S PA D. .  JNKl JNK2 JNK3  QHPYINVWYDPSEAEAPPPKIPDKQLDEREHTIEEWKEL1YKEVMDLEERTKNGVIRGQ R....T A Q.Y.A..E....A W...S.... VKD. A. V Q.Y T NS . . K VK. .  JNKlalphal JNKlalpha2 JNKlbetal JNKlbeta2 JNK2alphal JNK2alpha2 JNK2betal JNK2beta2 JNK3alphal JNK3alpha2  PSPLAQVQQ ....GAAVINGSQHPSSSSSVNDVSSMSTDPTLASDTDSSLEAAAGPLGCCR QQ ....GAAV1NGSQHPSSSSSVNDVSSMSTDPTLASDTDSSLEAAAGPLGCCR ..--..MQQ ..—DAAV-SSNATPSQSSSINDISSMSTEQTLASDTDSSLDASTGPLEGCR ..--..MQQ ..—DAAV-SSNATPSQSSSINDISSMSTEQTLASDTDSSLDASTGPLEGCR ...S...QQ ...SGAAVNSSESLPP-SSSVNDISSMSTDQTLASDTDSSLEASAGPLGCCR  Figure 1.5 - Alignment of the Isoforms of J N K . The predicted amino acid structure of human J N K l , JNK2 and JNK3. Splice variants are shown only where they differ from J N K l , JNK2 or JNK3 respectively. Where residues are identical to J N K l a 1 they are indicated by periods. Gaps were introduced to optimize the alignment and are indicated by dashes.  25  MKK4  ATF-2  MKK7  c-Jun  Elk-1  Figure 1.6 - Activators and Substrates of the JNK pathway.  26  is a specific activator of JNK (Foltz et al., 1998; Tournier et al., 1997). These kinases are in turn presumably activated by several mammalian homologues of Stel 1 (Fig. 1.4). The J N K protein kinases phosphorylate and interact with a number of substrates that are mainly transcription factors including c-Jun and JunD, ATF-2, Elk-1 and Sap-la.  The  phosphorylation of these transcription factors by J N K increases their transcriptional activity, cJun, a transcription factor that heterodimerizes with c-Fos to form AP-1, is phosphorylated on Ser63 and Ser-73 within its N-terminus. JNK interacts with a 16 amino acid motif N-terminal to these phosphorylation sites in c-Jun known as the delta domain (Adler et al., 1994; Dai et al., 1995). This motif is believed to repress c-Jun function as it is deleted in v-Jun, the viral oncogenic form of c-Jun. v-Jun is not phosphorylated eventhough it contains the corresponding serine residues of cJun, demonstrating the importance of the delta domain to direct J N K to its substrate  in  vivo.  However, v-Jun is transcriptionally active suggesting another role for the delta domain. Consistent with this notion, inactive J N K bound to c-Jun through the delta domain targets c-Jun for ubiquitinylation (Fuchs et al., 1996). In this model, phosphorylation by J N K not only increases its transcriptional activity, but also its half-life by preventing its ubiquitinylation. The delta domain interacts with a region between sub-domain IX and X near the catalytic pocket of J N K (Kallunki et al., 1996), and implies a functional role for the internal splicing event that generates the a and p isoforms of J N K (Gupta et al., 1996). While all isoforms phosphorylate the transcription factors c-Jun and ATF-2 in in vitro kinase assays, their interaction with these substrates differs (Gupta et al., 1996). The beta isoforms of JNK1 interact with both c-Jun and ATF-2 better than its alpha isoforms.  The alpha isoforms of JNK2 interacted with c-Jun better than its beta isoforms,  however the opposite is found for ATF-2.  It is important to note that the p isoform of JNK1  contains the same splice variant as the a isoform of JNK2 (Fig. 1.5), likely explaining their preferential interactions with c-Jun, and suggesting a rationale for a change in their nomenclature. While the in  vivo  relevance of these studies is not clear, the interaction between J N K and its  substrates must be important for  in vivo  substrate selection.  27  J N K has recently been shown to phosphorylate the anti-apoptotic protein Bcl-2 and the transcription factor p53. Bcl-2 and p53 are phosphorylated on the same sites in vivo, as are phosphorylated by J N K in vitro (Hu et al., 1997; Maundrell et al., 1997; Milne et al., 1995). However, whether these phosphorylation events are mediated by J N K in vivo has yet to be determined. The ability of J N K to phosphorylate p53 is supported by the findings that JNK and p53 are constitutively associated in vivo, and the phosphorylation of p53 by J N K stabilizes p53 by preventing its ubiquinitylation, in a similar manner as seen with c-Jun (Fuchs et al., 1998; Fuchs et al., 1998).  1.5.3. Overview of different p38 genes -  The p38 M A P K family is a group of  serine/threonine kinases encoded by four genes; p38oc/CSBP/RK/SAPK2a, p38p/SAPK2b, p38y/ERK6/SAPK3, and p385/SAPK4 (Fig. 1.7). The p38cc and p385 genes are expressed ubiquitously. The p38p gene is highly expressed in the brain, and to a lower degree in most other tissues. The p38y gene is specifically expressed in skeletal muscle. The p38a gene has two splice variants in humans known as CSBPl/p38al and CSBP2/p38a2 (Lee et al., 1994).  These  transcripts are generated by internal splicing of two different exons, without the addition or loss of an exon, as seen with the generation of the alpha and beta isoforms of JNK1 and JNK2. Mxi2 is potentially another splice variant of p38a, and is identical to p38a until sub-domain X I where it terminates after 16 unique amino acids (Zervos et al., 1995). The p38p gene also has two splice variants; p38pi andp38p2, that are generated by the addition of a small exon between subdomain V and VI (Kumar et al., 1997; Stein et al., 1997).  Splice variants encoding other functional  kinases from p38y or p385 have not yet been characterized. However, two transcripts of p388 with premature stop codons are known, but the significance of these splice variants is unknown in vivo (Stein et al., 1997).  1.5.4. A p38 MAPK inhibitor SB 203580 -  In a drug screen at Smith-Kline Beecham, a  compound that blocked the production of TNF and IL-1 by monocytic cells after treatment with  28  I  II  p3 8a p3 83 p3 8y  MSQERP TFYRQELNKTIWEVPERYQNLSPVGSGAYGSVCAAFDTKTGLRVAVKKLSR . . GP . A G V. . . . Q . L . G . R S . Y . ARLRQK . .SPP.ARSG VT. .A. . .RAV.RD.Q A. . S . V . G R . - AK. .1. . . Y . . . L I . - - K K G . .KQDV. . . A . . L . K T . V S P T H S . I . K R S . E K . .1  p3 8a  PFQSIIHAKRTYRELRLLKHMKHENVIGLLDVFTPARSLEEFNDVYLVTHLMGADLNNIV . . . .L. . .R L T.I.D.SE T . . . . ELF . . . A R DET . DD. TDF . . . MPF . . T . . GKLM . . . . E . F . . . A . . . . L Q S . . R N . YDF . . . MPF . QT . . QK . M  p388  I l l  p38P p3 8y p3 88  IV  V  VI  p3 8a p3 8(3  p38P2 p38y p388  VII  KCQKLT DDHVQFLIYQILRGLKYIHSADIIHRDLKPSNLAVNEDCELKILDF . . . AGAHQGARLAL . E V..L G V R.... ...A.S .E V..L G V R....  . H E . .G  E.RI. . .V. .M.K. .R. . .A.G  G  GME-FS  EEKI . Y . V . . M . K  G  *  *  VIII  GW IX  p3 8a  GLARHTDDEMTGYVATRWYRAPEIMLNWMHYNQTVDIWSVGCIMAELLTGRTLFPGTDHI . ...QA.E Q.KA....S.Y. . . . .QA.S V VI. . . .R.T MI . . K . . . K . S . . L A. A V VI . S M. . . K . . . K . K . YL  p3 8al  NQLQQIMRLTGTPPAYLINRMPSHEARNYIQSLTQMPKMNFANVFIGANPLAVDLLEKML D..KL.L..V...G.E.LKKIS.ES D. . K R . . E W . . . S P E V L A K I S . E H . . T PP. . QKDLSSI. R I . . .GR. . D. . K E . .KV E F V Q . L Q . D . .K.NMKG.PELE.KD. . S I L T N . S . . . .N D. . T . . L K V . .V.GTEFVQKLNDKA.KS P.T.RKD.TQL.PR.S.Q.A  p38P p3 8y p3 88  X  p38a2 p3 83 p38y p3 88 p3 8a  p38P p3 8y p3 88 p3 8a  p38P p3 8y p3 88  XI  VLDSDKRITAAQALAHAYFAQYHDPDDEPVAD-PYDQSFESRDLLIDEWKSLTYDEVISF Q.VS..E S E . . . E . E - . . . E . V . AKERTLE . . . E . . . Q . . L . . ...AEQ.V..GE....P..ESL..TE...QVQK-..D..DDV.RTL....RV..K..L.. E. .V. . .L T.PF.EPFR. .EE.TE.QQ.F.D.L.HEK.TV. . . .QHI.K.IVN. VPPPLDQEEMES K..EPPKPPGSLEIEQ K..RQLGARVSKETPL S.IARKDSRRR.GMKL  Figure 1.7 - Alignment of the Isoforms of p38. The predicted amino acid structure of human p38a, p38p, p38y and p38S. The splice variants p38a2 and p38p2 are only indicated where they differ from p38a and p38p respectively. Where residues are identical to p38a they are indicated by periods. Gaps were introduced to optimize the alignment and are indicated by dashes.  29  endotoxic lipopolysaccharide was identified. These drugs belong to a class of pyridinyl imidazoles and are exemplified by SB 202190 and SB 203580 (Fig. 1.8). The target of these compounds is CSBPl/p38al and CSBP2/p38a2 M A P K (Lee et al., 1994).  The drugs inhibit p38a and p38p\  but not p38y and p388, by acting as competitive inhibitors of p38 M A P K (Young et al., 1997). SB 203580 binds to the ATP-binding pocket of p38 M A P K , and inhibits the enzyme in vivo with an IC50 around 1 uM (Lee et al., 1994; Young et al., 1997). The crystal structure of p38 M A P K bound to SB 203580 demonstrates the binding of the drug to the ATP-binding pocket (Fig. 1.9) (Pav et al., 1997; Tong et al., 1997). These crystal structures reveal specific amino acids in the ATP-binding pocket involved in drug interaction. Mutation of three amino acids in p38a (Thrl06  ;  H i s l 0 7 Leu 108) to the corresponding residues in p38y (Met, Pro, Phe) converts p38a M A P K ;  into an SB 203580-resistant kinase without affecting its substrate specificity (Gum et al., 1998). These drugs were not only useful for the identification of p38 M A P K , but also have defined most of our knowledge of the substrate specificity and function of p38 M A P K . These drugs may inhibit Cyclooxygenase-2 and some isoforms of J N K at 10-fold or 40-fold higher concentrations respectively than required to ablate p38 M A P K activity.  1.5.5. Activators and Substrates of p38 MAPK  - The four p38 genes encode protein  kinases with a characteristic T G Y motif in their activation loop. Similar to J N K and E R K M A P K , these enzymes require dual phosphorylation on both threonine and tyrosine residues to be activated (Fig. 1.10).  This phosphorylation event is mediated by the dual specificity kinases M K K 3 or  M K K 6 , and at least in vitro by M K K 4 . M K K 3 and M K K 6 have many splice variants that differ in their length and their activity, with the longest isoforms of each being the most active. These two enzymes activate both p38a or p385 M A P K equally, whereas M K K 6 was more effective at activating p38(3 and p38y M A P K (Cuenda et al., 1997; Han et al., 1996; Jiang et al., 1996; Jiang et al., 1997; Wang et al., 1997b). Several kinases have been suggested to act upstream of M K K 3 and M K K 6 including T A K 1 , M E K K 5 / A S K 1 and M L K 3 (Fig. 1.4).  30  31  p38 M A P K bound to A T P  p38 M A P K bound to SB 203580  Figure 1.9 - Crystal Structure of p38 M A P K bound to A T P or SB 203580. The crystal structure of the ATP-binding pocket was resolved at 2.0 A bound to either ATP (left), or the p38 M A P K inhibitor SB203580 (right). This figure was adapted from Tong et al. 1997.  32  MKK3  MKK6  MAPKAPK5 Mnkl  CHOP  ATF-2 ATF-1 CREB  MAPKAPK2 MAPKAPK3 PRAK  Elk-1 Sap-la  Figure 1.10 - Activators and Substrates of p38 MAPK.  33  Downstream targets for p38a and p38(3 MAPK are fairly well established, however substrates for p38y and p388 are unknown. The p38 MAPK cascade was initially described as a pathway leading to the phosphorylation of Hsp25 and was thought to act through MAPKAP kinase-2 (Freshney et al., 1994). MAPKAP kinase-2, and the related kinase MAPKAP kinase-3, were proven to be substrates for p38a and p38p using SB 203580 (Clifton et al., 1996; McLaughlin et al., 1996). The phosphorylation of MAPKAP kinase-2 and MAPKAP kinase-3 by p38 MAPK occurs on identical residues observed in vivo, and activates these enzymes (Ben-Levy et al., 1995). Recently, p38-regulated and activated kinase (PRAK), or MAPKAP kinase-4, was also identified as a substrate for p38a and p38p MAPK (New et al., 1998). These three enzymes regulate the phosphorylation of Hsp25, an actin capping protein, providing a link between p38 MAPK activity and regulation of the actin cytoskeleton. These isoforms of p38 also regulate the activity of MAPKAP kinase-5 and Mnkl. Mnkl phosphorylates the RNA capping protein, elF4e, and may explain how p38 MAPK activity regulates the translation of several proteins including TNFa (Waskiewicz et al., 1997). As with the JNK pathway, the p38 MAPK pathway also regulates several transcription factors. The CREB family of transcription factors including activating transcription factor (ATF)1, and CREB is regulated by p38a or p38p, whereas ATF-2 is regulated by JNK and p38 MAPK. However, a study analyzing the E-selectin promoter with protein cross-linkers found that JNK was solely bound to c-Jun and ATF-2 was solely complexed with p38 MAPK, suggesting that p38 MAPK solely regulates ATF-2 in vivo (Read et al., 1997). ATF-1 and CREB are also regulated by the p38 MAPK pathway, and by a cAMP-responsive pathway (Tan et al., 1996). The regulation of ATF-1 and CREB is complicated as IL-3, an activator of p38 MAPK, activates ATF1 and CREB through p38 MAPK-independent mechanisms, whereas ceremide activates ATF-1 and CREB solely through p38 MAPK-dependent pathways (Scheid et al., 1999). The differences likely reflect discrete substrate specificities of p38a and p38p MAPK, or cell-specific signalling cassettes implying the existence of discrete pools of ATF-1 and CREB that respond to different stimuli.  34  The p38 MAPK pathway regulates the C/EBP and the Ets families of transcription factors. CHOP/GADD, a member of the C/EBP family of transcription factors, is phosphorylated by p38 MAPK on sites that, when phosphorylated, prevent adipocyte differentiation (Wang and Ron, 1996). The p38 MAPK pathway also regulates Elkl and Sap-la, two Ets family members. These proteins are ternary complex factors for the  c-fos  promoter, and depending on the  extracellular stimuli may be regulated by either p38 MAPK, JNK, or ERK MAPK pathways. Recently, the p38 MAPK and the ERK5 MAPK pathway were shown to regulate myocyteenhanced factor (MEF) 2C, a transcription factor involved in c-Jun transcription. Together, these results provide a mechanism whereby p38 MAPK might regulate AP-1 activity through transcriptional regulation of c-Fos and c-Jun.  1.5.6. Stimuli upstream of p38 MAPK and JNK -  The stress-activated protein kinases  were initially characterized as enzymes activated by physical, chemical or environmental stresses. These stress stimuli include protein synthesis inhibitors such as anisomycin; DNA damaging agents such as cis-platinum, etoposide, or 1-p-D-arabinofuranosylcytosine; UV irradiation; yirradiation; hyperosmolar!ty; heat shock; ischemia; reperfusion; alkalinization or acidification of cells; or free radicals such as hydrogen peroxide. These stimuli are very diverse in nature and the mode of JNK or p38 MAPK activation is not obvious. Other more conventional receptor-mediated activators of the stress-activated protein kinase cascades include the pro-inflammatory cytokines including IL-1 and TNFa, the canonical growth factors such as EGF or PDGF, and the hematopoietic growth factors GM-CSF, IL-3 and SLF.  Ligation of receptors that regulate the  immune system such as the receptor for the Fc fragment of immunoglobulin G or E, the B-cell receptor for antigen, the T-cell receptor, MHC class 1, Fas and CD40 also induce both JNK and p38 MAPK activation. These stimuli are all initiated by receptor dimerization or oligomerization at the cell surface. The sheer diversity of receptors that engage these kinases argues for many diverse biological roles for signalling by these enzymes.  35  The mechanism of JNK or p38 M A P K activation by stress stimuli is under investigation. U V irradiation was believed to activate the stress-activated protein kinases through DNA damage, however JNK is activated by U V irradiation in enucleated cells demonstrating that DNA damage is not required for a cell to respond to U V irradiation (Devary et al., 1993). U V irradiation and hyperosmolarity aggregate receptors on the cell surface, thereby activating J N K (Rosette and Karin, 1996). Pre-treatment of cells with either antibodies against the T N F a or IL-1 receptors to prevent receptor dimerization, or with T N F a or IL-1 to downregulate cell surface expression of their receptors, ablates the activation of J N K by U V irradiation (Rosette and Karin, 1996). Therefore the signal induced by U V irradiation or osmotic shock should vary between cells depending on the expression of different cell-surface receptors. Similarly, U V irradiation-induced apoptosis requires cross-linking of Fas on the cell surface (Rehemtulla et al., 1997). Thus, U V irradiation and hyperosmolarity induced J N K and p38 M A P K activation are, at least in part, receptor mediated events. Protein synthesis inhibitors also activate JNK and p38 M A P K through a mechanism that is presently being elucidated. Translational elongation of proteins involves the sequential binding of aminoacyl-tRNA, transferring of the peptidyl group to the nascent peptide, and translocation of the ribosome. Anisomycin targets the 3'-end of the 28S ribosomal R N A that is directly involved in these three steps and only activates J N K or p38 M A P K in the presence of transcriptionally active ribosomes (Iordanov et al., 1997). Antibiotics and ribotoxic agents that target the 28S ribosomal RNA also activate the JNK pathway in cells with transcriptionally active ribosomes (Iordanov et al., 1997). Together these findings indicate that anisomycin targets the 28S ribosomal R N A to activate the stress-activated protein kinase pathways, and likely explains why cycloheximide, a protein synthesis inhibitor that prevents ribosomal translocation, weakly activates the JNK or p38 M A P K pathway (Shu et al., 1996; Zinck et al., 1995). U V irradiation also damages the 28S ribosomal RNA, indicating that U V irradiation has at least two pathways that activate the stressactivated protein kinase pathways (Iordanov et al., 1998).  36  The activation of JNK and p38 MAPK by heat shock is poorly understood, and appears to require mitochondria and cytoplasmic proteins. Intact cells are not required for activation of JNK as heat shock of cell lysates greatly increases JNK activity (Adler et al., 1995). The overexpression of Hsp70, a chaperone protein involved in protein folding, or the addition of Hsp70 to crude lysates, abrogates JNK and p38 MAPK activation by heat shock (Gabai et al., 1997; Mosser et al., 1997). Conversely, immunoprecipitation of Hsp70 from cell lysates before heat shock enhances JNK and p38 activation (Gabai et al., 1997). Hsp70 maintains protein conformation during heat-shock, and prevents the conversion of procaspases into active caspases after heat shock (Buzzard et al., 1998; Mosser et al., 1997). Hsp70 may prevent the release of Cytochrome C from mitochondria, and therefore heat shock may reflect a sub-lethal activation of caspases leading to the activation of JNK and p38 MAPK. In support of this notion, cells lacking mitochondria fail to activate the stress-activated protein kinases after heat shock (Adler et al., 1995). Reactive oxygen species potently activate the stress-activated protein kinase pathways. TNFa, hydrogen peroxide and UV irradiation all activate JNK through a mechanism requiring reactive oxygen species and are prevented by antioxidants such as N-acetylcysteine (Adler et al., 1995; Gotoh and Cooper, 1998). Interestingly, TNFa and hydrogen peroxide dimerize and activate ASK1 in an anti-oxidant sensitive mechanism (Gotoh and Cooper, 1998).  The  dimerization of ASK1 is likely regulated by its association with thioredoxin. Thioredoxin interacts with the N-terminus of ASK1 inhibiting its kinase activity, and N-acetylcysteine maintains the association of ASK1 with thioredoxin (Saitoh et al., 1998). Together, these studies suggest that reactive oxygen species alter the affinity of thioredoxin for ASK1 promoting its activation, and thereby activating the JNK and p38 MAPK pathways. DNA damaging agents are potent activators of the stress-activated protein kinase pathways, but their mode of action is poorly characterized. 1-p-D-arabinofurosylcytosine or cis-platinum activate the JNK and p38 MAPK pathway downstream of the non-receptor tyrosine kinase c-Abl, as fibroblasts lacking c-Abl fail to activate JNK or 38 MAPK after these treatments (Kharbanda et  37  al., 1995; Pandey et al., 1996). In both instances, ectopic expression of c-Abl restored activation of JNK or p38 M A P K by these chemicals. The D N A alkylating agent methyl methanesulphonate (MMS) differs in its mechanism of J N K and p38 M A P K activation.  M M S activates the stress-  activated protein kinases normally in the absence of c-Abl, however this chemical requires the tyrosine kinase c-Src to activate JNK (Liu et al., 1996a). Receptors involved in immune regulation, including the B cell antigen receptor (BCR), the T cell receptor (TCR), or the Fc receptors (FcR) for IgG or IgE, contain immunotyrosine activation motifs and ligation of these receptors activates several members of the Src tyrosine kinase family, the ZAP70/Syk protein tyrosine kinase family, and the Tec protein tyrosine kinase family. These receptors also activate the stress-activated protein kinases, but how these tyrosine kinases regulate upstream activators of the stress-activated protein kinases is not completely determined. In T cells, ligation of CD3 and CD28 synergizes to activate the stress-activated protein kinases (Salmon et al., 1997; Su et al., 1994). Signalling from Syk, but not Lck, synergizes with Racl to activate J N K (Jacinto et al., 1998). CD28 recruits PI-3 kinase that is required to activate J N K and p38 M A P K by these receptors. The requirement for PI-3 kinase may exist on several levels as the Tec family tyrosine kinases, and all known exchange factors for Rac/Cdc42 contain P H domains. In support of this notion, cells lacking Btk, a Tec family kinase, are deficient in J N K activation downstream of the FcR for IgG and IgE, BCR, SLF and heterotrimeric G-proteins signalling through Gaq subunits (Bence et al., 1997; Kawakami et al., 1997, 1998; Jiang et al., 1998). Btk is required to maintain a sustained calcium flux, that may be required to activate calcineurin (Avraham et al., 1998; Fluckiger et al., 1998).  In this instance, Btk may be activating J N K and p38 M A P K  through the calcium-sensitive non-receptor tyrosine kinase Pyk2 (Tokiwa et al., 1996; Yu et al., 1996), or the calcium/calmodulin dependent protein kinase 4 (Enslen et al., 1996).  The activation  of JNK by the FcR for IgE, or by co-stimulation with CD3 and CD28, also uses a PKC-dependent pathway. The broad-specificity PKC inhibitor, Roche 3, partially blocks the activation of JNK in mast cells, and completely blocks the activation of J N K in mast cells lacking Btk after ligation of the FcR (Kawakami et al., 1998). In T cells,  PKC0 and calcineurin  38  synergize to activate the J N K  pathway (Avraham et al., 1998). These receptors may share a common mechanism for activating the stress-activated protein kinases that is yet to be elucidated.  1.6  Biological Roles of the Stress-Activated Protein Kinases  1.6.1. Role of Stress-Activated  Protein Kinases in Yeast.  Yeast must sense and  respond to osmotic changes to grow and survive. The budding yeast, S. cerevisiae, has evolved two distinct sensors to respond to hyperosmolarity, one involves the histidine kinase receptor Slnlp, and the other involves the transmembrane receptor Sholp. Both sensors activate the Ste7 homolog polymyxin B-sensitive kinase 2 (PBS2), and H o g l , or high osmolarity glycerol-1 (Maedaetal., 1995; Posas and Saito, 1997). H o g l , a homolog of mammalian p38 M A P K , was the first stress-activated protein kinase to be identified in yeast, and its activity is required for the growth of yeast in hyperosmotic media (Brewster et al., 1993). The genome of S. cereviseae is known, and lacks a gene encoding a homolog of mammalian J N K (Hunter and Plowman, 1997). However, both mammalian p38 M A P K and JNK can complement the loss of H o g l , and allow the growth of yeast on hyperosmolarity medium (Han et al., 1994; Kumar et al., 1995). Complementation requires phosphorylation of these enzymes and kinase activity (GalchevaGargova et al., 1994; Kumar et al., 1995). Yeast lacking Pbs2 and Hogl are not complemented by JNK or p38 M A P K alone, indicating that Pbs2 is upstream of p38 M A P K in yeast (GalchevaGargova et al., 1994; Kumar et al., 1995).  Interestingly, only p38al M A P K , but not p38a2  M A P K , is able to complement the Hogl-deficiency in yeast (Kumar et al., 1995). The inability of p38a2 M A P K to rescue Hogl-deficient yeast was unexpected, and is believed to reflect greater activity of p38cc2 M A P K leading to cell cycle arrest (Kumar et al., 1995). The fission yeast S. pombe also express a homolog of mammalian p38 M A P K , Spcl (Shiozaki and Russell, 1995).  Yeast lacking Spcl (Spcl") were identified as suppressors of  lethality due to loss of protein phosphatase 2C. Spcl" are sensitive to hyperosmotic medium and undergo a G2/M cell cycle arrest when grown on limiting nutrients. Expression cloning identified  39  Spel by its ability to rescue Spel" mutants grown on hyperosmotic medium. Another mutant Spc2" was also identified in the same screen as Spel", and was rescued by W i s l , a M A P K kinase (Shiozaki and Russell, 1995).  Yeast lacking Spel" and Spc2" are phenotypically identical,  suggesting these mutations lay on a single pathway (Degols et al., 1996; Shiozaki and Russell, 1995). Furthermore, over-expression of W i s l is lethal in wildtype fission yeast, but not in Spel" yeast. In support of the genetic evidence, W i s l directly phosphorylates and activates Spel in vitro (Shiozaki and Russell, 1995). The over-expression of the tyrosine phosphatases Pypl and Pyp2 induces a similar G2/M cell cycle arrest as seen in the Spel" mutants in hyperosmotic medium (Shiozaki and Russell, 1995). The cell cycle arrest is exacerbated in high osmolality, and does not occur on the Spel" background. Yeast lacking Pypl exhibit an increased phosphorylation of Spel, and the overexpression of Pypl leads to the dephosphorylation of Spel (Shiozaki and Russell, 1995). Yeast lacking both Pypl and Pyp2 fail to proliferate, probably due to excess Spel activity as yeast lacking P y p l , Pyp2 and Spel are viable (Degols et al., 1996; Shiozaki and Russell, 1995).  As seen for p38a2 M A P K in S. cerevisiae, excess Spel activity inhibits  proliferation indicating that p38 M A P K activity can function to either promote or inhibit cell cycle progression in yeast. In summary, W i s l , Pypl and Pyp2 regulate Spel activity, and the activation of Spel is critical for growth during nutrient deficiency and hyperosmolarity.  1.6.2. Role of Stress-Activated Protein Kinases in Drosophila.  Components of the  JNK and p38 M A P K signalling cascades are conserved in mammals and insects.  The best  established model for genetic analysis of the J N K signal transduction cascade is dorsal closure in D. melanogaster. Dorsal closure occurs at mid-embryogenesis and at this time, the dorsal region of the embryo is covered by amnioserosa. The embryonic ectoderm, which is located ventral to the amnioserosa, moves upward and completely covers the embryo. This process does not involve cell division or cell recruitment, but instead the ectodermal cells change shape and move coordinately as an epithelial sheet to surround the embryo (Glise et al., 1995). Hemipterous was identified by saturation mutagenesis looking for lethal mutations that blocked the morphogenetic  40  process of dorsal closure.  Hemipterous  is located on the X-chromosome and encodes dHep, a  Drosophila M A P K kinase (Glise et al., 1995).  A loss of dHep results in second generation  homozygous recessive lethality as maternal gene products from heterozygous parents is sufficient to get homozygous null flies through embryogenesis.  The role of dHep in dorsal closure was  confirmed by reintroducing dHep under constitutive or inducible promoters to rescue the mutant flies (Glise et al., 1995). M K K 7 , and to a much lesser extent M K K 4 , complements the genetic defect in dHep null flies, indicating that M K K 7 and dHep are orthologs (Holland et al., 1997). Drosophila M K K 4 (DMKK4) was recently identified, but its role in dorsal closure has not been addressed (Han et al., 1998). The Drosophila homolog of J N K (DJNK) was also identified in mutants that fail to undergo dorsal closure. In fact, two distinct mutations in the gene encoding DJNK, are known and lead to embryonic lethality.  basket  (bsk),  has a point mutation between sub-domain IX and  Bskl  X that prevents interaction with substrate, while bsk2 has a nonsense mutation introducing a premature stop codon in DJNK (Riesgo-Escovar et al., 1996; Sluss and Davis, 1997).  dHep  phosphorylates and activates DJNK, which in turn phosphorylates DJun. Genetic disruption of DJun also results in a dorsal closure defect that is rescued by over-expressing a constitutively active mutant of DJun (Riesgo-Escovar et al., 1996; Sluss and Davis, 1997).  Taken together,  dHep, D J N K and DJun mediate dorsal closure, and this provides genetic evidence that DJNK phosphorylates DJun in  vivo.  Loss of function mutations of DFos also result in embryonic lethality due to a failure in dorsal closure (Riesgo-Escovar and Hafen, 1997). As with mammalian c-Jun and c-Fos, DJun and DFos heterodimerize to form a functional AP-1 transcription factor. A P I activity is required to induce the expression of decapentaplegic  (Dpp),  a member of the TGFp family (Riesgo-Escovar  and Hafen, 1997). Expression of Dpp is abolished in the leading edge cells of embryos lacking Hep,  Bsk, DJun  or DFos,  and ectopic expression of dpp rescues the dorsal closure defect in  bsk  mutants (Glise and Noselli, 1997; Hou et al., 1997; Kockel et al., 1997; Riesgo-Escovar and Hafen, 1997; Sluss and Davis, 1997).  Furthermore, a constitutively active mutant receptor for  4 1  Dpp rescues embryos lacking DJNK or DJun (Hou et al., 1997; Riesgo-Escovar and Hafen, 1997) . Therefore, JNK signalling is required to induce Dpp expression for dorsal closure during Drosophila embryogenesis. The JNK and the p38 M A P K pathways have also been implicated in Drosophila immunity. As in mammalian systems, insect cells respond to bacterial lipopolysaccharide (LPS) in response to bacterial infection.  LPS induces the expression of a number of antimicrobial gene products  including attacin and cecropin (Han et al., 1998). These proteins are secreted into the hemolymph and function synergistically to lyse invading microorganisms.  Insect cells treated with LPS  activate DJNK as well as two isoforms of p38 M A P K , Dp38a and Dp38b (Han et al., 1998). The role of DJNK in insect immune responses has not been determined due to embryonic lethality. However, SB 203580 also inhibits Dp38a and Dp38b activity and allows analysis of the function of p38 M A P K in Drosophila immunity. Surprisingly, inhibition of p38 M A P K greatly enhances LPS-induced production of message encoding attacin, and to a lesser degree cecropin (Han et al., 1998) . Therefore, p38 M A P K activity appears to attenuate the insect immune response.  1.6.3.  Role of Stress-Activated Protein Kinases in Embryogenesis  and  Hematopoiesis. The generation of mice lacking the genes encoding enzymes in the stressactivated protein kinase pathways has enhanced our understanding of their biological functions. Mice lacking M K K 4 die in  utero  between day 12.5 and 14.5 (Yang et al., 1997b) due to an  inability to undergo normal hepatogenesis (Ganiatsas et al., 1998).  Livers of embryos lacking  M K K 4 are approximately normal size with a reduced total number of cells, that led to hemorrhaging during development (Ganiatsas et al., 1998).  As hematopoiesis in yolk sac  progenitors is normal in day 10.5 embryos, these embryos likely die of anemia from blood loss. Apoptosis in hepatocytes of M K K 4 null embryos was increased about 2-fold indicating an antiapoptotic function for M K K 4 in liver organogenesis (Ganiatsas et al., 1998). Mice with a targetted disruption of c-Jun have a similar defect in liver organogenesis (Hilberg et al., 1993; Johnson et al., 1993). Interestingly, mice deficient in c-Met or HGF die in  42  utero  with similar phenotypes as  the mice lacking M K K 4 or c-Jun (Schmidt et al., 1995). Cross-linking of the met oncogene, the receptor for hepatocyte growth factor (HGF), activates the JNK pathway suggesting a critical role of JNK downstream of c-Met (Rodrigues et al., 1997). Taken together, these studies indicate that M K K 4 regulates c-Jun transactivation findings.  in vivo,  providing genetic evidence to support biochemical  Mice lacking J N K l or JNK2 exhibit defects in cytokine production by T helper  subclasses of C D 4  +  T cells (Dong et al., 1998; Yang et al., 1998), but otherwise are  phenotypically normal, likely reflecting the functional redundancy of these enzymes.  Genetic  disruption of the JNK3 results in a defect in the apoptosis of neuronal cells after treatment with glutamate receptor agonists (Yang et al., 1997a). The genetic disruption of the genes encoding all stress-activated protein kinases is undoubtedly in progress, but has not as yet been described. Chimeric mice produced from MKK4-deficient embryonic stem cells and RAG2-deficient blastocysts have allowed the generation and analysis of T and B lymphocytes lacking M K K 4 . Rag2-deficient mice have a block of CD4-CD8"TCR- thymocyte progenitors to CD4+CD8+TCR+ immature thymocytes, and therefore fail to generate single or double positive T lymphocytes. MKK4-deficient blastocyst chimeric mice exhibit a profound block in the production of double positive T lymphocytes compared to wildtype M K K 4 blastocyst chimeric mice (Nishina et al., 1997) . Loss of M K K 4 sensitizes thymocytes to apoptosis induced by ligation of CD3 or Fas (Nishina et al., 1997). MKK4-deficient thymocytes are also defective in production of IL-2, and proliferation after ligation of CD28 and CD3 (Nishina et al., 1997).  In contrast, disruption of  M K K 4 by targetting either sub-domain VI, or the activation loop fails to reproduce the phenotypes of mice targetted in exon 2 outside the catalytic domain (Ganiatsas et al., 1998; Nishina et al., 1997; Yang et al., 1997b). Furthermore, thymocytes derived from transgenic mice expressing a dominant negative mutant of M K K 4 undergo Fas-induced apoptosis normally (Alberola-Ila et al., 1998) . The reason for these differences is not clear, but may represent other genetic differences between the ES cells used to generate the chimeric mice, or the expression of truncated forms of M K K 4 in ES cells targetting the kinase domain. The MKK4-deficient blastocyst chimeric mice that were targetted in the catalytic domain display lymphadenopathy and plasmacytosis between 2  43  and 6 months of age (Swat et al., 1998). These T and B lymphocytes are polyclonal in nature, and their accumulation only occurred in peripheral lymphoid tissues, suggesting a role for M K K 4 activity in the removal of T and B lymphocytes.  1.6.4. Role of Stress-Activated Protein Kinases in Cytokine Production and Inflammation. - Gene expression is regulated transcriptionally and post-transcriptionally. Posttranscriptional regulation includes mRNA processing, mRNA turnover, and translation.  p38  M A P K was identified as the target of the pyridine imidazole compound, SB 202190, a compound that blocks the production of IL-1 and T N F a in monocytes treated with LPS (Lee et al., 1994). Pyridine imidazole compounds inhibit the production of T N F a at the translational level, while the mRNA encoding TNFa is unaffected (Young et al., 1993). Translation is inhibited by preventing the entry of mRNA encoding T N F a into the polysomal compartment, and the initiation of protein translation (Prichett et al., 1995). Translational initiation is known to be regulated by eIF-4e, that is sequestered in unstimulated cells in a complex with PHAS-1, another substrate for p38 M A P K (Jiang et al., 1996; Wang et al., 1997b). Upon phosphorylation of PHAS-1, eIF-4e is released and initiates translation of specific mRNA (Lin et al., 1994).  M n k l , a kinase activated by p38  M A P K and E R K M A P K , phosphorylates eIF-4e and thereby the activation of the p38 M A P K pathway may co-ordinately derepress eIF-4E by phosphorylating PHAS-1, and activate eIF-4e through M n k l (Waskiewicz et al., 1997).  The role of p38 M A P K in T N F a production is cell-  type dependent. In MC/9 mast cells, the J N K pathway, but not E R K or p38 M A P K , is required for T N F a production after ligation of the Fc receptor for IgE (Ishizuka et al., 1997). However, ligation of the Fc receptor for IgE requires the E R K M A P K pathway, but not J N K or p38 M A P K activity, to produce TNFa in CPU or R B L cells (Csonga et al., 1998; Zhang et al., 1997). Despite the proposed roles of E R K or J N K in the production of T N F a , p38 M A P K regulates the production of T N F a in vivo. The lethality of LPS results from vascular failure associated with excess production of TNFa. Inhibitors of p38 M A P K reduce serum T N F a levels, and prevent the death of mice that have been injected with normally lethal doses of LPS (Badger et al., 1996).  44  The inhibition of p38 M A P K also antagonizes the biological actions of I L - l and T N F a , two pro-inflammatory cytokines that activate J N K and p38 M A P K .  Cells treated with these  cytokines may express IL-6, IL-8, Cyclooxygenase-2, or several metalloproteinases.  The p38  M A P K pathway is required for the production of IL-6, but not IL-8, in human umbilical vascular endothelial cells or gingival fibroblasts (Ridley et al., 1997). In contrast, the expression of IL-8 is regulated by p38 M A P K in peripheral blood mononuclear cells and THP-1 cells after hyperosmotic shock (Shapiro and Dinarello, 1995). This finding demonstrates that the function of p38 M A P K in cytokine production is cell-type dependent. In an animal model of adjuvant-induced arthritis, the production of IL-6 is prevented by SB 203580, demonstrating the importance of p38 M A P K activity for the production of IL-6 in vivo (Badger et al., 1996).  The p38 M A P K inhibitor  prevents the translation of mRNA encoding IL-6 without affecting the message, similar to I L - l or TNFa (Ridley et al., 1997).  These transcripts all contain AU-rich elements in their 3'-UTR,  suggesting these elements may regulate translation through a p38 MAPK-dependent mechanism. Besides cytokines, cells treated with I L - l or T N F a release arachidonic acid (AA) which is required for the biosynthesis of prostaglandins (PG), leukotrienes (LT), and lipoxins (LX). The production of P G is regulated by Cyclooxygenase (COX) -1 and COX-2.  Inhibition of p38  M A P K prevents I L - l induced production of PGE2 and PGF2a at 0.1 uM (Badger et al., 1996). SB 203580 was derived from a series of compounds that inhibit C O X and 5-Lipoxygenase, the enzyme required for L T production, and directly inhibits C O X activity at 10 uM (Ridley et al., 1997). The p38 M A P K inhibitor also prevents the expression of message encoding COX-2 at 0.1 uM, a concentration that specifically inhibits p38 M A P K (Ridley et al., 1997). A A is produced by specific cleavage of phosphatidylcholine by cytosolic phospholipase A2.  This enzyme is  phosphorylated, and possibly activated, by p38 M A P K suggesting that SB 203580 not only inhibits production of prostanoids by blocking transcription of COX-2, but also by inhibiting the biosynthesis of PG, L T and L X by preventing A A release (Borsch-Haubold et al., 1997; Kramer et al., 1996).  45  The p38 M A P K inhibitor reduces many symptoms of inflammation and arthritis, that in large part are due to the actions of T N F a and IL-1.  In murine models of collagen-induced or  adjuvant-induced arthritis, the p38 M A P K inhibitor reduces footpad inflammation and serum amyloid levels (Badger et al., 1996).  Inhibition of p38 M A P K also prevents the loss of bone  mineral density and bone mineral content associated with adjuvant-induced arthritis, possibly by preventing the activation of osteoclasts by IL-1 and T N F a (Badger et al., 1996). Therefore, the p38 M A P K pathway regulates many different inflammatory conditions. The infiltration of leukocytes into sites of inflammation is regulated by the upregulation of adhesion molecules such as E-selectin on endothelial cells after tissue injury. Mice lacking ATF-2 fail to express E-selectin, indicating the importance of ATF-2 for transcription of this protein (Reimold et al., 1996). ATF-2 is regulated by JNK and p38 M A P K in vivo and in vitro (Gupta et al., 1995; L i et al., 1996; Wang et al., 1997). Cells expressing constitutively active M E K K 1 , or treated with T N F a , upregulate E-selectin in both J N K and p38 MAPK-dependent fashions (Min and Pober, 1997; Read et al., 1997).  U V cross-linking studies using the E-selectin promoter  indicate that both ATF-2 and c-Jun constitutively bind to elements in the E-selectin promoter. These studies also indicate that c-Jun interacts solely with J N K , and surprisingly that ATF-2 interacts solely with p38 M A P K (Read et al., 1997). Therefore, the E-selectin promoter provides an example of how two M A P K pathways co-ordinate to regulate gene expression. Besides the production of T N F a , p38 M A P K activity is required for the production of Interferon-y (IFNy). Transgenic mice expressing dominant negative p38 M A P K exhibit reduced expression of IFNy and impaired T h i responses (Rincon et al., 1998).  Similarly, mice  overexpressing dominant active M K K 6 show an increased production of IFNy and a predominant Thi response (Rincon et al., 1998). Mice lacking JNK2 also fail to produce IFNy normally and exhibit a defect in T h i responses (Yang et al., 1998).  JNK1 is also involved in cytokine  production as CD4+ T cells derived from mice lacking JNK1 produce high levels of Th2 cytokines, even when differentiated to the T h i subset (Dong et al., 1998).  A failure in IFN-y  production may also reflect a defect in IL-12 production, as IL-12 induces the expression of  46  interferon-Y (IFNy) in T cells. However, the production of IFNy was found to be regulated directly by p38 M A P K activity as the production of IFNy was completely inhibited by SB 203580 in the presence of exogenous IL-12 (R. Salmon, unpublished observations).  Inhibition of p38  M A P K activity also prevents the production of IL-12 by splenic antigen presenting cells (R. Salmon, unpublished observations). These findings demonstrate the importance of p38 M A P K and JNK activity in regulating not only cytokine production, but also the differentiation of C D 4 T +  cells in mice. In T cells, the ligation of CD3 and CD28 synergizes for the activation of the stress-activated protein kinases and the production of IL-2. T lymphocytes lacking M K K 4 fail to produce normal levels of IL-2, indicating the importance of the JNK pathway in regulating IL-2 production  in vivo  (Nishina et al., 1997). The IL-2 promoter contains AP-1 sites, providing p38 M A P K and J N K responsive elements. The expression and transcriptional activation of c-Jun is directly regulated by JNK, and indirectly regulated by p38 M A P K through MEF2C.  Consistent with this notion,  inhibition of p38 M A P K activity by SB 203580 or by dominant negative M K K 6 inhibits the production of IL-2 at the promoter level (Matsuda et al., 1998). The J N K pathway also regulates the production of IL-2 by stabilizing the mRNA transcript through two cis-acting elements (Chen et al., 1998). In support of this notion, the IL-2 message is specifically stabilized by M E K K 1 or M K K 7 , but not by constitutively active M K K 6 or E R K (Chen et al., 1998).  The increase in  mRNA stability and transcription mediated by JNK and p38 M A P K likely explains the synergistic production of IL-2 after treatment with CD3 and CD28. r  1.6.5. Role  of  Stress-Activated Protein Kinases in  Proliferation and  Tumorigenesis - The stress-activated protein kinases are required for the proliferation of yeast grown in hyperosmotic media.  Similarly, these kinases regulate the proliferation and  tumorigenesis of mammalian cells. D N A synthesis of hematopoietic cells grown in IL-3, SLF, IL4, IL-7 or IL-2 is blocked by the p38 M A P K inhibitor (Crawley et al., 1997, I. Foltz, unpublished data). p38 M A P K may regulate mitogenesis through M A P K A P kinase 2, an Hsp25 kinase.  47  Hsp25 is an actin capping protein, and regulates the formation of F-actin from G-actin monomers (Lavoie et al., 1995). A role for the actin cytoskeleton in mitogenesis is not surprising, and is supported by the ability of cytochalsin D to block cell cycle progression (Sampath and Pollard, 1991). However, an SB 203580-resistant isoform of p38a MAPK restores MAPKAP kinase-2 activity in the presence of SB 203580, but fails to restore growth factor induced DNA synthesis in hematopoietic cells (I. Foltz, unpublished data). Therefore, either p38a MAPK and p38(3 MAPK exhibit different substrate specificities  in vivo,  or another target for SB 203580 is required for  DNA synthesis. The activation of the p38 MAPK pathway can also inhibit proliferation as cells overexpressing MKK3 and p38 MAPK fail to express cyclin Dl or progress through cell cycle. Similarly, the inhibition of p38 MAPK by SB 203580 increases the expression of cyclin Dl in CHO cells (Lavoie et al., 1996). The kinase activity of p38 MAPK also correlates with the arrest of cells in the M phase of the cell cycle. Nocodazole-treated cells arrest in M-phase due to the disruption of spindle fibers, and exhibit constitutive activation of p38 MAPK, but not JNK or ERK MAPK (Takenaka et al., 1998). The inhibition of p38 MAPK activity allows the cells to proceed through into the GI phase of cell cycle, supporting the notion that p38 MAPK negatively regulates progression through cell cycle (Takenaka et al., 1998). A role for the JNK pathway in mitogenesis is expected as ES cells lacking c-Jun are unable to proliferate unless they overexpress the middle T antigen of SV40 (Johnson et al., 1993). Proliferation induced by EGF is also inhibited by dominant negative c-Jun and by anti-sense oligonucleotides against JNKl and JNK2; however, the basal proliferation rates are unaffected. In contrast, inhibition of MEK1 prevents basal proliferation, but not EGF-induced proliferation, suggesting discrete roles for ERK and JNK in basal and growth-factor-induced proliferation respectively (Bost et al., 1997). Similarly, IL-3 induced proliferation is prevented by overexpression of M3/6 phosphatase, a JNK-specific phosphatase, that indirectly implicates JNK with mitogenesis (Smith et al., 1997). These findings support a critical role for JNK and c-Jun in proliferation.  4 8  Transformation and tumorigenesis require the stress-activated protein kinase pathways. Activated Ras potently induces the expression of c-Jun, and the over-expression of a dominant negative c-Jun inhibits Ras-induced transformation. Furthermore, activated Ras is unable to cause a loss of contact inhibition, anchorage independence, or tumorigenicity in fibroblasts lacking the expression of c-Jun (Johnson et al., 1996c).  Another potent transforming protein, v-Met,  constitutively activates the J N K pathway, and J N K activity is required for transformation (Rodrigues et al., 1997). Transformation of cells by the v-Crk oncogene also implicates the activation of J N K with transformation.  Co-expression of v-Crk and the guanine nucleotide  exchange factor C3G potentiates JNK activation, and enhances both the growth rate and anchorage independent growth of NIH 3T3 cells in a JNK-dependent fashion, supporting a role for J N K activity in transformation (Tanaka et al., 1997). The transformation of pre-B cells expressing the oncogene Bcr-Abl also requires the J N K pathway.  Cells transformed by Bcr-Abl exhibit  constitutive J N K and p38 M A P K activity, and the transformation is reversible by ectopic expression of dominant negative c-Jun. Enforced expression of JNK-inhibitor protein 1 (JIP1) also potently inhibits Bcr-Abl induced transformation (Dickens et al., 1997). JIP1 was initially described as a cytoplasmic inhibitor of the J N K pathway that acts by preventing nuclear translocation of J N K (Dickens et al., 1997).  Primary T cell leukemias, or T lymphocytes  transformed with H T L V - 1 , also exhibit constitutive activation of the J N K pathway.  Ectopic  expression of Tax-1, the transactivator of HTLV-1, only activates the JNK pathway in transformed T cells, and simply expressing Tax-1 is insufficient to activate J N K (Xu et al., 1996b). Another virus that transforms  human cells is Kaposi's sarcoma-associated  herpesvirus  (KSHV).  Transformation and tumorigenicity is induced by a constitutively active G-protein coupled receptor (ORF 74) that activates both the J N K and the p38 M A P K pathways, but not the E R K M A P K pathway, correlating the activation of these kinases with transformation (Bais et al., 1998). In contrast, M K K 4 may act as a tumor suppressor based on loss of function mutations in two cell lines derived from pancreatic and lung carcinomas (Teng et al., 1997).  Despite a role in  transformation, the JNK pathway also regulates apoptosis, and perhaps a loss of M K K 4 improves  49  the survival of transformed cells. Together, these findings support a role for J N K signalling in promoting the transformation of mammalian cells by oncogenes and viruses.  1.6.6. Role of Stress-Activated Protein Kinases in Apoptosis -  Apoptosis is the  process of programmed cell death, and cells undergoing apoptosis are characterized by cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation.  Apoptosis removes unwanted cells  without initiating an inflammatory response, and is critical for embryogenesis, organogenesis and for the regulation of the hematopoietic system. Many stimuli that induce apoptosis also activate JNK and p38 M A P K . However, the role of these enzymes in apoptosis is not straightforward. JNK and p38 M A P K were first implicated as effectors of neuronal apoptosis. Apoptosis induced by nerve growth factor is accompanied by an increase in J N K and p38 M A P K activity, and a decrease in E R K M A P K activity (Xia et al., 1995). Neuronal apoptosis is also induced by constitutive M E K K 1 activity in a JNK-dependent fashion, suggesting the J N K pathway is proapoptotic. Constitutive activation of p38 M A P K also weakly induces apoptosis in neuronal cells (Xia et al., 1995). Mice lacking JNK3 provides genetic evidence supporting a pro-apoptotic role of JNK activity in neuronal tissue (Yang et al., 1997).  These mice are resistant to apoptosis of  hippocampal neurons induced by the glutamate agonist kainic acid, but still undergo apoptosis normally during development indicating that JNK activity is not required for all neuronal apoptosis (Yang et al., 1997).  Consistent with these findings, the stimulation of cultured neurons with  kainic acid activates J N K and p38 M A P K , and induces apoptosis that is prevented by the p38 M A P K inhibitor (Kawasaki et al., 1997; Schwarzschild et al., 1997). Neuronal apoptosis is also prevented by insulin through the inhibition of p38 M A P K activity (Heidenreich and Kummer, 1996). Furthermore, the removal of insulin or NGF induces apoptosis in a p38 MAPK-dependent manner (Kummer et al., 1997).  Neuronal apoptosis correlates with the induction of c-Jun and  AP-1 activity (Herdegen et al., 1997), perhaps explaining how both enzymes can mediate neuronal apoptosis as both J N K and p38 M A P K regulate A P I activity (Han et al., 1997a; Hazzalin et al., 1996; Price et al., 1996). A n understanding of the role of JNK signalling in neuronal apoptosis is  50  important for the treatment of stroke patients. Ischemia induces neuronal apoptosis, and potently activates the JNK pathway. A recently identified inhibitor of the J N K pathway prevents apoptosis of cultured rat embryonic motoneurons after ischemia, providing evidence that inhibiting the J N K pathway may prevent the apoptosis of neurons in stroke patients (Maroney et al., 1998). The T N F receptor superfamily encodes proteins that directly influence apoptosis and activate the stress-activated protein kinases. The prototypical death receptor Fas, when crosslinked by Fas ligand, initiates a signal that rapidly induces apoptosis. The cytoplasmic domain of Fas associates directly with both F A D D , or Fas-associated death domain, and D A X X (Medema et al., 1997; Yang et al., 1997c). FADD and D A X X are adaptor proteins that recruit procaspase-8 and ASK1 to Fas (Chang et al., 1998; Ichijo et al., 1997). Signalling through Fas induces two phases of JNK and p38 M A P K activity (Salmon et al., 1997; Toyoshima et al., 1997). The early activation is caspase independent, and likely lies downstream of ASK1 (Toyoshima et al., 1997). The second phase is caspase-dependent and is caused by the process of apoptosis (Juo et al., 1997) . Cross-linking of Fas cleaves procaspase-8 to produce active caspase-8, which activates a cascade of proteases and effectively sentences a cell to death. Several proteins have been identified as substrates of the caspases including Raf-1, F A K , M E K K 1 and P A K . The cleavage of Raf-1 and F A K inactivates their respective signalling pathways in cells, effectively removing antiapoptotic signalling pathways (Widmann et al., 1998). However during apoptosis, the activation of the J N K and p38 M A P K results from the cleavage of M E K K 1 , and maybe P A K 2 , to form constitutively active kinases (Cardone et al., 1997; Rudel and Bokoch, 1997). The expression of dominant negative P A K 2 prevents the activation of J N K by Fas without affecting apoptosis, demonstrating that J N K activity is not required for Fas-induced apoptosis (Rudel et al., 1998). The activation of caspases cleaves M E K K 1 (Deak et al., 1998), and this cleavage product is critical for apoptosis after genotoxic stress and anoikis, the apoptosis that occurs after adherent cells lose their integrin-mediated contacts with the extracellular matrix (Cardone et al., 1997; Widmann et al., 1998) . Constitutively active M E K K 1 potently induces both apoptosis and the J N K pathway; however, co-expression of dominant negative mutants of J N K or c-Jun fails to prevent this  5  1  apoptosis, suggesting the J N K pathway is not critical to this apoptotic event (Johnson et al., 1996b). Similarly, apoptosis induced by Fas is not prevented by inhibiting J N K or p38 M A P K activity (Lenczowski et al., 1997; Salmon et al., 1997). In contrast, ceramide or Fas-induced apoptosis is prevented by over-expressing dominant negative mutants of Ras, Racl, p38 M A P K or JNK in Jurkat cells (Brenner et al., 1997). The apoptotic mechanisms may differ between cells and may partially explain the conflicting data on the role of JNK and p38 pathways in apoptosis. The pro-inflammatory cytokine T N F a also causes the activation of J N K and p38 M A P K pathways as well as apoptosis.  T N F a weakly induces apoptosis and pre-treatment of the cells  with protein synthesis inhibitors is required for effectively cell killing. These inhibitors prevent the induction of anti-apoptotic genes by the transcription factor NF-kB and maybe by transcription factors downstream of JNK or p38 M A P K . Apoptosis induced by TNFa occurs in the absence of JNK or p38 M A P K activity. Cells lacking TRAF2 fail to activate JNK or p38 M A P K , but still die by apoptosis in the presence of T N F a (Lee et al., 1997; Liu et al., 1996; Yeh et al., 1997). In fact, cells lacking TRAF2 are more sensitive to TNFa-induced apoptosis, suggesting a role for JNK in the prevention of apoptosis (Yeh et al., 1997).  Similarly, TNFa-induced apoptosis is  potentiated by inhibition of JNK or p38 M A P K activity by dominant negative mutants of M K K 4 , M K K 6 , or the p38 M A P K inhibitor (Roulston et al., 1998). Apoptosis and activation of the stress-activated protein kinases is also split as dominant negative FADD blocks apoptosis without affecting the activation of JNK or p38 M A P K (Natoli et al., 1997). Therefore, the activation of p38 and JNK by TNFa appears to be cytoprotective in some instances. Other stimuli such as heat shock cause concomitant activation of J N K and p38 M A P K and induction of apoptosis. In fact, cells selected by survival after heat shock are unable to activate JNK (Zanke et al., 1996). The overexpression of dominant negative M K K 4 also makes cells resistant to heat shock induced-apoptosis (Zanke et al., 1996). Therefore in this instance, J N K activity promotes apoptosis.  Heat shock potently drives the expression of heat shock proteins  (Hsp) including Hsp70 (Wu et al., 1985). Cells expressing high levels of endogenous Hsp70 fail to activate JNK or p38 M A P K after heat shock, and fail to undergo heat shock induced apoptosis  52  (Buzzard et al., 1998; Gabai et al., 1997; Meriin et al., 1998; Mosser et al., 1997). However the ectopic expression of Hsp70 does not prevent J N K or p38 M A P K activation by heat shock, even though it protects cells from heat shock-induced apoptosis (Buzzard et al., 1998; Mosser et al., 1997). These findings are contradictary, and suggest that JNK or p38 M A P K activity may provide a permissive signal for apoptosis only in the proper cellular context. Activation of J N K and p38 M A P K often correlates with apoptosis, and while their activation is not a death sentence, it may provide a permissive signal for apoptosis.  These  pathways either directly phosphorylate proteins that modulate apoptosis, or indirectiy regulate the expression of proteins that regulate apoptosis. The anti-apoptotic protein Bcl-2 is a substrate for JNK, but not p38 or E R K M A P K (Maundrell et al., 1997). Over-expression of a constitutively active mutant of Racl induces the phosphorylation of Bcl-2 on the identical sites that are phosphorylated by J N K in vitro. The constitutively active mutants of Racl or Cdc42 are strong inducers of apoptosis in a JNK-dependent fashion (Chuang et al., 1997).  Bcl-2 is believed to  inhibit caspases, and prevent the release of cytochrome C from mitochondria.  The  phosphorylation of Bcl-2 by J N K may prevent the inhibition of caspases by Bcl-2, or prevent its interaction with pro-apoptotic Bcl-2 family members, thereby promoting apoptosis.  A role for  phosphorylation of Bcl-2 was suggested in a mutant strain of WEHI-231 cells, that is unusual as it fails to undergo apoptosis after treatment with anti-IgM antibodies. The resistance to IgM-induced apoptosis was due to a deletion in Bcl-2 that removed the putative J N K phosphorylation sites (Chang et al., 1997).  Together, these findings suggest a role for J N K signalling in Bcl-2  dependent apoptotic pathways. The tumor suppressor p53 is also phosphorylated by J N K on a site that is known to be phosphorylated in intact cells (Hu et al., 1997; Milne et al., 1995). p53 constitutively associates with J N K (Hu et al., 1997), and the association of inactive J N K with p53 promotes its ubiquitinylation (Fuchs et al., 1998a).  In the absence of J N K activity, p53 and Mdm2 are  constitutively associated, with Mdm2 also targeting p53 for ubiquitinylation and destruction (Fuchs et al., 1998b).  The phosphorylation of p53 by J N K dissociates Mdm2 and p53, thereby  53  stabilizing p53 and increasing its transcriptional activity (Fuchs et al., 1998b). The importance of p53 phosphorylation in apoptosis is unclear.  However, the co-transfection of p53 and  constitutively active M E K K 1 into cells lacking p53 potentiates apoptosis compared to cells transfected with M E K K 1 alone, suggesting a role for J N K in the promotion of p53-dependent apoptosis (Fuchs et al., 1998b). The stress-activated protein kinases may also regulate apoptosis by upregulating proapoptotic proteins. The expression of Fas ligand (FasL) is increased by D N A damaging agents such as etoposide, an activator of JNK and p38 M A P K pathways (Kasibhatla et al., 1998). The ectopic expression of NFkB, or c-Fos and c-Jun is sufficient to drive the FasL promoter through kB or AP-1 sites respectively (Kasibhatla et al., 1998). Therefore, J N K or p38 M A P K activity may induce expression of FasL.  Consistent with this notion, over-expression of constitutively  active M E K K 1 induces the expression of Fas ligand through the AP-1 site in its promoter, and dominant negative c-Jun or ATF-2 prevents the expression of FasL (Faris et al., 1998). The expression of Fas ligand provides a mechanism by which the stress-activated protein kinases regulate gene expression to promote apoptosis. The JNK and p38 signalling pathways also prevent apoptosis in primary tissues. lacking M K K 4 die in  utero  Mice  of a failure in liver organogenesis that is due to the increased apoptosis  of hepatocytes (Ganiatsas et al., 1998). Furthermore, T lymphocytes lacking M K K 4 are more sensitive to apoptosis induced by crosslinking of CD3 or Fas (Nishina et al., 1997a). reports suggest a role for M K K 4 in the prevention of apoptosis  in vivo.  These  Mice lacking M K K 4 also  exhibit splenomegaly and lymphadenopathy that becomes evident with age. The cells represent a polyclonal expansion of both B and T-cell pools, suggesting a failure in the apoptosis of peripheral lymphocytes. In contrast, the inhibition of p38 M A P K has no effect on activation-induced cell death in T lymphocytes treated with anti-CD3 antibodies, or the prevention of apoptosis by CD28 (Salmon et al., 1997). U V irradiation potently induces apoptosis, and cells lacking c-Fos or c-Jun are more sensitive to UV-irradiation induced apoptosis (Haas and Kaina, 1995; Schreiber et al.,  54  1995). Together, these reports demonstrate that the J N K and p38 M A P K pathways serve a cytoprotective function in normal cells. Therefore it appears the stress-activated protein kinases may provide pro-apoptotic, or antiapoptotic signals in a cell-dependent and a stimulus-dependent fashion. These observations may be explained in part by the presense of other signals regulating apoptosis.  For example,  hematopoietic growth factors activate the stress-activated protein kinases (Foltz et al., 1997; Foltz and Schrader, 1997; Nagata et al., 1997a,b). These cytokines function to prevent apoptosis, further demonstrating that activation of JNK and p38 M A P K is not simply a signal for apoptosis. However, these cytokines also activate PI-3 kinase, A K T and the E R K M A P K pathways, and perhaps either JNK or p38 M A P K activity would provide an apoptotic stimulus in the absence of these anti-apoptotic pathways.  55  CHAPTER 2 - MATERIALS AND METHODS  Methods Used In Chapter 3 -  Antibodies and reagents. The anti-p38 M A P kinase anti-serum used for immunoprecipitation was raised against full-length CSBP2 as described (Lee et al., 1994). The anti-MAPKAP kinase-2 antibody (06-534) and anti-phosphotyrosine antibody 4G10 (05-321) were purchased from Upstate Biotechnology (Lake Placid, N Y ) . The anti-p38 M A P kinase antibody (sc-535) used for western blotting, the anti-p90 k antibody (sc-231), and the truncated ATF-2 (1-96) was rs  purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-p38 M A P kinase specific antibody (921 IS) was from New England Biolabs (Beverly, M A ) , the recombinant murine Hsp25 was from StressGen Biotechnologies (Victoria, BC), the myelin basic protein (MBP) was from Sigma (St. Louis, MO), and the recombinant murine CSF-1 was from R & D Systems (Windsor, ON). The monoclonal antibody 5A1, specific for CSF-1, was provided by H . Ziltener (BRC, Vancouver). The antibody 2.4G2 (anti-FcR) that binds to and blocks the Fey receptor II and III without inducing signalling was purchased from Pharmingen (San Diego, CA), and the polyclonal rabbit anti-rat Ig antibody (anti -Ig) used to crosslink the anti -FcR antibodies on the cell surface to induce FcR mediated signalling was a gift of Dr. H . Ziltener (Vancouver, BC). RPMI 1640 was purchased from Canadian Life Technologies (Burlington, ON) and fetal calf serum (FCS) was purchased from Intergen (Purchase, N Y ) . Recombinant SLF was provided by James Wieler and synthetic cytokines were provided by I. Clark-Lewis (BRC, Vancouver).  Cell culture and stimulation conditions.  RPMI 1640 was supplemented with 10% F C S ,  10 uM 2-mercaptoethanol (2-Me) and 100 Units of Penicillin/Streptomycin prior to using for cell culture. Primary mast cells were derived by culturing bone marrow cells from (C57BL/6 x DBA/2) F i hybrid mice in RPMI 1640 supplemented with 2% WEHI-3B conditioned medium as a source of IL-3 and 3% X063-mIL4 conditioned medium as a source of IL-4 for 3 weeks as  56  described (Welham et al., 1992). The factor-dependent hematopoietic cell lines MC/9 and Ba/F3 were passaged in RPMI 1640 supplemented with 2% WEHI-3B conditioned medium. FD-5/13R and FD-MACII cells were passaged in RPMI 1640 supplemented with 2% L-cell conditioned medium (a source of CSF-1) as previously described (Welham et al., 1994, Orchansky et al, 1998). HU-3 cells were cultured in RPMI 1640 supplemented with 3% gibbon IL-3 conditioned medium. Prior to stimulation, cells were cultured overnight in 0.2% IL-3 conditioned medium, 7 washed 3 times with phosphate-buffered saline (PBS) and then incubated at 10 cells/mL in serum-free  medium buffered  with  10 m M HEPES  (N-[2-hydroxyethyl]piperazine-N'-[2-  ethanesulfonic acid]) p H 7.2, for 1 hr. Cells were stimulated with the following doses of recombinant or synthetic growth factors: IL-4 - 10 ng/mL; G M - C S F - 10 ug/mL; IL-3 - 10 ug/mL; TNFa - 50 ng/mL. For FcR cross-linking, MC/9 mast cells were initially incubated with anti-FcR for 10 min and then were incubated for a further 10 min either untreated or with the addition of anti-Ig to cross-link the a-FcR antibody.  A l l stimulations had been shown in preliminary  experiments to give maximal levels of tyrosine phosphorylation of the respective receptors. Immunoprecipitation conditions. Cells extracts were prepared in solubilization buffer (50 m M Tris, pH 7.5, 150 m M NaCl, 5 m M EDTA, 1% (v/v) Nonidet P-40 (NP-40), 1 mM sodium molybdate, 200 m M sodium orthovanadate, 1 m M sodium fluoride, 50 m M p-glycerol phosphate, 10 ng/mL aprotinin, 10 ug/mL soybean trypsin inhibitor, 0.7 ug/mL pepstatin, 2 ug/mL leupeptin, and 40 ug/mL phenylmethylsulfonyl fluoride). To monitor the levels of tyrosine phosphorylation in the control and the treated samples, a portion of each cell lysate was routinely resolved by SDSP A G E and immunoblotted with the anti-phosphotyrosine antibody 4G10 (0.1 ug/mL). The remaining lysate was subjected to immunoprecipitation and the precipitate was analysed by immunoblotting or kinase assays.  5 7  Protein-kinase assays. p38 M A P kinase activity was measured using an immune complex kinase assay with a truncated form of ATF-2 as a substrate. The cell lysate was mixed with an anti-p38 M A P kinase anti-serum and 20 pL packed volume of protein-A Sepharose beads. After 2 hrs the beads were washed extensively with solubilization buffer and once with kinase assay buffer (25 m M HEPES pH 7.2, 25 m M magnesium chloride, 2 m M dithiothreitol, 0.5 m M sodium vanadate, and 25 pM ATP). The kinase reaction was initiated by the addition of 20 uL of kinase assay buffer containing 2 ug of ATF-2 and 10 pCi of [Y-^^P]ATP, and stopped after 20 min at 30°C by the addition of SDS-PAGE sample buffer. To determine the effect of the p38 M A P kinase inhibitor SB 203580 on the in vivo kinase activation of p38 or E R K M A P kinases, we investigated the effects of SB 203580 on activation of their respective putative downstream targets M A P K A P kinase-2 and p90 ^rs  We incubated 10^  cells/mL with or without 1 pM SB 203580 for 20 min prior to stimulation. To assay in vivo activation of p38 M A P kinase, M A P K A P kinase-2 was precipitated using 5 pg of anti-MAPKAP kinase-2 and 20 pL packed volume of protein-G Sepharose. The kinase assay was performed as described above, except that 5 pg of recombinant murine Hsp25 was used as substrate. To assay in vivo activation of the E R K M A P kinase, p 9 0 ^ was immunoprecipitated from aliquots of the rs  same cell lysate using 5 pg of anti-p90 k antibody bound to 20 pL of packed protein-G rs  Sepharose. The kinase assay was initiated by the addition of 20 pL of kinase assay buffer (20 mM HEPES, 5 m M magnesium chloride, 1 mM E G T A , 5 m M mercaptoethanol, and 2 m M sodium vanadate) containing 3 pg of M B P and 10 pCi of [y-^PJATP, and stopped after 10 min at 30°C by the addition of SDS-PAGE sample buffer. In all kinase assays the phosphorylated proteins were resolved by SDS-PAGE and visualized by autoradiography.  D N A synthesis - D N A synthesis was assayed by incorporation of H-thymidine. Cells were cultured in Terasaki plates at 200 cells per well with indicated doses of growth factors and SB 203580. After 24 or 48 hrs, cells were pulsed with H-thymidine for 4 hrs and then frozen to lyse  58  the cells. The lysate was harvested onto glass filters and rinsed once with both water and ethanol. 3  The amount of H-thymidine incorporated into D N A was determined by scintillation counting.  Methods used in Chapter 4 Preparation of G S T - c - J u n  - The N-terminus of c-Jun (1-169) fused to glutathione S-  transferase was expressed in bacteria and purified by affinity chromatography on glutathione Sepharose 4B beads (Pharmacia Biotech Inc., CA) accordingly to the manufacturers instructions. Briefly, a 5 mL culture of E. coli strain UT5600 containing the plasmid encoding GST-c-Jun was grown in 2xYT broth containing 100 ug/mL ampicillin overnight.  This culture was used to  inoculate a 1 L culture of 2xYT broth containing 100 |j,g/mL ampicillin the next day and incubated at 37°C until the culture reaches an OD 600 of 0.5-0.7. The culture was then placed at 4°C for 30 min followed by the addition of isopropylthiogalactoside to a final concentration of 0.1 m M . The induced culture was then incubated at 26°C for another 4 hrs. The cells were pelleted at 4k rpm for 10 min and the supernatant was discarded. The bacterial pellet was resuspended in 10 mL of resuspension buffer (5 mM Tris, pH 7.5, 150 m M NaCl, 5 m M 2-Me, 10 ug/mL of soybean trypsin inhibitor and 40 ug/mL of PMSF). The bacteria were then treated with 50 uL of 10 mg/mL lysozyme and incubated on ice for 30 min. The remaining bacteria were lysed with successive freeze/thaw cycles between a dry ice/ethanol bath and 37°C waterbath (the lysate was aliquoted into 2-3 tubes of approximately 4 mL each to speed the process). The lysate was then incubated with 50 uL of 1 M MgCl2 and 50 uL of 10 mg/mL DNAasel until the solution no longer appeared viscous. To improve the yield of the GST-c-Jun, 1 mL of 10% (v/v) NP-40 was added to the preparation and incubated at 4°C for 30 min. The lysate was then centrifuged at 15k ipm for 30 min.  The supernatant was retained and frozen in 1 mL aliquots at -70°C.  Aliquots were  subsequently thawed and incubated with 250 uL of a 50% slurry of glutathione Sepharose beads in a 15 mL Bio-Rad column at 4°C for 2 hrs. The supernatant was then eluted from the column and rinsed 3 times with ice-cold l x PBS containing 0.5 % NP-40, 100 m M NaCl and 5 mM 2-Me.  59  GST-c-Jun was then eluted from the glutathione beads in 5 aliquots (150 uL) of elution buffer (20 mM Glutathione, 150 m M Tris, pH 7.5, and 5 m M 2-Me).  The eluates were analyzed by  Coommassie Blue staining after SDS-PAGE to determine yield.  Antibodies. The antibodies specific for isoforms of JNK1 (sc-474) or JNK2 (sc-827) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-MKK4 specific antibody (915IS) was purchased from New England Biolabs (Beverly, M A ) .  Protein kinase assays. The kinase activity of S A P K was measured using an assay with GST-c-Jun as a substrate.  in vitro  kinase  The cell lysate was mixed with GST-c-Jun and 20 uL  packed volume of glutathione Sepharose beads. After 2 hrs the beads were washed extensively with solubilization buffer and once with kinase assay buffer (25 m M HEPES pH 7.2, 25 mM magnesium chloride, 2 mM dithiothreitol, 50 m M (3-glycerol phosphate and 0.5 mM sodium vanadate).  The kinase reaction was initiated by the addition of 20 uL of kinase assay buffer  containing 10 uCi of [y- P]ATP, and stopped after 20 min at 30°C by the addition of SDS-PAGE 32  sample buffer. For in-gel kinase assays, the cell lysate was mixed with an antibody specific for either JNK1 or JNK2 and protein A-sepharose beads for 2 hours. The beads were washed extensively in solubilization buffer and the immunoprecipitated proteins were eluted by boiling in SDS-sample buffer. The samples were analysed by SDS-PAGE using a 10% separating gel containing 1 mg of bacterially-expressed GST-c-Jun. The gel was then sequentially washed twice in buffer A (20% isopropanol and 50 m M Tris, pH 8) for 30 min, twice in buffer B (50 m M Tris, pH 8 and 5 m M 2mercaptoethanol) for 30 min, once in buffer C (6 M guanidine HCI, 50 m M Tris, pH 8 and 5 mM 2-Me) for 1 hour, several times in cold buffer D (0.05% (v/v) Tween-20, 50 m M Tris, pH 8 and 5 m M 2-Me) over 15-18 hours, and once in kinase buffer (40 m M HEPES, pH 7.2, 2 m M DTT, 15 m M magnesium chloride, 1 m M manganese chloride, 0.3 m M vanadate and 0.1 m M EGTA) for 30 min. The kinase assay was initiated by the addition of 20 mL of kinase buffer containing 100 uCi  60  of [y-32p]ATP and incubated for 1 hr. The gel was then rinsed with buffer E (5% trichloroacetic acid and 1% sodium pyrophosphate) repeatedly until radioactivity was undetectable by geiger counter. The gel was then dried using a gel dryer and phosphorylated GST-c-Jun was detected using autoradiography.  Methods used in Chapter 5 -  Molecular Cloning of MKK7.  An Expressed-Sequence Tag (EST) clone (aaO 19720)  containing the 3' end (464 bp) of M K K 7 was identified by screening the EST database (University of Washington-Merck EST project) with a degenerate oligonucleotide sequence based on a sequence conserved in other M K K s [a.a. F(x) F(x) CLxK(x) R(x) H]. We used oligonucleotide 6  6  4  8  primers based on this M K K 7 sequence to amplify the 5' end of the cDNA from a human fetal kidney R A C E cDNA library using Vent DNA polymerase for polymerase chain reaction (PCR). PCR products were randomly subcloned into pBSKS and blue-white colony screening, white colonies were selected.  A l l white colonies were transferred onto nitrocellulose and Southern  blotted with the EST fragment that had been radiolabeled by random priming.  We used this  technique to obtain a further 861 bp of M K K 7 a that contained an in-frame stop codon as well as a splice variant of M K K 7 (hMKK7(3). The cDNA encoding full length M K K 7 a was cloned from at least 2 separate PCR reactions and was sequenced on both strands. We used degenerate primers based on the human sequence to amplify the murine cDNA from Ba/F3 hematopoietic cell cDNA. The EST database was screened to identify a third splice variant of murine M K K 7 (mMKK7y). Published databases were screened for M K K s related to M K K 7 using the National Center for Biotechnology Information Advanced B L A S T search program.  Nucleotide sequence accession numbers. The nucleotide sequences presented in this thesis have been submitted to GenBank under the following accession numbers: h M K K 7 a - AF013588; hMKK7(3 - AF013589; m M K K 7 a - AF022112 and m M K K 7 y - AF022113. The M K K from C.  61  elegans  (cMKK7) was previously submitted to GenBank as part of the  C.  elegans  genome  sequencing project (Wilson et al., 1994) and was given the accession number U38377 (gene K08A8.1).  Preparation of Competent E. coli - A stab culture of E.  coli  (DH5a or UT5600 strains) was  incubated in 5 mL of 2xYT or L B broth at 37°C overnight. This culture was used to innoculate 1 L of medium the next morning. The cells were incubated at 37°C until the optical density (OD) 550 reached 0.3 to 0.4 (2 to 3 hrs). The cells were chilled at 4°C for 45 min and then pelleted at 4k rpm for 10 min at 4°C (Sorvall GS3 rotor). The cells were resuspended in cold 100 mM CaC12 and left on ice for 30 min. The cells were then pelleted as before and resuspended in 10 mL of cold 50 mM C a C l , and left on ice for 4 to 24 hrs. The cells were dispensed in 250 uL aliquots 2  and were frozen in a dry ice/ethanol bath after the addition of 1 mL of autoclaved 75% glycerol.  Transformation of E. coli - A n aliquot of heat-shock competent  E. coli  was thawed slowly on  ice for 30 min. The desired plasmid (1 uL) or ligation mixture (5 uL) was added to 100 uL of competent cells and left on ice for 45 min. The cell/DNA mixture was heat-shocked at 42°C for 1 min and 35 sec and then placed on ice for 2 min. Generally 2xYT broth (1 mL) was added to the heat-shocked cells and the cells were allowed to recover for 30 min at 37°C.  The cells were  pelleted at 4 k rpm for 30 sec and the supernatant was removed. The cells were resuspended in 200 uL of 2xYT and were plated onto either 2xYT or L B plates containing Ampicillin (40 ug/mL).  Purification of plasmid D N A - For the purification of D N A from small cultures (1 mL) of E. coli  containing plasmids smaller than 10 kbp the TELT method was typically used. The cells were  pelleted at 14k rpm for 1 min at room temperature and the supernatant was discarded. The cells were resuspended in 100 uL of TELT buffer (2.5 M L i C l , 50 mM Tris, pH 8.0, 4% (v/v) Triton X-100, 62.5 m M E D T A , pH 8.0) and an equal aliquot of TE (50 m M Tris, pH 8.0, 1 mM EDTA)saturated Phenol:Chloroform:isoamylalcohol (50:49:1 ratio).  62  This mixture was vortexed  vigorously for 1 min and then centrifuged at 14k rpm for 1 min. The upper phase containing the plasmid D N A was removed and added to 300 uL of 100 % ethanol. The D N A was precipitated at 70°C for 10 min and then pelleted at 14k rpm for 10 min at 4 °C. The ethanol was removed and the pellet was rinsed with 70% ethanol before the pellet was resuspended in 20 uL of TE containing 10 pg/mL RNAase A .  The STET method was used for plasmids larger than 10 kbp. The cells were pelleted at 14k rpm for 1 min and the supernatant was discarded. The cells were resuspended in 200 pL of STET buffer (8 % Sucrose, 20 mM Tris, pH 8.0, 50 m M EDTA, pH 8.0, 0.5 % Triton X-100) and boiled for 1 min. The mixture was centrifuged at 14k rpm for 5 min at 4°C and the pellet was removed from the bottom of the tube with a toothpick.  A 350 pL aliquot of TE and then  Phenol/Chloroform was added to the lysate and mixed vigorously. The mixture was centrifuged at 14k rpm for 5 min at 4°C and the upper phase (550 pL) containing the plasmid D N A was transferred to a new eppendorf tube. An aliquot (350 pL) of chloroform was added to the upper phase and vortexed vigorously. The mixture was centrifuged as before and the upper phase (500 pL) was removed and added to 1 mL of 100% ethanol in a clean eppendorf tube. The DNA was precipitated at -70°C for 10 min and then was pelleted at 14k rpm for 10 min at 4°C.  The ethanol  was removed and the pellet was rinsed with 70% ethanol before the pellet was resuspended in 20 uL of TE containing 10 pg/mL RNAase A . For medium scale purifications (100 mL cultures) of plasmid D N A to be used for DNA sequencing, the Qiagen method was used. The bacterial pellet was resuspended in one volume (4 mL for Q100 column; 10 mL for Q500 column) of Buffer PI (50 m M Tris, pH 8.0, 10 mM EDTA, 100 pg/mL RNAase A). One volume of Buffer P2 (200 m M NaOH, 1 % SDS) was added to the cells, mixed gently and the mixture was left at room temperature for 1 min. One volume of chilled Buffer P3 (3 M potassium acetate, pH 5.5) was added to the bacterial lysate and mixed gently. The insoluble material was removed by centrifugation at 4k rpm for 10 min at room temperature. The supernatant was filtered through a cheese cloth onto an equilibrated Q100 or  63  Q500 column and was eluted by gravity. The resin in the column was rinsed twice with Buffer QC (1 M NaCl, 50 m M MOPS, pH 7.0, 15% ethanol) and the bound plasmid D N A was then eluted with 5 mL of Buffer QF (1.25 M NaCl, 50 m M Tris, pH 8.5, 15% ethanol). An aliquot (3.5 mL) of isopropyl alcohol was added to the eluant and the D N A was left to precipitate at room temperature for 30 min. The D N A was pelleted at 4k rpm for 10 min at room temperature and the D N A pellet was rinsed with 70% ethanol twice before the pellet was resuspended in 100 uL of TE. The concentration of the D N A was determined by measuring the absorbance at 260 nm (where 1 OD is approximately equal to 50 ug/mL plasmid DNA). The cesium chloride method was used for large scale purifications (1 L cultures) of plasmid D N A to be used for transient and stable transfections. Briefly, the bacterial pellet was resuspended in 10 mL of Buffer 1 (25 m M Tris, pH 8.0, 10 mM EDTA, pH 8.0, 50 m M glucose, 5 mg/mL lysozyme). The cells were lysed by the addition of 20 mL of Buffer 2 (0.2 N NaOH, 1% SDS) and left on ice for 10 min. The lysate was neutralized by the addition of 15 mL of ice cold Buffer 3 (3 M potassium/ 5 M acetate) and left on ice for 10 min. The insoluble material was removed by centrifugation at 4k rpm for 10 min at room temperature. The supernatant was filtered with a cheese cloth and the D N A was precipitated by the addition of 0.6 times total volume of isopropyl alcohol. After 10 min at room temperature the DNA was pelleted by centrifugation at 4k rpm for 10 min at room temperature. The supernatant was discarded and the pellet was dried upside down. The pellet was resuspended in 8 mL of TE and then mixed with 8.8 g of cesium chloride and 400 uL of ethidium bromide. The solution was transferred to ultracentrifuge tubes and centrifuged at 60k rpm for 4 hrs at 15°C. The D N A was eluted from the tubes and was ultracenlrifuged as before. The lower D N A band was removed from the ultracentrifuge tube using a 21 gauge needle into a 50 mL conical tube. The eluant was diluted up to 7.5 mL with TE. The ethidium bromide was repeatedly extracted (5-6 times) with an equal volume of TE-saturated butanol until the TE (lower phase) appeared colourless. The T E containing the plasmid D N A was diluted to 10 mL with T E and then 200 uL of 5 M NaCl and 20 mL of 100% ethanol were added to precipitate the DNA. The D N A was pelleted at 4k rpm for 10 min at room temperature and the D N A pellet was  64  rinsed with 70% ethanol twice before the pellet was resuspended in 500 uL of TE.  The  concentration of the D N A was determined by taking an OD 260 as above.  Restriction analysis of plasmid D N A - Restriction analysis was used routinely for subcloning of D N A fragments. Typically about 1-2 ug of DNA in a total volume of 17 uL of water was used for each digestion. Then 2 uL of the appropriate 10 x Reaction buffer and 1 uL of the Restriction enzyme were added to the D N A solution. The reaction was generally incubated at 37°C for 1-2 hrs (1 Unit of restriction enzyme cuts 1 ug of D N A in an hr). The reactions were stopped by the addition of 5 uL of 5x Stop buffer (0.13% bromophenol blue, 0.13% xylene cyanol, 15% glycerol).  Reaction products were analyzed by separation through an agarose gel by  electrophoresis.  Ligations - After digesting the vector and desired insert with the appropriate restriction enzyme, the D N A fragments were run out on an agarose gel. The gels were stained with ethidium bromide and visualized with low U V light. The D N A fragments were excised from the gel and the DNA was extracted from the agarose using the Qiagen gel extraction kit. Briefly the gel was solublized in 600 uL of QX1 buffer at 55°C for 10 min. The mixture was then applied to a Qiagen gel extraction column and spun at 14k rpm for 1 min. The eluate was discarded and the column was rinsed with 750 uL of Buffer PE by centrifugation as before. The eluate was discarded and the column was re-centrifuged as before to remove all the remaining wash buffer.  The column was  then routinely left for 5 min to allow the column to dry before eluting the bound DNA with 25 uL of elution buffer (10 m M Tris, pH 8.5). A sample of the vector and the insert were then routinely analyzed on an agarose gel as before to determine the relative quantities of vector and insert. The ligations were then set up with a 5:1 ratio of insert to vector. The D N A mixture was brought up to a total volume of 8 uL and the reaction was initiated after addition of 1 uL of lOx Ligase buffer by the addition of 1 uL of T4 D N A Ligase. The reaction was left at 16°C for either 1 hr (sticky  65  overhang ligation) or 4 hrs (blunt ended ligation). The ligation was then transformed into competent bacteria by heat-shock as described above.  Blue/White colony selection - PCR products or other blunt ended D N A fragments were typically sub-cloned into the multiple cloning region of the pBluescript phagemid (pBSKS). The multiple cloning region of pBSKS is within the coding region of the a-subunit of (3-galactosidase and ligation of an insert into pBSKS will disrupt the reading frame of this enzyme. A l l transformed E. coli strain DH5a were plated onto 2xYT plates containing Amp and coated with 50 uL of 2% (w/v) Bluo-Gal (a compound related to galactose and conjugated to a blue chromophore) and 10 uL of 200 mg/mL Isopropyl-thiogalactoside (IPTG). The DH5a strain of E. coli only lack the a-subunit of p-galactosidase and cannot cleave galactose unless transformed with pBSKS encoding an intact a-subunit (a-complementation). P-galactosidase cleaves Bluo-gal to release the normally colorless chromophore and as a result the E. coli colonies that lack insert turn blue. Colonies failing to change color were mini-prepped and analyzed for the presence of the proper insert.  Oligonucleotide primers - Many oligonucleotide primers were used for the cloning and sequencing of M K K 7 . The sequences of the oligonucleotides involved in the RACE-PCR, RTPCR and epitope tagging are detailed below:  IF111: (anti-sense): 5'- G A T GTC A T A GTC C G G CTT -3' IF113: (anti-sense): 5'- T C A C G G C C G GTC TTC G C C A T G A C A TCC T -3' IF115: (anti-sense): 5'- T G A GCT C C A T G G C G A T G A A G A -3' IF117: (anti-sense): 5'- TGC C C A T G A GCT C C A T -3' IF122: (sense): 5'- A T C G G A TCC C A G C G C T A C C A G G C A G A -3' IF129: (anti-sense): 5'- T A C TCT A G A T C A GTC TTC GCC A T G A C A TC -3' IF134: (sense): 5'- C A T G G A TCC G G G A A A A T G G C G G C G T -3'  66  API (sense): 5'- C C A T C C T A A T A C G A C T C A C T A T A G G G C -3' AP2 (sense): 5'- A C T C A C T A T A G G GCT C G A G C G G C -3'  The underlined nucleotides represent the presence of a Restriction site introduced into the sequence to facilitate sub-cloning.  cDNA constructs. Human M K K 7 was amplified by PCR using oligonucleotides IF134 and IF129 to allow subcloning into the BamHl and Xbal sites in the pEFBOS-Nmyc3 vector to produce pEFBOS-Nmyc3-MKK7.  Oligonucleotide IF134 introduces an in-frame BamHl site at  the 5'-end and oligonucleotide IF129 introduces a Xbal site at the 3'-end of the cDNA encoding M K K 7 . Similarly human M K K 7 was amplified by PCR using oligonucleotides IF134 and IF113 to allow subcloning into the BamHl and Eagl sites in the pEBG vector to produce p E B G - M K K 7 . Oligonucleotide IF113 introduces an Eagl site in the cDNA of M K K 7 , and a partial Eagl digest of the PCR product was required for subcloning into pEBG as the cDNA encoding M K K 7 contains an Eagl site within its open reading frame. The pEBG vector was produced by digesting pEBGSEK1 with BamHl to release the cDNA encoding SEK1 followed by ligating the empty vector together. The vectors encoding p E B G - J N K l and p E B G - S E K l were the gifts of Dr. L . Zon. The mammalian expression vector encoding Flag-tagged CSBP2/p38 M A P K was provided by Dr. Peter Young. The vectors encoding constitutively active Ras  K61  V12  , Rac  V12  and Cdc42  were  received from Dr. R. Kay, Dr. F. McCormick and Dr. R. Cerione respectively.  Polymerase Chain Reaction (PCR) - PCR was used extensively for the cloning of M K K 7 from a RACE-cDNA library, to introduce restriction sites to facilitate sub-cloning and to analyze expression of mRNA encoding M K K 7 from several cells and cell lines. A PCR reaction mixture included l x ThermoPol Buffer, 200-250 uM of dNTP, 75 uM of each the sense and anti-sense oligonucleotides, and the D N A template in a 50 uL reaction volume. All PCR reactions were heated to 94°C for 5 min in a Perkin Elmer D N A Thermal Cycler and initiated by the addition of 1  67  uL of Vent DNA Polymerase. Reactions were carried out either for thirty cycles if cloning from cDNA derived from total R N A or ten cycles if amplifying the PCR products from plasmid D N A . One cycle involved heating the reaction up to 94°C for 45 seconds, cooling the reaction to 50-55°C for 45 seconds to allow the oligonucleotides to anneal, and then heating the reaction to 72°C for 1 min to allow elongation of the PCR products. All PCR products were visualized after separation on agarose gels by staining with ethidium bromide.  Southern blot analysis - Southern blotting was used to screen white colonies derived from 5'R A C E PCR products for the presence of an insert encoding M K K 7 .  White colonies picked and  streaked onto 2xYT plates. The colonies were allowed to grow for several hours and then were replica plated onto nitrocellulose. The nitrocellulose was soaked for 15 min in denaturation buffer (0.5 M NaOH, 1.5 M NaCl), dried and then soaked for 15 min in neutralization buffer (0.5 M Tris, pH 7, 1.5 M NaCl). The nitrocellulose was then baked at 80°C for 1 hr. The nitrocellulose was then soaked in wash buffer (0.1% SDS and 2x SSC) and the remnants of the bacterial colonies were scraped off the nitrocellulose.  The nitrocellulose was then blocked in pre-  hybridization buffer (33% deionized formamide, 40 mM sodium phosphate, pH 6, 4x SSC (20x Stock: 3 M NaCl, 0.3 M Sodium citrate, pH 7.0), 4 x Denhardt's solution (50x Stock: 1% Ficoll, 1% polyvinylpyrrolidone, and 1% BSA), 0.8% glycine and 125 ug/mL herring sperm DNA).  The  membrane was left to block for 20 min and then placed in hybridization buffer (33% deionized formamide, 25 m M sodium phosphate, pH 6, 4x SSC, 10% Dextran Sulphate, 100 ug/mL herring sperm DNA) with radioactive probe at around 1 million cpm/mL for 1 hr to over-night in a 42°C hybridization oven. The probe was removed and the nitrocellulose was rinsed 3 times in wash buffer (0.1% SDS, 2x SSC) at 42°C. The temperature was raised slowly from 42°C to 60°C in 3 degree increments. The radioactivity present on the membrane was analyzed at each increment and washing continued until radioactivity was no longer detectable on the membrane using a Geiger counter (typically around 55°C). The membrane was then wrapped in saran wrap and exposed to o  autoradiographic film at -70 C in a film cassette with intensifying screens.  68  Probes were routinely prepared as follows using approximately 200 ng of DNA for each Klenow reaction. The D N A was brought up to 27 pL in water and then boiled for ten min. The DNA was then immediately placed onto ice followed by the addition of 15-18 uL of oligo mix lacking dCTP (10 uL of 1 M HEPES, pH 6.6, 0.5 uL of 1 M MgCl2, 0.5 uL of 0.1 M DTT, 2 uL of 2.5 m M dNTP, 3 uL of 5 mg/mL random hexamers and 2 uL of 10 mg/mL BSA), 2 uL of Klenow D N A Polymerase (10-20 Units) and 5 uL of P - d C T P (50 uCi). The reaction was 32  incubated for 1-4 hours at room temperature before stopping by addition of 130 pL of l x SET (10 mM Tris, pH 7.5, 5 mM EDTA and 1% SDS) and 20 uL of 0.25% bromophenol blue. The radiolabeled probe was separated from unincorporated radioactivity over a Sephadex G-50 column and the eluate was boiled for 5 min before addition to the hybridzation solution at the desired concentration.  Northern Blot Analysis  - A Northern blot of mRNA from multiple human tissues was  purchased from ClonTech Biotechnology (San Diego, CA). The blot was hybridized with a probe from the 5' end of M K K 7 [1-660 bp] that was radiolabelled by random priming as above. The probe was derived from pEFBOS-Nmyc3-MKK7 by restriction digestion with BamHl and Ncol. Hybridization and washing were performed according to manufacturer's specifications and the mRNA encoding M K K 7 was visualized using autoradiography.  Preparation of GST-JNK1 - The prokaryotic expression vector for GST-JNK1 was produced from p E B G - J N K l by restriction digestion with BamHl and NotI followed by subcloning the cDNA encoding JNK1 into the in-frame BamHl and NotI sites in the polylinker of pGEX4T-3. Both GST-JNK1 and GST-c-Jun were expressed in E. coli strain UT5600 and were purified by affinity chromatography on glutathione Sepharose beads as described in Chapter 4.  Purification of total RNA from mammalian cells - Typically 1 x 1 0 cells were 6  centrifuged at 14k rpm for 1 min and the supernatant was discarded. For adherent cells a 6 cm  69  dish at 50% confluency was used. The cells were then resuspended in 1 mL of Trizol (Gibco B R L , Grand Island, N Y ) . If the Trizol appeared cloudy then 500 uL of the mixture was transferred to a new eppendorf and another 500 uL of Trizol was added. The mixture was diluted with 200 uL of chloroform and vortexed for 1 min. The samples were centrifuged at 14k rpm for 10 min at 4°C. The top aqueous phase (350 uL) was transferred to a new eppendorf tube and the R N A was precipitated with 500 uL of isopropyl alcohol. This was generally left at -20°C for one hr to overnight. The R N A was then pelleted by centrifugation for 10 min at 14k rpm and 4°C. The pellet was rinsed once with 70% ethanol and resuspended in 25 uL of distilled water. The concentration of the R N A was determined by taking an absorbance at 260 nm (where 1 OD is approximately equal to 40 ug/mL of total RNA).  Reverse transcription - To investigate the expression of mRNA encoding M K K 7 , total R N A from various cell lines was reverse transcribed using an anti-sense oligonucleotide present in both human and murine M K K 7 for subsequent RT-PCR (Table 5.4). Briefly, 1.5 pig of total R N A was primed with the oligonucleotide IF113 at a final concentration of 20 pmol/uL in a 10 uL reaction volume.  The mixture was heated to 70°C for 10 min and chilled on ice to allow the  oligonucleotides to anneal. The following reagents were added to the RT reaction (4 uL of First strand synthesis buffer (250 m M Tris-HCl, pH 8.3, 375 m M potassium chloride, and 15 mM magnesium chloride), 2 uL of 0.1 M dithiothreitol, 1 uL containing dTTP, dATP, dCTP and dGTP at 10 mM, and 1 uL of Moloney Murine Leukemia Virus-Reverse Transcriptase) and the resulting mixture was incubated at 37°C for 1 hour.  Conditions for Transient Transfections in adherent cells - HeLa cells were transiently transfected using SuperFect reagent (Qiagen, CA) according to manufacturer's recommendations. Briefly, HeLa cells were plated in a 100 mm dish, such that the cells would be at 50-80% confluency the next day.  The desired combination of D N A (10 ug total) was combined and  brought up to 300 uL in serum-free, antibiotic-free medium D M E M . The D N A solution was then  70  mixed with 60 uL of SuperFect Transfection Reagent and incubated for 5 to 10 min at room temperature to allow complexes to form. During this time the cells were rinsed once with l x P B S . Then 3 mL of D M E M containing 10% FCS and antiboitics was combined with the D N A mixture, and immediately transferred to the cells. The cells were incubated with the DNA complexes for 2 hrs at 37°C before the supernatant was replaced with normal growth medium. Often the cells from a single transfection were split into several smaller dishes and used to analyze over-expressed proteins with several different stimuli. The cells were harvested 20-24 hrs post-transfection.  Conditions for Transient Transfections in non-adherent cell lines - Transient transfection of Ba/F3 cells was routinely performed to analyze the activation of exogenously expressed M K K 7 by hematopoietic growth factors.  Ba/F3 cells (1-1.5 x 10 /sample) were 7  transiently transfected with 15-25 ug of D N A using electroporation (300 V , 960 uF) in 400 uL of serum-free medium containing 10 pg/mL of DEAE dextran (Pharmacia Biotech Inc., C A ) . After electroporation, the cells were placed in 20 mL of room temperature RPMI 1640 containing 2% IL3 conditioned medium and incubated for 20 min at room temperature before returing the cells to the 37°C incubator. The cells were harvested 20-24 hrs post-transfection.  Conditions for Stable Transfections in non-adherent cell lines - Ba/F3 clones that stably expressed M K K 7 were generated by co-electroporation of a vector encoding myc-tagged M K K 7 (pEFBOS-mycMKK7) and a vector encoding a puromycin resistance gene (pGK-puro). Cells were plated in 96 well microtiter plates at 1.5 x 10 cells/well and selected in 1 pg/mL 4  puromycin (Calbiochem, CA). After two weeks, we observed 90 wells with drug-resistant clones. We selected 3 wells at random and recloned cells by limit dilution. Clones arising from each of these wells expressed readily detectable levels of myc-tagged M K K 7  as determined by  immunoblotting lysates with an anti-Myc monoclonal antibody 9E10 (Santa Cruz, CA).  Antigen preparation for immunization of rabbits - Peptides corresponding to the N -  7 1  terminus and the C-terminus of M K K 7 were synthesized as C-terminal fusions with the T-cell epitope from Tetanus Toxin (Valmori et al., 1992). The sequence of the N-terminal peptide was O Y I K A N S K F I G I T E L K K S S L E O K L S R L E A K L K O E N R E A R R and the C-terminal peptide was OYIKANSKFIGITELKKKDVMAKTESPRTSGVLSOP.  The New Zealand White rabbits were  initially injected with 250 ug of peptide in complete Freund's Adjuvant. The second injection (167 Ug peptide) and all subsequent injections (83 ug peptide) were prepared with incomplete Freund's adjuvant. The appropriate dose of antigen was dissolved in 1 mL of PBS and added to 2 mL of Freund's adjuvant. The adjuvant was emulsified through 18-gauge needles on glass syringes and left at 4°C overnight. The rabbits were injected intramuscularly for the initial injection and subcutaneous for all subsequent injections. The rabbits received two injections before the first ear bleed and then the rabbits were either injected or bled every second week. The N-terminal peptide or the C-terminal peptide was injected into rabbits IF3/IF4 or IF5/IF6 respectively.  ELISA Protocol - The antigen (Ag) was dissolved at 10 ug/ml in 0.5% sodium azide in PBS and was stored at 4°C.  Antigen was coated onto non-sterile 96 well microtitre plates at 50 uL of  Ag to each well of the plate using a multichannel pipetter. All samples were analyzed in triplicate ie. coating 3 lanes per sample. The ELISA trays were stored in a tupperware container with damp paper towels covering the bottom of the container. The plates were incubated at room temperature from 2 to 24 hrs to ensure efficient binding of the antigen to the plate. The Ag was removed from the wells, and the wells were blocked with 0.5% skim milk powder dissolved in l x PBS (about 1 L per plate to be washed). Briefly the milk solution was added to the tupperware container until it covered the ELISA plate (ensuring all the wells are filled). The milk solution was removed and the plate was dried on a paper towel. The block and dry steps were repeared two more times and the ELISA plate was air dried upside down on a clean paper towel. The antibody samples were typical diluted 1:100 for first bleeds and 1:200 for all subsequent bleeds. The antiserum was diluted in 0.5% milk in PBS and antibodies were incubated in the wells for 1 to 24 hrs in a tupperware container. Generally the antibodies were diluted in 1:2 steps. Every column of the plate contained  72  50 uL of 0.5% milk except the first column to which 100 uL of the appropriate dilution of antiserum in 0.5% milk in PBS was added. A Socorex pipettor was used to transfer 50 uL from the first well to the second well, from the second well to the third well, etc. leaving the last column as a control containing 0.5% milk alone. The primary antibody was removed from the wells and the plate was rinsed three times in 0.5% milk in l x PBS as before.  A secondary Horseradish  peroxidase (POD)-coupled Goat anti-Rabbit antibody (DAKO) was used for detection of primary antibody bound to the ELISA plate. The POD antibody was diluted 1:2000 in 0.5% milk in PBS and 50 ul was added to each well. The secondary antibody was incubated for 1 to 2 hours at room temperature in the tupperware container. The secondary antibody was removed from the wells and the plate was rinsed three times in 0.5% milk in PBS followed by three washes with distilled water (dH20). The citrate buffer was prepared by dissolving 1.29 grams of citric acid and 1.37 grams of Na2PO4»2H20 into 100 mL of dH20.  The substrate solution (1 mg/mL A B T S , 0.006%  hydrogen peroxide in citrate buffer) was freshly prepared before use. The substrate solution (50 uL) was added to each well and the plate was incubated for 30 min at room temperature.  The  absorbance of each well was determined with an ELISA plate reader using dual mode between OD 405 and OD 490.  Antibodies. The anti-MKK4 polyclonal antibody (sc-964) used for immunoprecipitation and kinase assays was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The M2 (cc-Flag) monoclonal antibody was obtained from Intersciences (Markham, ON).  Rabbit polyclonal  antibodies recognizing M K K 7 were raised against peptides corresponding to the N-terminus (a.a. 4-26) and the C-terminus (a.a. 394-413) of M K K 7 . Anti-MKK7 antibodies were purified from serum of rabbits IF4 and IF6 using protein A-Sepharose.  Immunoblotting procedures were  performed as described previously (Duronio et al., 1992; Welham et al., 1994; Welham and Schrader, 1992).  Cell Culture Conditions. The factor-dependent hematopoietic cell lines MC/9 and Ba/F3 were  73  passaged in RPMI 1640 supplemented with 2 % WEHI-3B conditioned medium. HeLa cells were grown in D M E M supplemented with 10% FCS and 10 pM 2-mercaptoethanol.  The B-cell  lymphoma cell line WEHI-231 was cultured in RPMI 1640 supplemented with 10% FCS and 10 pM 2-mercaptoethanol.  Stimulation and Immunoprecipitation  Conditions. Prior to stimulation, Ba/F3 or MC/9  cells were cultured in 0.2% WEHI-3B conditioned medium for 16 to 20 hrs. Cells were washed and  equilibrated  in serum-free  RPMI  1640  buffered  with  10  m M HEPES  hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), pH 7.2 prior to stimulation.  (N-[2-  Cells were  stimulated with 0.2 M NaCl, 50 pg/mL anisomycin, 100 ng/mL T N F - a , 100 ng/mL E G F , or optimal doses of synthetic IL-3 or synthetic IL-4 (10 pg/mL) for the indicated times. For FcR cross-linking, MC/9 mast cells were initially incubated with anti-FcR for 10 min and then were incubated for a further 10 min either untreated or with the addition of anti-Ig to cross-link the antiFcR antibody. For U V irradiation, HeLa and Ba/F3 cells were exposed for 20 min in 60 mm dishes containing 1 mL of serum-free medium from a 30 W U V light. HeLa cells were heat shocked by incubating for 20 min at 42°C.  After stimulation, cells were lysed in solubilization  buffer (50 m M Tris, pH 7.5, 150 m M NaCl, 200 m M L i C l , 5 m M E D T A , 1% (v/v) Nonidet P-40, 1 mM sodium molybdate, 50 mM sodium orthovanadate, 1 m M sodium fluoride, 50 m M (3glycerol phosphate, 10 pg/mL aprotinin, 10 pg/mL soybean trypsin inhibitor, 0.7 ug/mL pepstatin, 2 pg/mL leupeptin, and 40 pg/mL phenylmethylsulfonyl fluoride). Samples for subsequent assays were normalized for total protein using a Pierce protein assay or for expression of transfected proteins by assaying co-transfected (3-galactosidase activity.  Proteins tagged with GST were  affinity precipitated by incubating cellular extracts with 20 pL of a 50% slurry of glutathione Sepharose beads for 1 hr at 4°C. Endogenous M K K 7 was immunoprecipitated using the rabbit polyclonal antibody raised against the N-terminus of M K K 7 and endogenous M K K 4 was immunoprecipitated with an oc-MKK4 antibody. Flag-tagged p38 M A P K was immunoprecipitated using the M 2 antibody and protein G-Sepharose.  74  MKK7 Kinase Assays.  Beads with associated kinases were washed extensively with  solubilization buffer and once with kinase assay buffer (25 m M HEPES pH 7.2, 25 mM magnesium chloride, 2 m M dithiothreitol, 50 mM (3-glycerol phosphate and 0.5 mM sodium vanadate). M K K 7 activity was measured directly by the ability of M K K 7 to phosphorylate 1 ug of GST-JNKl in the presence of an excess of free GST protein to prevent the substrate from binding to the beads. M K K 7 activity was also assessed indirectly by first incubating M K K 7 with 1 ug of GST-JNKl in the presence of 50 uM unlabelled ATP for 30 min and then removing an aliquot and determining the activity of GST-JNKl in an in vitro kinase reaction using 1 ug of GST-c-Jun as substrate. A l l kinase reactions were initiated by the addition of kinase assay buffer containing the appropriate substrate and 10 uCi of [y-32p]ATP, and stopped after 20 min by the addition of SDSsample buffer. For p38 M A P kinase assays, Flag-tagged p38 M A P K was immunoprecipitated using a-Flag monoclonal antibodies and protein-G Sepharose beads. The beads were washed and kinase activity was assessed as described above. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography.  Densitometry was performed using an HSI scanning  densitometer.  75  CHAPTER 3 - Activation of p38 MAPK in hematopoietic cells.  3.1  Introduction  Our interest in p38 M A P K began with the work of Dr. Melanie Welham and her studies on tyrosine phosphoproteins induced by interleukin-3 and Steel locus factor in primary bone marrow mast cells. Using 2-dimensional gel electrophoresis and the monoclonal antibody 4G10, she observed phosphoproteins unique to each stimulus, and several phosphoproteins induced by both growth factors (Fig. 3.1). Phosphoproteins unique to each cytokine were observed at the predicted electrophoretic mobility of their respective receptors. Interestingly both stimuli induced phosphoproteins at 70 kDa (p70), 55 kDa (p55), 46 kDa (p46), 44 kDa (p44), 42 kDa (p42) and 38 kDa (p38). Subsequent work identified p70 as the tyrosine phosphatases SHP1 and SHP2, p55 and p46 as different isoforms of She, and p44 and p42 as the M A P kinases, ERK1 and ERK2. However the tyrosine phosphorylated p38 kDa protein remained to be identified. Several independent groups identified murine p38, a new M A P K family member, as the mammalian homolog of H O G l of S. cereviseae. Dr. Peter Young and Dr. John Lee at SmithKline Beecham also identified CSBP2, the human p38 M A P K , as the target for a pyridinyl imidazole compound that inhibits I L - l and TNFa production. They demonstrated that p38 M A P K ran at the same electrophoretic mobility and isoelectric point as the p38 phosphoprotein observed by Dr. Welham.  Hypothesis: (1) The tyrosine phosphoprotein p38 was p38 M A P K , and that its activity would be regulated by both IL-3 and SLF in normal bone marrow mast cells. (2) The activity of p38 M A P K is required to activate M A P K A P kinase-2, an enzyme previously demonstrated to be activated by IL-3 and GM-CSF. (3) The activity of p38 M A P K is required for some biological functions of these cytokines.  76  SLF - 2 min  Untreated I  ^^ki  I  IL-3 - 1 0 min M W >T*"—- T  '  'TP  »  -200  •116 -97 -66  *  ....  + -  + -  ~ ' +  Figure 3.1 - Analysis of phosphoproteins induced by IL-3 and S L F using 2 D S D S - P A G E . Murine bone marrow-derived mast cells were lyzed after being treated with SLF or IL-3 for the indicated times. Whole cell extracts were analyzed using 2 dimensional SDS-PAGE, and the proteins were immunoblotted with an anti-phosphotyrosine antibody 4G10. The position of the phosphoprotein putatively corresponding to p38 M A P K is indicated by arrowheads. This figure was adapted from Welham et al., 1992.  77  Objectives: (1) We initiated a collaboration with Dr. Young to determine if p38 M A P K was indeed the same p38 phosphoprotein. Dr. Young provided us with antisera that recognized full-length p38 M A P K , that would be used to immunoprecipitate p38 and determine if it was tyrosine phosphorylated and activated by IL-3 and GM-CSF. (2) Dr. Young also provided us with the specific inhibitor of p38 M A P K , SB 203580. We would use this inhibitor to determine if M A P K A P kinase-2 was indeed a substrate of p38 M A P K or the E R K M A P K , which were previously identified as activators of M A P K A P kinase-2. (3) Hematopoietic growth factors are required for the proliferation, differentiation and the prevention of apoptosis in hematopoietic cells. We would test the role of p38 M A P K in these biological functions using the p38 M A P K inhibitor to acutely deprive a cell of p38 M A P K activity.  3.2  3.2.1  Results  Identification  of p38 MAPK  - an unknown phosphoprotein.  To determine  whether p38 M A P kinase was involved in responses to IL-3 or SLF, we stimulated primary bonemarrow derived mast cells with these factors, immunoprecipitated p38 M A P kinase and assessed its tyrosine phosphorylation by immunoblotting with the anti-phosphotyrosine specific antibody 4G10.  As expected, both S L F and IL-3 induced tyrosine phosphorylation of p38 M A P kinase  (Fig. 3.2).  3.2.2  Activation of p38 MAPK by IL-3, GM-CSF or SLF but not IL-4 or IL-13.  To demonstrate that the tyrosine phosphorylation of p38 M A P kinase in cells treated with hematopoietic growth factors correlated with increased kinase activity, we immunoprecipitated p38 M A P kinase from either untreated MC/9 cells, or cells treated with saturating doses of G M - C S F , SLF or 0.2 M NaCl. We then assessed the activity of p38 M A P kinase in an using a truncated form of ATF-2 as substrate (Fig. 3.3).  78  in vitro  kinase assay  p38 M A P kinase immunoprecipitated  -  2  10 10  5  min IP:  anti-p38  IB: 4G10 IB: a n t i - 3 8  Figure 3.2 - I L 3 or S L F , but not I L - 4 , induce tyrosine phosphorylation of p38 M A P K in bone marrow-derived mast cells. Cells were incubated at 37 °C in RPMI 1640 for 1 hr and then left untreated ( C O N ) , or treated with saturating doses of IL-3 (IL-3), IL-4 (IL4), or SLF ( S L F ) for the indicated times. Immunoprecipitated p38 M A P kinase was analyzed after SDS-PAGE by immunoblotting (IB), initially with the anti-phosphotyrosine antibody 4G10 ( 4 G 1 0 ) , and subsequently with an anti-p38 M A P kinase antibody (anti-p38) to quantitate loading. The position of p38 M A P K is indicated by an arrow.  79  £p  cf <f #  IP: anti-p38  £ <J> ATF-2 IB:  4G10  IB: anti-p38  Figure 3.3 - Activation of p38 M A P K by hematopoietic growth factors i n M C / 9  cells. Cells were incubated in RPMI 1640 at 37 °C for 1 hr. Equal numbers of cells were then left untreated (CON), or stimulated with saturating doses of IL-4 (IL-4), murine GM-CSF (GM) or SLF (SLF) or 0.2 M NaCl (NaCl) for the indicated times. The cells were lysed and p38 MAP kinase was immunoprecipitated with anti-p38 MAP kinase anti-serum. The relative kinase activity was assessed by an immune complex kinase assay using a truncated form of ATF-2 as substrate. SB 203580 (10 uM) was included in one sample (SB) to inhibit the in vitro kinase activity of p38 MAP kinase. The reaction products were resolved by SDS-PAGE and transferred onto nitrocellulose. Phosphorylation of ATF-2 was assessed by autoradiography (Top). The membrane was immunoblotted (IB) initially with the anti-phosphotyrosine antibody 4G10 (4G10) to assess tyrosine phosphorylation of p38 MAP kinase (Middle) and with an anti-p38 MAP kinase antibody (anti-p38) to assess loading (Bottom).  80  from cells stimulated with G M - C S F or S L F exhibited increased levels of kinase activity, which correlated with the levels of tyrosine phosphorylation of the enzyme. The activity of p38 M A P kinase was completely abolished by the inclusion of SB 203580 during the in vitro assay (Fig. 3.3). To examine the kinetics of S L F and G M - C S F induced activation of p38 M A P kinase in MC/9 cells, we used an antibody specific for the activated form of p38 M A P kinase. This antibody recognizes p38 M A P kinase when phosphorylated on the tyrosine of the T G Y activation motif. The activation of p38 M A P kinase is dependent on dual phosphorylation on both threonine and tyrosine residues as previous demonstrated (Doza et al., 1995; Raingeaud et al., 1995). Treatment with growth factors resulted in a rapid increase in the levels of tyrosine phosphorylation of p38 M A P kinase (Fig. 3.4). Cells treated with S L F exhibited detectable phosphorylation of p38 M A P kinase as early as 2 minutes and maximal phosphorylation of p38 M A P kinase between 5 and 10 minutes. Cells treated with GM-CSF had maximal phosphorylation of p38 M A P kinase at around 10 minutes. In both instances the phosphorylation of p38 M A P kinase was transient and returned to almost basal levels by 30 minutes. It was of particular interest to investigate the effect of IL-4 as we had previously shown that it failed to activate E R K M A P kinases (Welham et al., 1992).  IL-4 failed to induce tyrosine  phosphorylation of p38 M A P kinase in primary mast cells (Fig. 3.2), in MC/9 (Fig. 3.3) or in FD5/13R (Fig. 3.5). As predicted from these results, IL-4 also failed to stimulate enzymatic activity of p38 M A P kinase (Fig. 3.3).  Interestingly, treatment with IL-4 resulted in a small but  reproducible reduction in both the levels of tyrosine phosphorylation and the enzymatic activity of p38 M A P kinase (Fig. 3.3) compared with untreated cells. The related cytokine IL-13 also failed to induce tyrosine phosphorylation of p38 M A P K (Fig. 3.5). Thus, both IL-4 and IL-13 fail to activate either p38 M A P K or E R K M A P K (Welham et a l , 1995)  3.2.3  Activation of p38 MAPK by CSF-1 in FD-MACII  cells.  To test the effects of  CSF-1 or G M - C S F on the activity of p38 M A P kinase, we used murine factor-dependent cell lines that respond to these factors (Welham et al., 1992; Welham et al., 1994).  8  1  Stimulation of the  Min:  0 2 5 10 30 60  GM-CSF •  GM-CSF  SLF  o 73  SLF  c  o 0  IB: anti-phospho-p38  2  5  10  30  60  Minutes  Figure 3.4 - Kinetics of tyrosine phosphorylation of p38 M A P kinase by G M - C S F or S L F . After 1 hr in RPMI 1640 at 37 °C, MC/9 cells were left untreated, or were stimulated with saturating doses of G M - C S F or S L F for the indicated times. The cells were lysed and the NP-40 soluble fraction was analysed by SDS-PAGE followed by immunoblotting using an antibody specific for activated p38 M A P kinase. Levels of activation of p38 M A P kinase as detected by this antibody were quantitated using densitometry.  82  •  >  p p  IB: anti-phospho-p38 IB: 4G10  Figure 3.5 - IL-13 fails to activate p38 M A P Kinase in F D 5 / I L 1 3 R c e l l s .  Cells were incubated in RPMI 1640 at 37 °C for 1 hr. Equal numbers of cells were then left untreated (CON), or stimulated with saturating doses of IL-4 (IL-4), IL-13 (IL-13) or 0.2 M NaCl (NaCl) for 10 min. The cells were lysed and the cellular lysate was resolved by SDS-PAGE and transferred onto nitrocellulose. The membrane was immunoblotted (IB) initially with the anti-phospho-p38 antibody (anti-phospho-p38) to assess tyrosine phosphorylation of p38 MAP kinase (right). The membrane was also immunoblotted with 4G10 to demonstrate the cells responded to IL-4 and IL-13 (left).  83  macrophage-like FD-MACII cells with CSF-1 resulted in rapid tyrosine phosphorylation of p38 M A P kinase (Fig. 3.6). This effect was seen following treatment with either recombinant murine CSF-1  or a source of natural murine CSF-1, L-cell conditioned medium.  The tyrosine  phosphorylation of p38 M A P kinase was abrogated by the presence of a monoclonal antibody that neutralizes CSF-1 activity (Fig. 3.6), demonstrating that the induced phosphorylation of p38 M A P kinase was due to CSF-1 and not, for example, to contamination by endotoxin or to osmotic stress.  3.2.4  Activation  Immunoglobulin  of p38 G.  MAPK  by cross-linking  of the Fc Receptor  for  Besides hematopoietic growth factors, MC/9 mast cells also respond to  ligation of the Fc receptor for Immunoglobulin G (FcR). We used two approaches to determine if p38 M A P K was tyrosine phosphorylated after cross-linking of the FcR. The first involved the use of antigen»antibody complexes, and the second involved cross-linking an anti-FcR antibody bound to FcR on the cell-surface with a secondary antibody against a-FcR (a-Ig). We found that treating MC/9 cells with either K L H (Ag) or an antibody to K L H (Ab) failed to induce tyrosine phosphorylation of p38 M A P K . However, tyrosine phosphorylation of p38 M A P K was observed after the cells were treated with Ag»Ab complexes (Fig. 3.7). Signalling through the FcyRII and FcyRIII can be specifically prevented with 2.4G2 (a-FcR), a monoclonal antibody that binds to these FcR without signalling (Unkeless, 1979).  Pretreatment of the MC/9 cells with a-FcR  blocked the ability of Ag»Ab complexes to tyrosine phosphorylate p38 M A P K , indicating the complexes were specifically signalling through the FcR to activate p38 M A P K . Importantly, cells treated with either a-FcR or a-Ig alone failed to induce tyrosine phosphorylation of p38 M A P K . However, cells pretreated with a-FcR followed by treatment with a-Ig to cross-link a-FcR bound to the cell surface also induced the tyrosine phosphorylation of p38 M A P K (Fig. 3.7).  3.2.5 Hematopoietic growth factors activate MAPKAP  kinase-2.  M A P K A P kinase-2  has been reported to be a substrate of p38 M A P kinase (Freshney et al., 1994; Raingeaud et al.,  84  CSF-1:  -  cc-CSF-1:  LCCM:  +  -  -  -  +  +  -  +  -  »  IP*- anti-p38 -  +  +  +  —  IB: 4G10 IB:anti-p38  Figure 3.6 - Colony Stimulating Factor ( C S F ) - l induces tyrosine phosphorylation of p38 M A P K in F D M A C I I cells. Cells were incubated in  RPMI 1640 for 1 hr prior to stimulation, and then treated with CSF-1 (CSF-1), L-cell conditioned medium ( L C C M ) as a source of natural CSF-1, a neutralizing antibody (5A1) against CSF-1 (oc-CSF-1), or with indicated combinations of these for 5 min. Immunoprecipitated p38 MAP kinase was analyzed after SDS-PAGE by immunoblotting (IB) with the anti-phosphotyrosine antibody 4G10 (4G10), and subsequently with an anti-p38 MAP kinase antibody (anti-p38) to quantitate loading.  85  —  —  IB:  4G10  I B : anti-p38  Figure 3.7 - Cross-linking of the F c R for I g G induces tyrosine phosphorylation of p38 M A P K i n M C / 9 mast cells. MC/9 mast cells were pre-  treated with a-FcR (a-FcR) for 10 min as indicated, and then incubated for 10 min in medium alone, or with a rabbit a-rat immunoglobulin antibody (a-Ig). MC/9 cells were also pre-treated for 10 min with an anti-keyhole limpet hemocyanin (KLH) antibody (Ab), and then incubated with or without K L H (Ag) for 10 min as indicated. Cellular lysates were immunoprecipitated with antibodies recognizing p38 MAPK. Immunoprecipitated p38 MAP kinase was analyzed after SDS-PAGE by immunoblotting (IB) with the antiphosphotyrosine antibody 4G10 (4G10), and subsequently with an anti-p38 MAP kinase antibody (anti-p38) to quantitate loading.  86  1995), and to be activated in cells stimulated with G M - C S F or IL-3 (Ahlers et al., 1994). To determine whether the activation of M A P K A P kinase-2 by hematopoietic growth factors was due to activation of p38 M A P kinase, we imrnunoprecipitated M A P K A P kinase-2 from MC/9 cells that had been stimulated with IL-4, GM-CSF, S L F or 0.2 M NaCl and assessed its activity in an in vitro kinase assay using recombinant murine Hsp25 as substrate. As shown in the previous report (Ahlers et al., 1994), treatment with G M - C S F resulted in activation of M A P K A P kinase-2 (Fig. 3.8). SLF also induced strong activation of M A P K A P kinase-2 (Fig. 3.8). However, consistent with our finding that IL-4 failed to stimulate the enzymatic activity of p38 M A P kinase, IL-4 failed to induce activation of M A P K A P kinase-2 (Fig. 3.8). Indeed, treatment with IL-4 reduced the activity of M A P K A P kinase-2 to below the levels seen in untreated cells (Fig. 3.8), consistent with the reduction in p38 M A P kinase activity seen in IL-4 treated cells (Fig. 3.3). IL-4 was active on these cells as demonstrated by the induced tyrosine phosphorylation of a protein known to be IRS2, or pl70 (Fig. 3.8) (Welham et al., 1997). Thus the ability of hematopoietic growth factors to activate M A P K A P kinase-2 correlated with their ability to activate p38 M A P kinase.  3.2.6 GM-CSF  or SLF activate MAPKAP  kinase-2 via p38 MAPK.  MAPKAP  kinase-2 has been reported to be activated by members of the E R K M A P kinase family (Stokoe et al., 1992). GM-CSF, IL-3 and SLF induce activation of both E R K (Welham et al., 1992) and p38 M A P kinases (Fig. 3.2 and 3.3). To investigate which of these kinases was responsible for the activation of M A P K A P kinase-2 by these hematopoietic growth factors, we used SB 203580, a specific inhibitor of p38 M A P kinase activity (Cuenda et al., 1995; Lee et al., 1994). Pre-treatment of cells for 20 minutes with 1 uM SB 203580 abrogated the ability of IL-3 (Fig. 3.9), G M - C S F (Fig.  3.10) or S L F (Fig. 3.11) to induce activation of M A P K A P kinase-2.  In in vitro  experiments, the enzymatic activity of ERK-2 was not inhibited by concentrations of SB 203580 that abolished the enzymatic activity of p38 M A P kinase (Cuenda et al., 1995). To confirm that SB 203580 did not affect the activity of the E R K M A P kinases in vivo, we stimulated cells with GM-CSF or SLF and investigated the effects of the compound on the activation of p90 k, which rs  87  4  Mil  Hsp25 IP: a n t i - M A P K A P K 2  IB: 4G10  Figure 3.8 - Activation of M A P K A P kinase-2 by hemopoietic growth factors. MC/9 cells were incubated at 37°C in RPMI 1640 for 1 hr prior to simulation. Cells were then left untreated ( C O N ) , or treated with maximal doses of IL-4 (IL-4), G M - C S F ( G M - C S F ) , S L F (SLF) or 0.2 M NaCl (NaCl) for the indicated times. Cells were lysed and the majority of the lysate was used for a M A P K A P kinase-2 immune complex kinase assay with Hsp25 as a substrate. Autoradiograph of Hsp25 phosphorylation after SDS-PAGE (right). Immunoblotting of the proteins in the remaining cell lysate with an anti-phosphotyrosine antibody (4G10) after SDS-PAGE (left).  88  Untreated IL-3:  1 uM IP: anti-MAPKAPK2 SB203580  - 15 30 60 - 15 30 60 Hsp25  Figure 3.9 - I L - 3 activates M A P K A P kinase-2 via p38 M A P K .  Ba/F3 cells were incubated at 37°C in RPMI 1640 with or without 1 uM SB 203580 (SB 203580) for 20 minutes prior to stimulation. The cells were then left untreated, or were stimulated with saturating doses of IL-3 (IL-3) for the indicated times. M A P K A P kinase-2 was immunoprecipitated and its activity was assessed in immune complex kinase assays using recombinant murine Hsp25 as substrate. Reaction products were resolved by SDS-PAGE and the levels of phosphorylation of Hsp25 was assessed by autoradiography.  8 9  anti-p90 GM-CSF SB203580  + -  -  anti-MAPKAPK2  rsk  + +  + -  -  + + Hsp25  MBP  Figure 3.10 - G M - C S F activates M A P K A P kinase-2 via p38 M A P K . MC/9 cells were incubated at 37°C in RPMI 1640 with or without 1 uM SB 203580 (SB 203580) for 20 min prior to stimulation. The cells were then left untreated, or were stimulated with a saturating dose of G M - C S F ( G M - C S F ) for 10 min. M A P K A P kinase-2 or p 9 0 was immunoprecipitated from aliquots of cell lysates and their activities assessed in immune complex kinase assays using recombinant murine Ffsp25 or M B P as the respective substrates. Reaction products were resolved by SDS-PAGE and the levels of phosphorylation of Hsp25 or M B P were assessed by autoradiography. rsk  90  anti-p90 SLF SB203580  anti-MAPKAPK2  rsk  - -+ +  - - ++ - + - +  - + - +  Hsp25  MBP  Figure 3.11 - S L F activates M A P K A P kinase-2 via p38 M A P K .  MC/9 cells were  incubated at 37°C in RPMI 1640 with or without 1 uM SB 203580 (SB 203580) for 20 min prior to stimulation. The cells were then left untreated, or were stimulated with a saturating dose of SLF (SLF) for 5 min. MAPKAP kinase-2 or p 9 0 was immunoprecipitated from aliquots of cell lysates and their activities assessed in immune complex kinase assays using recombinant murine Hsp25 or MBP as the respective substrates. Reaction products were resolved by SDS-PAGE and the levels of phosphorylation of Hsp25 or MBP were assessed by autoradiography. rsk  9 1  is known to be a downstream target of the E R K M A P kinases (Blenis, 1993). As shown in Fig. 3.10 and 3.11, pretreatment of cells with SB 203580 did not affect the activation of p 9 0  r s k  by  G M - C S F or SLF, implying that the E R K M A P kinase pathway was not affected by SB 203580 and that p38 M A P kinase did not activate p 9 0 . rsk  3.2.7 p38a is sufficient for activation of MAPKAP  kinase-2.  Two different p38  M A P K genes, p38a and p38(3, encode protein kinases that are sensitive to inhibition by SB 203580 (Gum et al., 1998; Jiang et al., 1997; Kumar et al., 1997; Lee et al., 1994; L i et al., 1996). A mutant of p38cc M A P K was produced that was resistant to inhibition by SB 203580 (SBR-p38) (Gum et al., 1998). We made a stable line of Ba/F3 cells that expressed about 1/3 as much mutant p38cc M A P K compared to endogenous p38oc M A P K (Fig. 3.12, bottom). To test if SB 203580-resistant p38a M A P K was sufficient to restore M A P K A P kinase-2 activity in vivo, we stimulated both wild-type and SB 203580-resistant p38a MAPK-expressing Ba/F3 cells with 0.2 M NaCl for 15 minutes. This expression of p38a M A P K was sufficient to completely restore the activation of M A P K A P kinase-2 after hyperosmotic shock (Fig. 3.12, top).  3.2.8 Role of p38 MAPK in DNA synthesis. To evaluate the physiological significance of the activation of p38 by these growth factors, we utilized SB 203580 to inhibit p38 M A P K activity. A series of experiments employing two factor-dependent hematopoietic cell lines, MC/9 and Ba/F3, indicated that SB 203580 inhibited cell growth. As shown in Fig. 3.13, MC/9 cells that were cultured with titrated amounts of SB 203580 in the presence of IL-3 exhibited a dosedependent inhibition of ^H-thymidine incorporation with an  IC50  °f 3-5 u M . Experiments using  the IL-3 dependent myeloid cell-line Ba/F3, grown in the presence of IL-3, showed a similar inhibition of D N A synthesis (Fig. 3.14). We then examined the proliferation of Ba/F3 cells stably over-expressing the SB 203580-resistant form of p38a M A P K .  However, the SB 203580-  resistant mutant of p38a M A P K was unable to rescue D N A synthesis in the presence of SB 203580 (Fig. 3.14).  92  WT-p38 NaCl:  SB203580:  -  -  SBR-p38  + +  - + - +  - - +  +  - + -  + Hsp25  SBR-p38 WT-p38  Figure 3.12 - p38a M A P K is sufficient for activation of M A P K A P kinase-2.  Ba/F3 cells stably transfected with either the empty vector (WT-p38) or a mutant isoform of p38a (SBR-p38) were incubated at 37°C in RPMI 1640 with or without 1 uM SB 203580 (SB 203580) for 20 min prior to stimulation. The cells were then left untreated, or were stimulated with 0.2 M NaCl (NaCl) for 15 min as indicated. M A P K A P kinase-2 was immunoprecipitated and its activity was assessed in immune complex kinase assays using recombinant murine Hsp25 as substrate. (Top) The phosphorylation of Hsp25 was assessed by autoradiography. (Bottom) The relative level of expression of SBR-p38 and endogenous p38 are indicated.  93  Figure 3.13 - p38 MAPK activity is required for DNA synthesis in hematopoietic cells. ^.Thymidine incorporation in MC/9 cells was measured at 48 hours, and is shown plotted against the concentrations of SB 203580. Triplicate 15 uL cultures contained 10 cells in medium with or without saturating IL-3, 10% FCS and the indicated concentrations of SB 203580 diluted in culture media. Mean values of cpm in cultures lacking SB 203580 were 2,969 cpm in the absence of IL-3 and 19,750 cpm in the presence of IL-3. 3  94  Figure 3.14 - p38a MAPK fails to rescue the inhibition of DNA synthesis in hematopoietic cells. ^.Thymidine incorporation in Ba/F3 cells was measured at 24 hours, and is shown plotted against the concentrations of SB 203580. Triplicate 15 uL cultures contained 10 cells in medium with or without saturating IL-3, 10% FCS and the indicated concentrations of SB 203580. 3  95  3.3  Discussion  These experiments demonstrate that a series of hematopoietic growth factors which regulate the normal development and function of cells of the immune system stimulate tyrosine phosphorylation and enzymatic activation of p38 MAP kinase. This was the case for both those hematopoietic growth factors that signal through tyrosine kinase receptors, namely SLF (Fig. 3.2 and 3.3) and CSF-1 (Fig. 3.6), as well as those that signal through receptors of the hematopoietin receptor superfamily, namely IL-3 (Fig. 3.2) and GM-CSF (Fig. 3.3). Ligation of the Fc receptor for Immunoglobulin G, a receptor containing immunotyrosine activation motifs, also induces the tyrosine phosphorylation of p38 MAPK (Fig. 3.7).  The observed correlation of tyrosine  phosphorylation and activation of p38 MAP kinase (Fig. 3.3) is consistent with other evidence that phosphorylation of the tyrosine of the TGY activation motif of p38 MAP kinase is required for enzymatic activity (Doza et al., 1995; Raingeaud et al., 1995). The activation of p38 MAP kinase by GM-CSF and SLF in hematopoietic cells was rapid and transient (Fig. 3.4), and of the same order as that induced by hyperosmotic stress (Fig. 3.3).  This contrasts with reports that  stimulation of HeLa cells with epidermal growth factor activated p38 MAP kinase only weakly (Raingeaud et al., 1995) and that nerve growth factor (NGF) failed completely to activate p38 MAP kinase (RK) in PC-12 cells (Rouse et al., 1994). This may reflect differences in the cell types used in these studies.  It should be noted that p38 MAP kinase was activated by  hematopoietic growth factors in both cell lines and in mast cells from primary cultures. Given that MAPKAP kinase-2 is known to be an in vivo substrate of p38 MAP kinase (Freshney et al., 1994; Rouse et al., 1994), our observations that p38 MAP kinase is activated in response to hematopoietic growth factors are consistent with the earlier report that IL-3 and GMCSF activate MAPKAP kinase 2 (Ahlers et al., 1994). Our results confirm this observation and extend it by demonstrating that treatment of cells with SLF also induces MAPKAP kinase-2 activity (Fig. 3.8).  In that the specific inhibitor of p38 MAP kinase, SB 203580, completely  abrogated MAPKAP kinase-2 activation by IL-3 (Fig. 3.9), GM-CSF (Fig. 3.10) and SLF (Fig.  96  3.11), our data also extend current knowledge by indicating that the activation of M A P K A P kinase-2 by G M - C S F or S L F depended on p38 M A P kinase activity. Thus, despite the fact that treatment of cells with GM-CSF or SLF activates both E R K (Welham et al., 1992) and p38 M A P kinases (Fig. 3.3), SB 203580 specifically inhibited the in vivo activation of M A P K A P kinase-2 but not of p 9 0  r s k  (Fig. 3.10 and 3.11). These data demonstrate that SB 203580 fails to inhibit the  activity of E R K M A P kinases in vivo, consistent with in vitro data that SB 203580 fails to inhibit ERK-2 or p 9 0  r s k  (Cuenda et al., 1995). These results are also consistent with experiments in  which in vivo activation of E R K M A P kinases did not result in in vivo activation of M A P K A P kinase-2 (Rouse et al., 1994). Despite the fact that both E R K and p38 M A P kinases can activate M A P K A P kinase-2 in vitro  (Freshney et al., 1994; Rouse et al., 1994; Stokoe et al., 1992), and both are activated in cells  treated with G M - C S F or SLF, p38 M A P kinase activity was essential for activation of M A P K A P kinase-2 in vivo (Fig. 3.10 and 3.11). This evidence that M A P K A P kinase-2 is activated by p38 M A P kinase and not by E R K M A P kinase, suggests differences in molecular localization of these enzymes. Another possibility is that inactive p38 M A P kinase has a greater affinity for M A P K A P kinase-2 than the E R K M A P kinases, and that, in vivo,  M A P K A P kinase-2 is complexed with  inactive p38 M A P kinase, and not with E R K M A P kinases. In keeping with this notion, p38 M A P kinase has been shown to form a complex in vivo with human M A P K A P kinase-3, a close homologue of M A P K A P kinase-2 (70% amino acid identity) (McLaughlin et al., 1996). The failure of SB 203580 to inhibit the in vivo activation of p 9 0  r s k  in the same cells in  which it inhibited the in vivo activation of M A P K A P kinase-2 (Fig. 3.10 and 3.11) is consistent with the notion that p 9 0  r s k  is activated only by the E R K M A P kinases and not by p38 M A P  kinase. These results confirm the suggestion from unpublished in vitro experiments that p38 M A P kinase (RK) was unable to activate p 9 0  r s k  (Rouse et al., 1994).  It should be noted that the anti-MAPKAP kinase-2 antibody that we used was raised against a 16 amino-acid peptide from mouse M A P K A P kinase-2. This peptide differs by only two closely spaced, conservative amino acid substitutions from the corresponding peptide of human  97  MAPKAP kinase-3. If there is a murine homologue of human MAPKAP kinase-3, we would probably have precipitated it with this antibody. In the human, both MAPKAP kinase-2 and MAPKAP kinase-3 are activated by p38 MAP kinase (CSBP) (McLaughlin et al., 1996). Moreover both enzymes act as Hsp25 kinases  in vitro,  although whether both phosphorylate  Hsp25 in vivo is unclear. The functional significance of activation of the p38 MAP kinase pathway by growth factors and its relationship to actions of growth factors such as promotion of cell-cycle progression or suppression of apoptosis is unclear.  Two possible roles for p38 MAP kinase relate to the  regulation of actin polymerization and the activation of transcription factors. Actin polymerization appears to be regulated by phosphorylation of Hsp25, which lowers the affinity of its interaction with the barbed ends of filamentous (F) actin, thus allowing polymerization and the accumulation of F-actin (Lavoie et al., 1995). Growth factors and serum induce phosphorylation of Hsp25 on the same residues that are phosphorylated in response to stress (Landry et al., 1992; Saklatvala et al., 1991), consistent with the involvement of the same enzyme, most likely MAPKAP kinase-2 or MAPKAP kinase-3. Our results with hematopoietic growth factors demonstrate that inhibition of p38 MAP kinase completely blocks activation of MAPKAP kinase-2 (Fig. 3.10 and 3.11), suggesting that growth factor stimulated phosphorylation of Hsp25 reflects activation of p38 MAP kinase. Furthermore, the over-expression of an SB 203580-resistant isoform of p38a MAPK was sufficient to restore MAPKAP kinase-2 activation (Fig. 3.12). The role of actin polymerization in growth factor action is unclear although cell growth is inhibited by cytochalasin D (Lavoie et al., 1995) which, like unphosphorylated Hsp25, prevents elongation of F-actin (Sampath and Pollard, 1991). Over-expression of wild-type Hsp25, but not mutant Hsp25 that is unable to become phosphorylated, renders cells relatively resistant to the growth inhibitory effect of cytochalasin D (Lavoie et al., 1995). This implies a requirement for Hsp25 phosphorylation and F-actin accumulation for cell cycle progression.  Our observations that cells treated with SB 203580  underwent a rapid and reversible change in cellular morphology (F. Lee, unpublished data) were also consistent with the notion that p38 MAP kinase regulated actin polymerization.  98  Transcription factors regulating cell growth may be regulated by the p38 M A P kinase pathway through several mechanisms.  In vivo  activation of p38 M A P kinase increases the  transcriptional activity of Elk-1 (Raingeaud et al., 1996), implying a role for p38 M A P kinase in the transcriptional regulation of proteins such as c-Fos. Interestingly p38p, but not p38a M A P kinase, has been shown to phosphorylate and activate ATF-2  in vivo  (Jiang et al., 1996),  suggesting differential regulation of transcription factors by the p38 M A P kinase family members. Further experiments will be required to determine whether all family members are activated by hematopoietic growth factors.  Recently p38 M A P kinase has also been shown to regulate the  phosphorylation of CREB and ATF-1 through M A P K A P kinase 2 (Tan et al., 1996). In addition, p38 M A P kinase has been shown to regulate the production of IL-1, T N F a , IL-6 and G M - C S F (Beyaert et al., 1996; Lee et al., 1994), which are all encoded by mRNAs containing AU-rich motifs.  It is possible that the production of the transcription factors c-Myc, c-Fos and c-Jun,  which are translated from mRNA containing the same AU-rich motifs characteristic of cytokine mRNA (Chen and Shyu, 1995), may also be regulated in a p38 M A P kinase-dependent fashion. The notion that activation of the p38 M A P kinase pathway has a critical role in cell cycle progression is suggested by our observations that the specific p38 M A P kinase inhibitor SB 203580 inhibited D N A synthesis (Fig. 3.13 and 3.14). However the over-expression of an SB 203580-resistant isoform of p38a M A P K was unable to rescue D N A synthesis in Ba/F3 cells (Fig. 3.14), even though M A P K A P kinase-2 activation was restored, suggesting that M A P K A P kinase activity was not sufficient to rescue D N A synthesis.  However it is possible that  M A P K A P K 2 activity is not completely restored under normal cell culture conditions, and such possibilities require further investigation.  These findings also suggest that there may be  differences in the substrate specificity of p38a and p38p  in vivo,  or that the inhibition of DNA  synthesis was due to a non-specific effect of SB 203580. The correct interpretation of these data requires the analysis of an identical SB 203580-resistant mutant of p38p M A P K .  It will be  important to develop precise genetic tools to explore the role of p38 M A P kinase in growth factor action and cell cycle progression in mammalian cells.  99  A recent report implicated p38 M A P kinase in the induction of apoptosis (Xia et al., 1995). Withdrawal of nerve growth factor from PC-12 cells led to activation of p38 M A P kinase and the c-Jun N-terminal kinases (JNK), followed by apoptosis.  Over-expression of a constitutively  active M K K 3 , which at least in some cells activates p38 M A P kinase (Raingeaud et al., 1995; X i a et al., 1995), promoted apoptosis, whereas over-expression of a dominant negative M K K 3 inhibited apoptosis (Xia et al., 1995). However, these results do not discriminate between roles for p38 M A P kinase and JNK, as over-expression of a dominant negative M K K 3 appears to block activation of both of these kinases (Raingeaud et al., 1995). In contrast, our results show that p38 M A P kinase is activated by stimulation by growth factors (Fig. 3.2 and 3.3), not by their withdrawal. Moreover, in that hematopoietic growth factors suppress apoptosis (Williams et al., 1990), our findings indicate that, at least in hematopoietic cells, activation of p38 M A P kinase correlates with the suppression of apoptosis rather than its induction. Finally, treatment of cells with concentrations of SB 203580 that we have shown to inhibit p38 M A P kinase activity in vivo (Fig. 3.8) failed to inhibit the apoptosis induced by withdrawal of hematopoietic growth factors (I. Foltz, unpublished data). Interleukin-4 and IL-l3 are notable in being the only growth factors we investigated that failed to induce tyrosine phosphorylation (Fig. 3.2, 3.3 and 3.4) or activation of p38 M A P kinase (Fig. 3.3). The inability of IL-4 to activate p38 M A P kinase accounts for its failure to activate M A P K A P kinase-2 (Fig. 3.8) and correlates with its failure to activate Ras (Duronio et al., 1992; Satoh et al., 1991), and E R K M A P kinase (Welham et al., 1992; Welham et al., 1994). Further experiments will be required to determine whether the activation of p38 M A P kinase by growth factors in hematopoietic cells is Ras dependent. Interestingly, IL-4 consistently reduced the state of tyrosine phosphorylation and enzymatic activity of p38 M A P kinase to levels below those observed in control cells (Fig. 3.3). Likewise, treatment of cells with IL-4 reduced the in vivo activity of M A P K A P kinase-2 to levels below those in control cells (Fig. 3.8). The basis for the inhibition of the p38 M A P kinase pathway by IL-4 is under further investigation.  100  In conclusion, these experiments demonstrate that p38 M A P kinase participates in the responses to hematopoietic growth factors that interact with two structurally distinct classes of receptor. They demonstrate that p38 M A P kinase is not only involved in responses to stresses, but also in the action of growth factors that regulate the development and function of hematopoietic cells.  101  CHAPTER 4 - Activation of JNK in Hematopoietic Cells.  4.1  Introduction  While working to characterize the hematopoietic growth factors IL-3, GM-CSF and S L F as activators of the p38 M A P K pathway, it became apparent that the activation of p38 M A P K strongly correlated with the activation of the J N K pathway. Stimuli such as IL-1, T N F a , and U V light were characterized as activators of both p38 M A P K and JNK.  In fact, a stimulus capable of  separating the activation of p38 M A P K and J N K had not been described as yet.  Previous  unpublished data in our laboratory conducted by James Wieler had established that IL-3. G M - C S F and SLF induced mRNAs encoding c-Jun. However, IL-4 was unable to induce the expression of c-Jun. JNK had been identified as the relevant kinase for increasing c-Jun transactivation through N-terminal phosphorylation, thereby increasing AP-1 activity. The promoter of c-Jun contains AP-1 sites and therefore the literature suggested the induction of c-Jun was indicative of J N K activity.  Hypothesis: (1) IL-3, G M - C S F and SLF would increase the activity of JNK1 and JNK2. (2) These cytokines would induce the phosphorylation of M K K 4 .  (3) IL-4 would fail to activate either isoform of JNK or induce the phosphorylation of M X K 4 .  Objectives: (1) The transcription factor c-Jun had been described as having affinity for J N K through the delta peptide. We used GST-c-Jun to affinity precipitate J N K and perform in vitro kinase assays to determine the effect of cytokine stimulation on the activation of JNK. We also intended to identify commercial antibodies that could be used for immunoprecipitation of J N K for use in gel kinase assays, or immune-complex kinase assays.  102  (2) A t this time the only known activator o f JNK was the dual specificity kinase, M K K 4 .  There  were no antibodies commercially available to immunoprecipitate M K K 4 for direct analysis o f kinase activity.  However, New England Biolabs had just started producing antibodies that  specifically recognized M K K 4 when phosphorylated on an activating threonine residue. We decided to use this approach to correlate the activation of JNK with the activation state of M K K 4 . (3) Stimuli capable of separating the activation of p38 M A P K and JNK had not yet been described, and as such we did not expect IL-4 to be able to induce JNK activation. Both in gel kinase assays and immune complex kinase assays o f cell extracts from cells treated with I L - 4 would be conducted to determine i f IL-4 could activate JNK or its activator M K K 4 .  4.2  Results  4.2.1 Activation of JNKl  and JNK2 by Hematopoietic Growth Factors with the  Exception of IL-4 or IL-13. T o determine whether J N K was involved in responses to I L - 3 , GM-CSF or SLF, we stimulated MC/9 mast cells with these factors and immunoprecipitated J N K , using antibodies that precipitated both the 46 and 55 kDa isoforms o f either J N K l or JNK2. The precipitate was subjected to SDS-PAGE followed by renaturation and J N K activity was assessed using an "in gel" kinase assay. We found that stimulation with GM-CSF, I L - 3 or SLF induced activation of both the 46 and 55 kDa isoforms o f J N K l (Fig. 4.1). It was o f particular interest to investigate the effect o f IL-4 on J N K l activity, as our laboratory had previously shown that IL-4 failed to activate Ras (Duronio et al., 1992; Welham et al., 1994), ERK-1/2 M A P kinases (Welham et al., 1992; Welham et al., 1994) or p38 M A P kinase (Foltz et al., 1997).  Unlike the other  hematopoietins, I L - 4 and IL-13 failed to activate either J N K l (Fig. 4.1 and 4.2) or JNK2 (Fig. 4.2 and 4.3). We also examined the activation o f J N K 2 , and found that GM-CSF or SLF induced the activation of both 46 and 55 kDa isoforms of JNK2 (Fig. 4.3). To examine the kinetics o f the activation o f JNK induced by treatment o f MC/9 cells with SLF, IL-3 and GM-CSF, we used GST-c-Jun to preferentially immunoprecipitate activated J N K  103  A  w-  < f  10  •  Figure 4.1  10  N  c^V 10  -  5  ^flfl  «»  IP: anti-JNKl i  f  15  m  - Activation of JNK1 by hematopoietic  J P  46  growth factors with the  exception of IL-4 in MC/9 cells. Cells were incubated at 37°C in RPMI 1640 for 1 hr and then left untreated (CON), or treated with saturating doses of IL-3 (IL-3), IL-4 (IL-4), GM-CSF (GM), SLF (SLF) or 0.2 M NaCl (NaCl) for the indicated times. Immunoprecipitated JNK1 was analyzed using an in gel kinase assay after SDS-PAGE with a separating gel containing GSTc-jun. The phosphorylation of c-Jun was assessed by autoradiography and the presence of 46 and 55 kDa splice variants of JNK1 are indicated.  1 0 4  AP:GST-c-Jun  GST-c-Jun  Figure 4.2 - IL-13 fails to activate J N K l in F D 5 / I L 1 3 R cells. FD5 cells stably expressing the IL-13Ra chain (FD5/IL13R) were incubated at 37°C in RPMI 1640 for 1 hr and then left untreated (CON), or treated with saturating doses of IL-4 (IL-4), IL-13 (IL-13) or 0.2 M NaCl (NaCl) for 10 min. J N K was affinity precipitated (AP) using GST-c-Jun, and J N K activity was analyzed in a kinase assay with GST-c-Jun as substrate. The phosphorylation of cJun was assessed by autoradiography.  105  A  IP: anti-JNK2  p55 p46  Figure 4.3 - Activation of J N K 2 by G M - C S F and S L F , but not I L - 4 , in M C / 9 cells. Cells were incubated at 37°C in RPMI 1640 for 1 hr and then left untreated (CON), or treated with saturating doses of IL-4 (IL-4), G M - C S F (GM), SLF (SLF) or 0.2 M NaCl (NaCl) for the indicated times. Immunoprecipitated JNK2 was analyzed after SDS-PAGE with a separating gel containing GST-c-Jun, using an in gel kinase assay. The phosphorylation of c-Jun was assessed by autoradiography and the presence of 46 and 55 kDa splice variants of JNK2 are indicated.  106  (Adler et al., 1994; Dai et al., 1995; Kallunki et al., 1996). Treatment with S L F or G M - C S F resulted in a rapid increase in the activation of JNK (Fig. 4.4). Cells treated with S L F exhibited detectable activation of JNK after 2 minutes, with maximal activation of JNK occurring around 10 minutes (Fig. 4.4). In cells treated with GM-CSF the activation of JNK again peaked at around 10 minutes (Fig. 4.4). In both instances the activation of J N K was transient and was returning to basal levels by 30 minutes (Fig. 4.4).  4.2.2  Threonine Phosphorylation of MKK4 is induced by GM-CSF  not IL-4.  or SLF, but  At the time these experiments were done, M K K 4 was the only established upstream  activator of JNK (Derijard et al., 1995; Lin et al., 1995; Sanchez et al., 1994; Yan et al., 1994). Therefore we determined whether there was a correlation between activation of J N K by hematopoietic growth factors and activation of M K K 4 .  We immunoblotted cell extracts from  MC/9 cells that had been stimulated with GM-CSF, IL-4 or S L F (Fig. 4.5, top), or G M - C S F , SLF or 0.2 M NaCl (Fig. 4.5, bottom) with an antibody that specifically recognized the activated forms of human and murine M K K 4 . It was evident (Fig. 4.5, bottom) that stimulation of cells with GM-CSF, SLF or NaCl induced rapid phosphorylation of M K K 4 . This phosphorylation was transient and was returning to basal levels by 10 minutes. On shorter exposures, it was evident that GM-CSF was a weaker activator of M K K 4 than S L F (Fig. 4.5, top). These data indicate the activation of M K K 4 preceded the activation of JNK, consistent with a role in activation of JNK by these stimuli. In keeping with our finding that IL-4 failed to stimulate the enzymatic activity of JNK, IL-4 failed to induce phosphorylation of M K K 4 (Fig. 4.5). The activation of J N K in all cases correlated with activation of M K K 4 .  4.3  Discussion  These experiments utilizing MC/9 mast cells demonstrate that a series of hematopoietic growth factors which regulate the normal development and function of cells of the immune system  107  Figure 4.4 - Kinetics of J N K activation by G M - C S F or S L F i n M C / 9 cells. After 1 hr in RPMI 1640 at 37°C, MC/9 cells were left untreated, or treated with saturating doses of G M CSF ( G M - C S F ) , SLF (SLF), or 0.2 M NaCl (NaCl) as a positive control for the indicated times in minutes. Cell extracts were prepared and GST-c-Jun was used as both affinity reagent and substrate for an in vitro kinase assay. The phosphorylation of c-Jun was assessed by autoradiography (left). The fold activation of JNK in cells treated with SLF or GM-CSF compared to untreated cells was quantitated using densitometry (right).  108  cf  ^  &  -  10  10  &  5  15 —  fl  6  SLF 5  10  NaCl 5  10  phospho-MKK4  GM-CSF 5  10  Figure 4.5 - Threonine phosphorylation of M K K 4 by SLF or GM-CSF, but not IL-4. M C / 9 cells were incubated at 37°C in RPMI 1640 for 1 hr prior to simulation. Cells were then left untreated (CON), or (Top) treated with maximal doses of IL-4 (IL-4), G M - C S F (GMCSF) or S L F (SLF) or (Bottom) treated with maximal doses of G M - C S F (GM-CSF), S L F (SLF) or 0.2 M NaCl (NaCl) for the indicated times. After lysis, cell extracts were analysed by SDS-PAGE, and were immunoblotted with an antibody that specifically recognizes the activated form of S E K 1 / M K K 4 .  109  activate both JNK1 (Fig. 4.1) and JNK2 (Fig. 4.3). One of these hematopoietic growth factors, SLF, signals through a tyrosine kinase receptor, while the others, IL-3 and GM-CSF, signal through receptors of the hematopoietin receptor superfamily (Fantl etal., 1993; Taniguchi, 1995). Cells treated with GM-CSF, IL-3 or S L F exhibited increased activity of 46 and 55 kDa proteins corresponding to different splice variants of JNK1 (Fig. 4.1). Similarly, cells treated with G M CSF or S L F activated both 46 and 55 kDa splice variants of JNK2 (Fig. 4.3).  Together our  results indicate that IL-3 (Fig. 4.1), G M - C S F (Fig. 4.1 and 4.3) and S L F (Fig. 4.1 and 4.3) stimulate the activation of 46 and 55 kDa isoforms of both JNK1 and JNK2 in MC/9 cells. The kinetics of JNK activation in MC/9 cells treated with G M - C S F or S L F resembled that observed for activation of E R K (Welham et al., 1992; Welham et al., 1994) and p38 M A P K (Foltz et al., 1997) by these factors.  The activation of J N K by S L F showed similar kinetics to that  induced by G M - C S F (Fig. 4.4). In all cases the activation of J N K following stimulation with these factors was rapid and transient (Fig. 4.4).  We also used an antibody specific for the  activated form of M K K 4 to analyze the ability of these growth stimuli to activate M K K 4 , the only known upstream activator of JNK (Derijard et al., 1995; Lin et al., 1995; Sanchez et al., 1994; Yan et al., 1994). The phosphorylation of M K K 4 induced by treatment of cells with SLF, G M CSF or 0.2 M NaCl was evident at 5 minutes and was declining by 10 minutes when levels of JNK activity were peaking. However, the phosphorylation of M K K 4 by S L F was greater than that observed when cells were treated with GM-CSF.  The earlier kinetics of phosphorylation of  M K K 4 , compared with activation of JNK, are consistent with M K K 4 being upstream of JNK in the cellular response to these stimuli.  These results suggest that M K K 4 is involved in the  activation of JNK in response to S L F and GM-CSF.  There have been reports of other activators  of JNK (Moriguchi et al., 1995), but these proteins had not been characterized at the time these experiments were done. Interleukin-4 and IL-13 were unique among the hematopoietins that we examined in this study in failing to increase the activation of JNK1 (Fig. 4.1) or JNK2 (Fig. 4.3). As noted, the inability of IL-4 to activate JNK correlated with its inability to induce phosphorylation of M K K 4  110  (Fig. 4.5). This failure of IL-4 to activate J N K correlates with its inability to activate either the E R K (Welham et al., 1992; Welham et al., 1994) or the p38 M A P kinase pathway (Foltz et al., 1997). Interleukin-4 is also notable for its inability to activate Ras (Duronio et al., 1992; Satoh et al., 1991). We hypothesize that hematopoietic growth factors activate p38 M A P K and J N K in a Ras-dependent fashion.  Indeed, other growth factors activate J N K through Ras-dependent  mechanisms as dominant inhibitory mutants of Ras have been shown to prevent the activation of JNK in response to E G F and N G F (Minden et al., 1994).  Furthermore, J N K activity was  increased by the expression of constitutively active mutants of Ras (Minden et al., 1995). However, dominant inhibitory mutants of Cdc42 or Racl prevent J N K activation by Ras (Minden et al., 1995), and constitutively active mutants of Cdc42 or Racl increased J N K activity (Bagrodia et al., 1995; Coso et al., 1995; Minden et al., 1995).  These findings suggest that Ras was  mediating JNK activation through either Cdc42 or Racl. Observations that the activation of J N K by TNFa is not inhibited by over-expression of dominant-negative Ras however suggest that there may also be other Ras-independent pathways of JNK activation (Minden et al., 1995). Further studies using dominant inhibitory mutants of Ras and the Rho family of small GTPases will be required to elucidate the hierarchy of proteins leading to J N K activation by hematopoietic growth factors. Hematopoietic growth factors promote cell cycle progression in cells of the immune system. The functional significance of the activation of J N K l and JNK2 by hematopoietic growth factors, for example in the promotion of cell cycle progression, is unknown. Clearly the best argument for a role of JNK in cell cycle progression is their ability to phosphorylate and activate the transcription factors including c-Jun, ATF-2 and Elk-1 (Cavigelli et al., 1995; Derijard et al., 1994; Gupta et al., 1995; Kyriakis et al., 1995; Minden et al., 1994). J N K may have an obligate role in cell cycle regulation as J N K are the only known kinases capable of phosphorylating c-Jun on serines 63 and 73 and thereby increasing its transcriptional activity (Derijard et al., 1994; Kyriakis et al., 1995; Minden et al., 1994).  The observation that c-Jun is required for the  1 11  transformation of fibroblasts by activated Ras implies a role for JNK in the Ras-mediated control of growth (Johnson et al., 1996). Hematopoietic growth factors not only provide growth signals that promote growth of hematopoietic cells, but also signals that suppress apoptosis.  In some cells activation of J N K  correlates not with suppression of apoptosis but with its induction (Chen et al., 1996; Xia et al., 1995). It is possible that activation of the JNK pathway in the absence of other signals such as E R K activity results in a pro-apoptotic signal, whereas the integrated activation of E R K , J N K and p38 M A P K pathways, stimulated by factors such as GM-CSF, IL-3 and SLF, delivers a signal for the suppression of apoptosis. However, our data indicates that the activation of ERK, JNK or p38 M A P K pathways is not essential for the suppression of apoptosis as IL-4, which we have shown fails to activate any of these kinases, nevertheless suppresses the apoptosis of many hematopoietic cells, including the factor dependent cell line MC/9 used in these experiments. In conclusion, we have demonstrated that JNK1 and JNK2 are activated in response to IL3, G M - C S F and SLF, but not IL-4. Furthermore our results suggest that S A P K activation is mediated by M K K 4 , as both SLF and GM-CSF, but not IL-4, activated M K K 4 prior to activation of JNK. Importantly, our results demonstrate that JNK activity is involved not only in response to stress, but also in signaling by hematopoietic growth factors that in some cases, such as S L F , regulate the normal growth and development of the hematopoietic system.  112  CHAPTER 5 - MKK7 - A Specific Activator of JNK.  5.1  Introduction  The activation of JNK by G M - C S F or S L F followed very similar kinetics. However, the induction of threonine phosphorylation of M K K 4 by these cytokines was much more pronounced with S L F than with G M - C S F or NaCl. These findings suggested the existence of another J N K kinase that was regulated by GM-CSF. Several other lines of evidence pointed to the existence of another M K K capable of activating the J N K family. Moriguchi et al. identified at least one J N K kinase activity in addition to the activity of M K K 4 in rat fibroblastic 3Y1 cells stimulated by hyperosmotic shock (Moriguchi et al., 1995). Similarly Meier et al. identified two J N K kinase activities operationally termed S K K 4 and S K K 5 in K B cells, a human oral carcinoma cell line, following treatment with hyperosmolarity or anisomycin (Meier et al., 1996). The generation of embryonic stem (ES) cells in which the M K K 4 gene was disrupted provided conclusive evidence for the existence of another J N K kinase. ES cells lacking M K K 4 still exhibited increased J N K activity following hyperosmotic shock or U V irradiation, but failed to exhibit J N K activation after treatment with anisomycin or interleukin-1 (Meier et al., 1996; Nishina et al., 1997; Yang et al., 1997). Together these studies suggested the existence of another JNK kinase.  Hypothesis: (1) MC/9 cells express a novel M K K that acts as a direct activator of JNK. (2) The unknown M K K is activated by GM-CSF, IL-3, and NaCl.  Objectives: (1) Screen the expressed sequence tags (EST) database to identify a novel M K K . The sequences of the known M K K were aligned, and a consensus sequence at the C-terminus of these enzymes  113  was determined (Fig. 5.1). The resulting peptide was converted to a degenerate oligonucleotide (Fig. 5.2), and the oligonucleotide was used to screen the EST database. (2) Determine the expression pattern of any unknown M K K that was identified. (3) Characterize the enzymes for their substrate specificity and upstream activators.  5.2  Results  5.2.1  Cloning strategy to discover unknown MKK.  We screened the EST (expressed  sequence tags) database using a degenerate oligonucleotide sequence based on the amino acids conserved in the C-terminus of all previously characterized mitogen-activated protein kinase kinases (Fig. 5.1) (Lin et al., 1995). We identified 7 clones that encoded known M K K family members and one clone (aa019720) that contained a novel human sequence (Table 5.2).  This  clone contained 464 base pairs (bp) of coding region and approximately 1200 bp of 3' untranslated region.  We used 5'-RACE (Rapid Amplification of cDNA Ends) to isolate overlapping 5'-  fragments of this novel cDNA from a human fetal kidney R A C E cDNA library (Fig. 5.2). We amplified several over-lapping clones of M K K 7 using a fully nested PCR with the adaptor primers AP1/AP2 and primers IF113/IF111 or IF117/IF115 for the first or second/third screens respectively (Fig. 5.3). Using this technique we cloned a further 861 bp of human M K K 7 (hMKK7a) that contained an in-frame stop codon. When theoretical translation was initiated at the first methionine, this cDNA encoded a 419 amino acid protein (Fig. 5.4) that contained a putative protein kinase domain. We also isolated a splice variant of human M K K 7 (MKK7(3) that contained an additional 126 base pairs encoding an insert of 42 amino acids (Fig. 5.5). We screened the yeast and invertebrate databases for other M K K related to M K K 7 and although we failed to identify a yeast homolog, we did find an ortholog of M K K 7 in D.  melanogaster  (dHep - 69%  identity) (Glise et al., 1995). In C. elegans we identified an ortholog with 54% identity we termed c M K K 7 (Wilson et al., 1994). We compared the catalytic domains of M K K 7 with dHep and c M K K 7 (Fig. 5.6) or all other known human M K K (Fig. 5.7). M K K 7 was orthologous with  114  Alignment of M K K : MKK4: MKK3: MEK2: MEK1: PBS2:  FSPSFINFVNLCLTKDESKRPKYKELLKHPF ...E.VD.TAQ..R.NPAE.MS.L..ME... .T.D.QE...K..I.NPAE.ADL.M.TN.T. ..LE.QD...K..I.NPAE.ADL.Q.MV.A. ..SDAQD..S...Q.I.ER..T.AA.T...W  Consensus:  F  F . . . C L . K . . . .R  L. .H. .  Nucleotide Sequence: TT[TC].(.)16TT[TC].(.)7.TG[TC].T....AA[AG].(.)11.G.(.)15.T.(.)5.CA Figure 5.1 - Designing a probe to screen the EST database. The C-terminii of the  primary structures of human M K K 4 , M K K 3 , M E K 2 , M E K 1 and 5. cerevisiae PBS2 were aligned to determine a consensus motif to identify novel M K K . The amino acid motif was back-translated into degenerate nucleotides according to the human genetic code (Table 5.1). The resulting nucleotide sequence was used to screen the EST database.  Table 5.1 - The Genetic Code. T T  C  A  G  TTT TTC TTA TTG CTT CTC CTA CTG ATT ATC ATA ATG GTT GTC GTA GTG  -  F F L L L L L L I I I M V V V V  C TCT TCC TCA TCG CCT CCC CCA CCG ACT ACC ACA ACG GCT GCC GCA GCG -  S S S S P P P P T T T T A A A A  A TAT - Y TAC - Y TAA - * TAG - * CAT - H CAC - H CAA - Q CAG - Q AAT - N AAC - N AAA - K AAG - K GAT - D GAC - D GAA - E GAG - E  115  G TGT - C TGC - C TGA - * TGG - W CGT - R CGC - R CGA - R CGG - R AGT - S AGC - S AGA - R AGG - R GGT - G GGC - G GGA - G GGG - G  Table 5.2 - Analysis of Clones derived from the EST database. EST Clone aa008333 aa019720 n98702 rll022 r51239 ricr2291a t23364 wl6504 W29331 W78467  aa050011 aa026071 aa071840  Protein MKK3 Novel - MKK7 MKK4 MEK2  Species Human Human Human Human  Novel Novel GCK-like Novel MKK5 MKK3 MKK3  Rice Human Grain Human Human Mouse  MKK4  Mouse  -  -  -  -  EST clone: aa019720 ATG  TAG Step 1: Nested PCR Probe  A P I AP2  I  1  Adapter  i Step 2: Blunt end sub-cloning of PCR products into pBSKS  1 1 I  Step 3: Blue/White Colony Selection  Step 4: Colony Blot with Specific Probe from EST Clone  Step 5: Sequence Putative Positive Clones Figure 5.2- Strategy for cloning full length MKK7.  116  EST clone: aa019720 -1000 -900 -800 -700 -600 -500 -400 -300 -200 -100 I  I  l  I  I  I  I  I  I  i  — —  ATC  IF115IF117  0  100  200  300  400  L  — IF111  IF113 T A G  Primary Screen: Clone 10 Clone 24 Secondary Screen: Clone 7 Clone 17 Tertiary Screen: ^  —  ^  —  ^  C  l  o  n  e  FI  Figure 5.3- Amplification of fragments of MKK7 using 5'-RACE PCR. A human fetal kidney cDNA library was used to amplify M K K 7 . The adapter primers A P I and AP2 were used for all PCR amplifications as depicted in Figure 5.2. A fully nested PCR using either the primers IF113 and IF 111, or the primers IF117 and IF115, was used for the primary screen, or the secondary and tertiary screens respectively. The positive clones found in these screens are depicted to scale with respect to the full length human M K K 7 a , and the EST clone aaO 19720.  117  GGCGGTGTTTGTCTGCCGGACTGACGGGCGGCCGGGCGGTGCGCGGCGGCGGTGGCGGCCGGGGAAA ATGGCGGCGTCCTCCCTGGAACAGAAGCTGTCCCGCCTGGAAGCAAAGCTGAAGCAGGAGAACCGGGA M A A S S L E Q K L S R L E A K L K Q E N R E GGCCCGGCGGAGGATCGACCTCAACCTGGATATCAGCCCCCAGCGGCCCAGGCCCACCCTGCAGCTCC A R R R I D L N L D I S P Q R P R P T L Q L CGCTGGCCAACGATGGGGGCAGCCGCTCGCCATCCTCAGAGAGCTCCCCGCAGCACCCCACGCCCCCC P L A N D G G S R S P S S E S S P Q H P T P P GCCCGGCCCCGCCACATGCTGGGGCTCCCGTCAACCCTGTTCACACCCCGCAGCATGGAGAGCATTGA A R P R H M L G L P S T L F T P R S M E S I E GATTGACCAGAAGCTGCAGGAGATCATGAAGCAGACGGGCTACCTGACCATCGGGGGCCAGCGCTACC I D Q K L Q E I M K Q T G Y L T I G G Q R Y AGGCAGAAATCAACGACCTGGAGAACTTGGGCGAGATGGGCAGCGGCACCTGCGGCCAGGTGTGGAAG Q A E I N D L E N L G E M G S G T C G Q V W K ATGCGCTTCCGGAAGACCGGCCACGTCATTGCCGTTAAGCAAATGCGGCGCTCCGGGAACAAGGAGGA M R F R K T G H V I A V K Q M R R S G N K E E GAACAAGCGCATCCTCATGGACCTGGATGTGGTGCTGAAGAGCCACGACTGCCCCTACATCGTGCAGT N K R I L M D L D V V L K S H D C P Y I V Q GCTTTGGGACGTTCATCACCAACACGGACGTCTTCATCGCCATGGAGCTCATGGGCACCTGCGCTGAG C F G T F I T N T D V F I A M E L M G T C A E AAGCTCAAGAAGCGGATGCAGGGCCCCATCCCCGAGCGCATTCTGGGCAAGATGACAGTGGCGATTGT K L K K R M Q G P I P E R I L G K M T V A I V GAAGGCGCTGTACTACCTGAAGGAGAAGCACGGTGTCATCCACCGCGACGTCAAGCCCTCCAACATCC K A L Y Y L K E K H G V I H R D V K P S N I TGCTGGACGAGCGGGGCCAGATCAAGTTCTGCGACTTCGGCATCAGCGGCCGCCTGGTGGACTCCAAA L L D E R G Q I K F C D F G I S G R L V D S K GCCAAGACGCGGAGCGCCGGCTGTGCCGCCTACATGGCACCCGAGCGCATTGACCCCCCAGACCCCAC A K T R S A G C A A Y M A P E R I D P P D P T CAAGCCGGACTATGACATCCGGGCCGACGTATGGAGCCTGGGCATCTCGCTGGTGGAGCTGGCAACAG K P D Y D I R A D V W S L G I S L V E L A T GACAGTTTCCCTACAAGAACTGCAAGACGGACTTTGAGGTCCTCACCAAAGTCCTACAGGAAGAGCCC G Q F P Y K N C K T D F E V L T K V L Q E E P CCGCTTCTGCCCGGACACATGGGCTTCTCGGGGGACTTCCAGTCCTTCGTCAAAGACTGCCTTACTAA P L L P G H M G F S G D F Q S F V K D C L T K AGATCACAGGAAGAGACCAAAGTATAATAAGCTACTTGAACACAGCTTCATCAAGCGCTACGAGACGC D H R K R P K Y N K L L E H S F I K R Y E T TGGAGGTGGACGTGGCGTCCTGGTTCAAGGATGTCATGGCGAAGACTGAGTCACCGCGGACTAGCGGC L E V D V A S W F K D V M A K T E S P R T S G GTCCTGAGCCAGCCCCACCTGCCCTTCTTCAGGTAGCTGCTTGGCGGCGGCCAGCCCCACAGGGGGCC V L S Q P H L P F F R * AGGGGCATGGCCACAGGCCCCCCTCCCCACTTGGCCACCCAGCTGCCTGCCAGGGGAGACCTGGGACC TGGACGGCCACTAGGACTGAGGACAGAGAGT  Figure 5.4 - Nucleotide and primary amino acid sequence of human  M K K 7 . The nucleotides of the cDNA encoding human MKK7a. The initiating methionine is underlined and stop codons are shown in bold. The primary amino acid sequence is shown below the nucleotide sequence as predicted from the human codon usage chart (Table 5.1).  118  hMKK7 a mMKK7a mMKK7y  MAAS SLEQKLSRLEAKLKQENREARRRIDLNLDIS PQRPRPT  LQ  hMKK7a mMKK7a  LPLANDGGSRSPSSESSPQHPTPPARPRHMLGLPSTLFTPRSMESIEIDQKLQEIMKQTG T  hMKK7a hMKK7(3 mMKK7a  YLTIGGQ RYQAEINDLE VPPSLWRGEGGGPARLDPSWERQWGAGGGGRAPGTLQPSLSSQ  IIVITLSPAPAPSQRAA  hMKK7a mMKK7a  I II III IV NLGEMGSGTCGQVWKMRFRKTGHVIAVKQMRRSGNKEENKRILMDLDWLKSHDCPYIVQ I  hMKK7 a mMKK7a  CFGTFITNTDVFIAMELMGTCAEKLKKRMQGPIPERILGKMTVAIVKALYYLKEKHGVIH .V.  hMKK7a mMKK7a  VI VII * * VIII RDVKPSNILLDERGQIKLCDFGISGRLVDSKAKTRSAGCAAYMAPERIDPPDPTKPDYDI  hMKK7a  IX X RADVWSLGISLVELATGQFPYKNCKTDFEVLTKVLQEEPPLLPGHMGFSGDFQSFVKDCL  hMKK7a  XI TKDHRKRPKYNKLLEHSFIKRYETLEVDVASWFKDVMAKTESPRTSGVLSQPHLPFFR  Figure 5.5 - Alignment of primary structures of human and murine isoforms of  M K K 7 . Predicted amino acid sequence of human MKK7oc (hMKK7a) and MKK7(3 (hMKK7(3) and of the N-termini of murine M K K 7 a (mMKK7a) and M K K 7 y (mMKK7Y). Where residues are identical with those of hMKK7cc, they are indicated by periods. hMKK7(3 and mMKK7y are shown only where they differ from M K K 7 a . Gaps were introduced to optimize the alignment and are indicated by dashes.  119  dHep  MSTIEFETIGSRLQSLEAKLQAQNESHDQIVLSGARGPWSGSVPSARVPPLATSASAA  hMKK7 dHep  MAAS SLEQKLSRLEAKLKQENREARRRIDLNLDIS PQRPRPTLQ TSATHAPSLGASSVSGSGI.IAQRPAPPVPHATLRS PSAS S S S S SRSAFR.AAPATGLRW  hMKK7 dHep CMKK7  LPLANDGDSRSPSSESSPQHPTPPARPRHMLGLPSTLFTPRSMESIEIDQKLQEIMKQTG TYTPPTTRVSRATPTLPMLSSG.GGDVECTRPVILP.P..PHPPVS.T.M..KI..E... MER.F.LGMGRPGGLGGLGGE.IMQQMPQPA.HHPSRSSNDHNVKNLM.QA.—ENS.  hMKK7 dHep CMKK7  I II YLTIGGQRYQAEINDLENLGEMGSGTCGQVWKMRFRKTGHVIAVKQMRRSGNKEENKRIL K. N . N. RQ . PTD .... KH .. DL . N.. S . N... . MHLSSNTI T. .A ....L.N.RK.DLKE.QFVEDI.H.S..T.T.C.YKSV--IM...T.P.TS.SY.MS...  CMKK7  Ill VI V MDLDWLKSHDC PYIVQCFGTFITNTDVFIAMELMGTCAEKLKKRMQGPIPERILGKMTV K...K.L.C. VRDP . .W.C. . . . SM. FD . . L . LSKK .V. .Q. . . .V. . ICL.F R...Y....F.. RVC . . C . A. . LDR. LI . IKQ I. . LS .  hMKK7 dHep CMKK7  VI VII * * AIVKALYYLKEKHGVIHRDVKPSNILLDERGQIKLCDFGISGRLVDSKAKTRSAGCAAYM .T.N..S...D..G I....N N S.I...H...T..QIM WS.V A...IE.R. HSKQ . . . PL. .  hMKK7 dHep CMKK7  VIII IX X APERIDPPDPTKPDYDIRADVWSLGISLVELATGQFPYKNCKTDFEVLTKVLQEEPPLLP .K..K T ARS . . EG . N DS . . . C . . G. . .L. .NNFDS— ....S....F.VT QYP.AG--.E.DMMS.I.ND. . .R.D  hMKK7 dHep CMKK7  XI GHMG--FSGDFQSFVKDCLTKDHRKRPKYNKLLEHSFIKRYETLEVDVASWFKDVMAKTE YGE.YN..QQ.RD..IK....N.QD....PE..AQP..RI..SAK...PN..QSIKDNDC PA K. . P . . CQL . ES . . QR. PTM. . N. DM. . Q . P . WHH . KI. T . . EE . . A. . . G-EC  hMKK7 dHep CMKK7  SPRTSGVLSQPHLPFFR GQWRSNAPEVT G  hMKK7 dHep  Figure 5.6 - Alignment of human M K K 7 with its orthologs in C. elegans and D. melanogaster. Predicted amino acid sequence of human M K K 7 a (hMKK7), D. melanogaster M K K 7 (dHep) and C. elegans M K K 7 (cMKK7). Where residues are identical with those of h M K K 7 , they are indicated by periods. Gaps were introduced to optimize the alignment and are indicated by dashes.  120  MKK7 MKK4 MKK6 MKK 3 MEK5 MEK2 MEKl  I II III DLENLGEMGSGTCGQVWKMRFRKTGHVIAVKQMRRSGNKEENKRILMDLDWLKSHDCPY . .KD. . .I.R.AY.S.N. .VHKPS.QIM. . .RI.STVDEK.Q.QL ..MR.S. . . . ...PIM.L.R.AY.V.E...HVPS.QIM...RI.ATV.SQ.Q..L ISMRTV...F . .VTIS.L.R.AY.V.E.V.HAQS.TIM. . . R I . ATV. SQ . Q . . L INMRTV. . . . .IRYRDTL.H.NG.T.Y.AYHVPS.KIL...VILLDITL.LQ.Q.MSE.EI-.YKC.SS. .F.RIS.L.A.NG.V.T.VQH.PS.LIM R . L I H L E I K P A I R N Q . I R E . Q . - . H E C N S . . .F.KIS.L.A.NG.V.F.VSHKPS.L.M.R.LIHLEIKPAIRNQ.IRE.Q.-.HECNS..  MKK7 MKK 4 MKK 6 MKK 3 MEK5 MEK2 MEKl  VI V IVQCFGTFITNTDVFIAMELMGT-CAEKLKK RMQGPIPERILGKMTVAIVKALYYL . . .FYALFREG.CW c. S.-SFD.FY YVYSVLDDV. . .E. .. . I . L . T . . . NH T.TFY ALFREG..W c. D.-SLD.FY QVIDK -GQT. . .D. .. . I A . S . . . . EH D.-SLD.FYRKVLDK -NMT. . .D. ...IA.S..R. EH T.TFY ALFREG..W c. --RKM . .HV. .RIA..V..G T. . IGFY A . F V E N R I S CT F DGGSLDVY-. . GFY A.YSDGEIS C. H DGGSLDQVL E -AKR. . .E. ...VSI.VLRG A. -A.R. . .Q. . . . V S I . V I . G T. . .GFY A.YSDGEIS C. H DGGSLDQVL K  MKK7 MKK 4 MKK 6 MKK 3 MEK5 MEK2 MEKl  * * VIII VI VII KEKHGVIHRDVKPSNILLDERGQIKLCDFGISGRLVDSKAKTRSAGCAAYMAPERIDPPD ..NLKI T . . .D. RP. . . _g . I. . ,RS N •Q V Y I D . .KP... ....N.-E HSKLS V INAL V M. . .Y HSKLS.,, . .V INKE HV M. . V . .MD. .KP... ....N.-E WS-LKIL M VNTR . V . .V TQ .N I . . Y-V TN.... ....SGEQ . ,V • Q T . M NSF-V TRS... ...LQGTH R...QIM VNSR E , R...KIM . .V • Q I . M NSF-V TRS... ...LQGTH VNSR E.  MKK7 MKK 4 MKK6 MKK3 MEK5 MEK2 MEKl  IX PTKPDYDIRADVWSLGISLVELATGQFPYKNCKT ASRQG ..V.S T.Y R... PKWNS LNQKG.SVKS.1 TMI. . . I L R . . .DSWG. LNQKG.NVKS.V TMI.M.ILR. . .ESWG. .GIHS FM.IQ . SVQS . I . . M. L V. RY . I P P P D A K E L E A I F G R P W D G E E G E P H S I S P R F .SVQS.I..M.L....M.V.RY.IPPPDAKELELMFGCQV EGDAAETPPRF  MKK 7 MKK 4 MKK 6 MKK 3 MEK5 MEK2 MEKl  DFEVLTKVLQEEPPLLPGHMG-- FSGDFQSFVKDCLTKDHR .ES DQ • Q VKGD. Q. SNSEERE . .PS I N .NL p QQ KQ VE PS Q . ADK . .AE VD TSQ .K NSK p QQ KQ VE PS Q .ADR . . PE VD TAQ .R NPA KNQGSLMPLQL QCIVD DS .V . VGE . .EP VH ITQ MR QPK RPPGRPVSGHGMDSRPAMAI . L DYIVN P. K . NGV .TP. .E .NK . I NPA RTPGRPLSSYGMDSRPPMAI .L DYIVN P. K . SGV . .LE .D .NK . I NPA  MKK7 MKK4 MKK6 MKK3 MEK5 MEK2 MEKl  XI KRPKYNKLLEHSF KE . . K . P . E..T.PE.MQ.P. E.MS.LE.M..P. E..APEE.MG.P. E.ADLKM.TN.T. E.ADLKQ.MV.A.  _  v  Figure 5.7 - Alignment of the catalytic domains of known human M K K . The  predicted amino acid sequence of known human M K K . Where residues are identical with those of M K K 7 , they are indicated by periods. Gaps were introduced to optimize the alignment and are indicated by dashes.  121  dHep and c M K K 7 and was more closely related to these than to the known human M K K (Fig. 5.8). We used primers derived from the human sequence of M K K 7 to amplify a 869 bp fragment of cDNA from the murine Ba/F3 hematopoietic cell line. Sequencing indicated that this cDNA encoded the N-terminus and most of the kinase domain of a murine homologue of M K K 7 (mMKK7oc). We screened the EST database for cDNA encoding the N-terminus and identified another splice variant of m M K K 7 (mMKK7Y) that contained an additional 48 base pair exon or inframe intron (Fig. 5.5). We have not yet identified a human homologue of mMKKTy.  These  splice variants may encode alternative forms of M K K 7 or may represent incompletely processed mRNA. By far the most highly represented form of M K K 7 in a human fetal kidney cDNA library was M K K 7 a and therefore we chose this isoform for continued analysis of M K K 7 function.  5.2.2  Expression of MKK7 in human and murine cells.  To investigate expression of  endogenous and exogenous M K K 7 we immunoblotted lysates of MC/9 mast cells, Ba/F3 cells and a clone of Ba/F3 cells that we generated stably expressing M K K 7 (Ba/F3-MKK7) with an antiserum raised against the N-terminus of M K K 7 . We identified an immunoreactive protein of 47 kDa, consistent with the size predicted from the cDNA of MKK7cc or M K K 7 y (Fig. 5.9). Thus Ba/F3 and MC/9 cells expressed the N-terminus predicted by translation of the cDNA encoding m M K K 7 a or mMKK7Y . We were unable to detect an immunoreactive protein at the size (53 kDa) predicted to correspond to the hMKK7p splice variant in murine cells. These experiments indicated that the levels of exogenous M K K 7 expressed in Ba/F3-MKK7 cells were at least 30-fold greater than endogenous M K K 7 (Fig. 5.9). This was interesting as the Ba/F3-MKK7 cells exhibited no obvious abnormalities in growth and remained dependent on IL-3 for growth and survival. Moreover, over-expression of human M K K 7 did not appear to affect the expression of endogenous murine M K K 7 , as 5 x 10 cells of both untransfected and Ba/F3-MKK7 cells 5  expressed comparable levels of endogenous M K K 7 (Fig. 5.9).  122  MKK5  F i g u r e 5.8 - D e n d r o g r a m of k n o w n h u m a n M K K and the orthologs of MKK7. A radial dendrogram (Fitsch method) depicting the phylogeny of the catalytic domains of M K K 7 , dHep, c M K K 7 and other known human M K K . The percent identities within the kinase domain of other M K K with human M K K 7 a were: dHep - 69%; M K K 4 - 56%; c M K K 7 - 54%; M K K 6 49%; M K K 3 - 47%; MEK1/2 - 35% and M E K 5 - 30%.  123  Ba/F3  MKK7 e tf) N  50 kDa  e  Cells x IO  -4  Ift  M  myc-MKK7  _  MKK7  Figure 5.9 - Expression of M K K 7 in Ba/F3 and M C / 9 cells.  Immunoblot of endogenous M K K 7 in lysates of Ba/F3 and MC/9 hematopoietic cells and endogenous and exogenous M K K 7 in lysates of a clone of Ba/F3 cells that expressed human myc-tagged M K K 7 . The indicated numbers of cells were lysed and proteins were separated by SDS-PAGE. An antiserum raised against the N-terminus of M K K 7 (a.a.4-26) was used for immunoblotting. The position of endogenous M K K 7 and myc-tagged exogenous M K K 7 are indicated by arrowheads.  124  We used Northern blotting to investigate the expression of M K K 7 in multiple human tissues.  The probe contained the unique N-terminus of M K K 7 and the 5'-end of the kinase  domain which exhibits little sequence homology with other known M K K family members. The probe bound to a single transcript of around 4 kbp in all tissues tested, with highest levels of hybridization occurring with mRNA from skeletal muscle (Fig. 5.10). We also used RT-PCR to examine expression of M K K 7 in a number of human and murine cells and confirmed the P C R products as being derived from M K K 7 by sequencing and restriction analysis. M K K 7 mRNA was present in all tested cell lines corresponding to a number of cell lineages (Table 5.3). Thus, M K K 7 is widely expressed.  5.2.3  MKK7 specifically activates JNK, but not p38 MAPK.  It has been previously  shown that the transient co-expression of mitogen-activated protein kinases with the appropriate upstream M K K leads to in vivo activation of the M A P K . We investigated the substrate specificity of M K K 7 by co-expression of M K K 7 with either J N K l or p38a M A P kinase. Co-expression of M K K 7 with J N K l in HeLa epithelial cells (Fig. 5.11) or Ba/F3 hematopoietic cells (data not shown) without any deliberate stimulation of the cells, resulted in easily detectable activation of JNKl.  Stimulation of these co-transfected cells with hyperosmotic shock (0.2 M NaCl) or U V  light resulted in even greater J N K l activation (Fig. 5.11).  These results suggested that M K K 7  was upstream of J N K l and was activated by stimulation of cells by U V light or hyperosmotic shock. In contrast, co-expression of p38a M A P K with M K K 7 had no effect on activation of p38oc M A P K (Fig. 5.12).  Nor did co-expression of M K K 7 with p38 M A P K result in increased  activation of p38oc M A P K when the transfected cells were stimulated by hyperosmotic shock or U V light (Fig. 5.12). Thus M K K 7 , unlike M K K 4 , specifically activated J N K l , but not p38a M A P kinase.  5.2.4  MKK7 is activated by TNF or multiple stress stimuli.  The c-Jun N-terminal  kinases have been shown to be activated in response to a number of different stimuli including U V  125  in 3  g CU  fl  CJ  cm cu fl "C  Ji  eg .= a  fl •r  .5  u<•>  s .3  S  8 cu C cu °5  kbp  -  1.85  Figure 5.10 - Tissue Distribution of m R N A encoding M K K 7 . Northern blot of M K K 7 mRNA using poly-(A) mRNA isolated from multiple human tissues. A probe was prepared by random priming a 600 bp fragment from the 5'-end of M K K 7 . +  Table 5.3 - Expression determined by R T - P C R .  Cell Line HeLa HepG2 Jurkat Daudi H E K 293 C937 129/J E S cells Ba/F3 MC/9  Species Human Human Human Human Human Human Mouse Mouse Mouse  of m R N A  encoding  MKK7  in various  cell  Cell Lineage Epithelial (Cervical carcinoma) Hepatocarcinoma Acute T cell leukemia B lymphoma Embryonic kidney cell Monocytic Embryonic stem cell IL-3 dependent hematopoietic cell IL-3 dependent mast cell  126  lines  as  pEFBOS JNK1  MKK7 JNK1  c-Jun  Figure 5.11 - Co-expression of M K K 7 with J N K 1 activates J N K 1 . HeLa cells were transfected with constructs encoding GST-JNK 1 and either myc-tagged M K K 7 or the empty vector pEF-BOS (pEF). Transfected cells were split into 3 plates and either left untreated (Con) or stimulated with 0.2 M NaCl (Na) or U V irradiation (UV) for 2 0 min. GST-JNK1 was affinity purified with glutathione-Sepharose and its kinase activity was determined using GST-c-Jun as substrate. The phosphorylation of c-Jun was visualized after SDS-PAGE using autoradiography.  127  U  Z  P  IP: anti-Flag  MKK7 p38  pEFBOS p38  y  z  p  ATF-2 —  «—  — — —  IB:anti-p38  Figure 5.12 - Co-expression of M K K 7 with p38a M A P K activates p38a M A P K . HeLa cells were transfected with constructs encoding Flag-tagged CSBP2/p38a and either myctagged M K K 7 or the empty vector pEF-BOS (pEF). Transfected cells were split into 3 plates and either left untreated (Con) or stimulated with 0.2 M NaCl (Na) or U V irradiation (UV) for 20 minutes. Flag-tagged p38oc M A P K was immunoprecipitated with the M2 antibody and its activity determined using ATF-2 as substrate. The phosphorylation of ATF-2 was visualized after SDSP A G E using autoradiography.  128  light, hyperosmotic shock, protein synthesis inhibitors, heat shock, the pro-inflammatory cytokines IL-1 and TNFa and hematopoietic growth factors (Foltz and Schrader, 1997; Raingeaud et al., 1995).  We tested the ability of these stimuli to activate G S T - M K K 7  transiently over-expressed in HeLa or Ba/F3 cells.  that had been  GST or G S T - M K K 7 was purified from  extracts of cells subjected to various stimuli, and its ability to phosphorylate GST-JNK1 was assessed in an in  vitro  kinase assay. The activity of M K K 7 was increased by treatment of cells  with hyperosmotic shock (11 fold), heat shock (7.5 fold), U V light (5.5 fold), or T N F a (3.5 fold), but not by E G F (Fig. 5.13, top). The ability of these stimuli to activate M K K 7 and to in turn activate JNK1  in vitro  was also assessed. We observed that the ability of M K K 7 to activate  JNK1 was increased in transfected cells treated with T N F a (4 fold), hyperosmotic shock (5 fold) or anisomycin (5 fold - 30 min; 6.5 fold - 45 min) (Fig. 5.13, bottom).  Thus M K K 7 was  activated by all these known activators of JNK.  5.2.5  Activation of MKK7 by IL-3, but not IL-4, in Ba/F3 cells.  We and others  have previously demonstrated the activation of J N K in response to stimulation by the hematopoietic growth factor IL-3 in both Ba/F3 hematopoietic cells and MC/9 mast cells (Foltz and Schrader, 1997; Nagata et al., 1997; Terada et al., 1997). In contrast, the related cytokines IL-4 and IL-13 failed to activate J N K (Foltz and Schrader, 1997). We transiently expressed GSTM K K 7 in Ba/F3 cells, subjected them to various stimuli and assayed the ability of M K K 7 , that was activated  in vivo,  to phosphorylate GST-JNK1  in vitro.  As seen in Fig. 5.14, M K K 7 was  strongly activated in cells treated with IL-3 (5.5 fold), 0.2 M NaCl (4.5 fold) or U V light (3.5 fold). In contrast, M K K 7 was not activated in cells treated with IL-4 (Fig. 5.14), correlating with its inability to activate JNK (Foltz and Schrader, 1997).  5.2.6  Activation of endogenous MKK4  and MKK7  by IL-3, but not IL-4. We  next investigated the activation of endogenous M K K 7 in response to physiological stimuli. In parallel we also investigated the activation of the known J N K activator M K K 4 in order to identify  129  GST  GST-MKK7  GST-JNK1 IB: anti-MKK7  B  GST G S T - M K K 7 r © § B s cj »H «a o « o Z s S cs  U ZU H  «< Z c-Jun IB: anti-MKK7  Figure 5.13 - Activation of M K K 7 by various stimuli. Cells were transiently transfected with vectors encoding GST or GST-MKK7 and aliquots were stimulated as follows. (A) HeLa cells were either left untreated (Con), or stimulated with 100 ng/mL EGF (EGF) for 5 min, 0.2 M NaCl (Na) for 20 min, U V irradiation (UV) for 20 min, 100 ng/mL TNF-a (TNF) for 20 min or heat shocked (Heat) at 42 °C for 20 min. (B) HeLa cells were left untreated (Con), or stimulated with 100 ng/mL TNF-a (TNF) for 15 min, 50 ug/mL anisomycin (Aniso) for 30 or 45 min, or 0.2 M NaCl (Na) for 20 min. In all cases, transiently expressed proteins were affinity precipitated using glutathione Sepharose from samples of lysates that had been normalized for total protein using the Pierce assay. M K K 7 activity was determined by assaying its ability to phosphorylate 1 ug of G S T - J N K l , or by measuring its ability to activate J N K l in vitro by incubating it with 1 ug of GST-JNKl and 50 m M unlabelled ATP for 30 min and determining the ability of an aliquot of this reaction mixture to phosphorylate GST-c-Jun. Phosphorylated substrates were visualized after SDS-PAGE by autoradiography. The precipitated proteins were immunoblotted after SDS-PAGE with an anti-MKK7 antibody (a.a.4-26) to quantitate loading. The position of M K K 7 is indicated by an arrowhead. 130  GST G S T - M K K 7  Z UP P Z P c-Jun IB: a n t i - M K K 7  Figure  5.14 - Activation of MKK7 by I L - 3 , but not I L - 4 , in BaF3 cells. Cells were transiently transfected with vectors encoding GST or G S T - M K K 7 and aliquots were stimulated as follows. Ba/F3 cells were left untreated (Con), or stimulated with 10 ug/mL synthetic IL-3 ( I L 3) for 5 min, 10 ug/mL synthetic IL-4 ( I L - 4 ) for 10 min, 0.2 M NaCl (Na) for 20 min or U V irradiation (UV) for 20 min. In all cases, transiently expressed proteins were affinity precipitated using glutathione Sepharose from samples of lysates that had been normalized for total protein using the Pierce assay. M K K 7 activity was determined by assaying its ability to activate J N K l in vitro by incubating it with 1 ug of G S T - J N K l and 50 uM unlabelled A T P for 30 min and determining the ability of an aliquot of this reaction mixture to phosphorylate GST-c-Jun. Phosphorylated substrates were visualized after SDS-PAGE by autoradiography. The precipitated proteins were immunoblotted after SDS-PAGE with an anti-MKK7 antibody (a.a.4-26) to quantitate loading.  131  potential functional differences.  The antibodies used for immune complex kinase assays  specifically recognized M K K 4 or M K K 7 (Fig. 5.15). We then examined the ability of IL-3, IL-4, anisomycin and 0.2 M NaCl to activate M K K 7 in Ba/F3 cells. Consistent with the results of experiments with transiently expressed M K K 7 , cells treated with IL-3 exhibited an increased activation (8 fold) of endogenous M K K 7 (Fig. 5.16). Cells treated with IL-3 also exhibited an increase in M K K 4 activity (3 fold). In contrast, cells treated with IL-4 failed to activate M K K 7 (Fig. 5.16). Anisomycin and hyperosmotic shock also activated endogenous M K K 7 (4 and 14 fold).  5.2.7  Activation of endogenous MKK4 and MKK7 by TNF. The pro-inflammatory  cytokine T N F a is a well described activator of the J N K pathway. We examined the ability of TNFa to activate M K K 4 and M K K 7 in HeLa cells. As seen in Fig. 5.17, we observed an increase in the activity of both M K K 4 (5 fold) and M K K 7 (2.5 fold).  5.2.8  Activation of endogenous MKK4  receptor for IgG.  and MKK7  by cross-linking the Fc  Activation of JNK has also been shown to follow cross-linking of the  receptor for the Fc fragment of Immunoglobulin G (FcR) on myeloid cells.  The monoclonal  antibody 2.4G2 (a-FcR) binds to both FcgRII and FcgRIII and when cross-linked by a secondary antibody (a-Ig) induces signalling through the FcR.  As seen in Fig. 5.18, cells either left  untreated or incubated with either a-FcR or a-Ig alone exhibited baseline activities of M K K 4 or M K K 7 . However, when cells were pre-treated for 10 minutes with a-FcR and then incubated with a-Ig to aggregate the a-FcR/FcR complexes, an increase in both M K K 4 (7 fold) and M K K 7 (8 fold) activity was observed.  5.2.9  Constitutively active GTPases activate MKK4  and MKK7.  Expression of  constitutively active mutants of the Ras and Rho family of small GTPases activates the JNK family of protein kinases (Bagrodia et al., 1995; Coso et al., 1995; Derijard et al., 1994; Hibi et al., 1993;  132  IP Tt  h  w  ^  ^  o  a  a  igH^  M K K 4 " *"  IP  g  » » «  t  h>  H  |2  bd  y  a  a  g : ^  K  7  •  Figure 5.15 - Specificity of M K K 4 and M K K 7 antibodies. A lysate of WEHI-231 cells was split and immunoprecipitated with antibodies recognizing either M K K 4 , the N-terminus of M K K 7 , or with an irrelevant Ab (Con). Aliquots of immunoprecipitates were analysed by SDS-PAGE and immunobotted with either an antibody recognizing M K K 4 (left) or the C-terminus of M K K 7 (right). A sample containing 5 x 10 cell equivalents of whole cell extract (WCE) was analysed in parallel. The position of M K K 4 (2 isoforms) and M K K 7 are indicated by arrowheads. 5  133  MKK4  MKK7  c-Jun  Figure 5.16 - Activation of endogenous M K K 4 and M K K 7 by I L - 3 , but not I L - 4 . Ba/F3 cells were left untreated (Con), or stimulated with 10 ug/mL synthetic IL-3 (IL-3) for 5 min, 10 ug/mL synthetic IL-4 (IL-4) for 10 min, 0.2 M NaCl (NaCl) for 20 min or 50 ug/mL anisomycin (Aniso) for 30 min. Cellular lysates were immunoprecipitated with antibodies recognizing M K K 4 or M K K 7 . The activity of M K K 4 or M K K 7 was determined by incubating 1 ug of G S T - J N K l with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 ug of GST-c-Jun. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography.  134  MKK4 fl  5  TNF 5 10 mm  MKK7  3  a  TNF 5 10  « m m  c-Jun  Figure 5.17 - T N F a activates endogenous M K K 4 and M K K 7 i n H e L a cells. HeLa cells were left untreated (Con), or stimulated with 100 ng/mL T N F ( T N F ) for 5 or 10 min. Cellular lysates were immunoprecipitated with antibodies recognizing M K K 4 or M K K 7 . The activity of M K K 4 or M K K 7 was determined by incubating 1 ug of GST-JNK1 with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 ug of GST-c-Jun. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography.  135  MKK4  MKK7  c-Jun  Figure 5.18 - Cross-linking of the Fc receptor for IgG activates M K K 4 and M K K 7 in M C / 9 mast cells. MC/9 mast cells were pre-treated with a-FcR (a-FcR) for 10 min as indicated, and then incubated for 10 min in medium alone, or with a rabbit a-rat immunoglobulin antibody (a-Ig). Cellular lysates were immunoprecipitated with antibodies recognizing M K K 4 or M K K 7 . The activity of M K K 4 or M K K 7 was determined by incubating 1 ug of GST-JNK1 with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 ug of GST-c-Jun. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography.  136  Minden et al., 1995). We confirmed that co-expression of GST-JNK1 and Ras  , Rac  or  V12  Cdc42  activated the J N K pathway in HeLa cells (data not shown).  We then co-expressed  G S T - M K K 7 with each of these mutant GTPases, affinity-precipitated the G S T - M K K 7 and assessed its ability to activate G S T - J N K l  in vitro,  using phosphorylation of GST-c-Jun as a  readout of J N K l activity. We found that co-expression of activated mutants of Ras, Rac or Cdc42 and M K K 7 in HeLa cells resulted in marked activation of M K K 7 , demonstrating that this kinase could be activated by signals downstream of each of these GTPases (Fig. 5.19). Similar results were obtained when G S T - M K K 4 was co-expressed with these GTPases in HeLa cells (Fig. 5.19). We also performed similar experiments in the murine hematopoietic cell line Ba/F3. Co-expression of G S T - J N K l and the constitutively active mutants of Rac and Cdc42 resulted in activation of J N K l in Ba/F3 cells; however co-expression of activated Ras with J N K l failed to activate J N K l (data not shown). In keeping with these findings, co-expression of M K K 7 and Rac or Cdc42 in Ba/F3 cells activated M K K 7 ; however the co-expression of activated Ras with M K K 7 was not sufficient to activate M K K 7 (Fig. 5.20).  5.3 Discussion  Our results indicate that human and murine M K K 7 are highly conserved (99% identity, Fig. 5.5) and are most closely related to dHep, a M K K in  Drosophila  ,  exhibiting 69% identity in  the kinase domain (Fig. 5.6). M K K 7 was less related to M K K 4 , M K K 6 or M K K 3 (-45-55% identity), the other mammalian M K K known to be activators of J N K and p38 MAPE1, and was even less similar to MEK1 or M E K 2 (-30-35% identity), the activators of E R K M A P K (Fig. 5.7 and 5.8). We conducted extensive searches of the yeast and invertebrate databases and although we failed to identify M K K 7 in  S.  cerevisiae,  we did identify a highly related C.  elegans  MKK  (cMKK7) that exhibited 54% identity to M K K 7 within the kinase domain (Fig. 5.6) (Wilson et al., 1994). A more detailed inspection of alignments revealed a series of residues or motifs that were conserved among dHep, M K K 7 and c M K K 7 , that were not present in M K K 3 , M K K 4 and  137  GST fl C  CZ  GST-MKK7 fl C  CS  C*5 C3  U C3  GST-MKK4 W  "C  U Z U Z f t J C c J U  fl O  S  S  C « U W S  U Z PJ «  u c-Jun  Figure 5.19 - Activation of M K K 7 or M K K 4 by the small GTPases Ras, Rac and C d c 4 2 . HeLa cells were transiently co-transfected with a vector encoding either GST, GSTM K K 7 or G S T - M K K 4 and pEF-BOS that encoded the constitutively active mutants of Ras (Ras), Rac (Rac), Cdc42 (Cdc42) or an empty vector as a control. In every case, a reporter construct encoding (3-galactosidase in the same pEF-BOS vector was also co-transfected and (3-galactosidase activity was used to normalize the lysates for the expression of transfected genes. Cells transfected with the empty vector and M K K 7 were left unstimulated (Con), or stimulated with 0.2 M NaCl (Na) for 20 min as controls for the kinase assay. The activity of affinity purified M K K 7 or M K K 4 was determined by incubating 1 ug of G S T - J N K l with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 ug of GST-c-Jun. Phosphorylated proteins were visualized after SDS-PAGE by autoradiography.  138  G S T S3  O u  GST-MKK7 S3  03  z  O o  s C3  S3  S3  z  *0  u  c-Jun  Figure 5.20 - Activation of M K K 7 by the small GTPases Rac and C d c 4 2 . Ba/F3 cells were transiently co-transfected with a vector encoding either GST or G S T - M K K 7 and pEFBOS that encoded the constitutively active mutants of Ras (Ras), Rac (Rac) or Cdc42 (Cdc42) or an empty vector as a control. In every case, a reporter construct encoding p-galactosidase in the same pEF-BOS vector was also co-transfected and P-galactosidase activity was used to normalize the lysates for the expression of transfected genes. Cells transfected with the empty vector and M K K 7 were left unstimulated (Con), or stimulated with 0.2 M NaCl (Na) for 20 minu as controls for the kinase assay. The activity of affinity purified M K K 7 was determined by incubating 1 ug of G S T - J N K l with 50 uM of unlabelled ATP for 30 min and testing the ability of an aliquot of this reaction to phosphorylate 1 pig of GST-c-Jun. Phosphorylated proteins were visualized after SDSP A G E by autoradiography.  139  M K K 6 , and vice versa. Particularly striking are differences in the activation loops, with M K K 7 , dHep, and c M K K 7 being characterized by a basic amino acid (S[K/R]AKT), at the same position where in M K K 3 , M K K 4 , and M K K 6 a hydrophobic residue (S[17V]AKT) is found. Overall these patterns of conservation of specific motifs support the notion that M K K 7 is orthologous with dHep and c M K K 7 , and is more distantly related to M K K 4 , M K K 3 and M K K 6 (Fig. 5.6 and 5.7).  It  will be interesting to determine the functional significance of the conserved residues that characterize M K K 7 , dHep and c M K K 7 , and distinguish them from M K K 4 , M K K 3 and M K K 6 . Tournier et al. have recently reported the cloning of two splice variants of murine M K K 7 and the N-terminus of human M K K 7 a (Tournier et al., 1997). The amino acid sequence predicted from their murine M K K 7 a clone is identical to the sequence that we report here, with the exception that it lacks the N-terminus that was present in all of our predicted human and murine M K K 7 splice variants. We were able to immunoblot endogenous M K K 7 in lysates of Ba/F3 hematopoietic cells and MC/9 mast cells using an antiserum raised against the N-terminus of our human and murine M K K 7 sequence (Fig. 5.9), and therefore we are confident that the form of M K K 7 predicted from our human and murine cDNA is indeed expressed in murine cells. While this work was being completed, a number of articles describing the cloning of M K K 7 were published (Holland et al., 1997; Lawler et al., 1997; L u et al., 1997; Moriguchi et al., 1997; Wu et al., 1997; Yao et al., 1997). Holland et al. identified two isoforms of murine M K K 7 , one corresponding to murine M K K 7 a and the other having a unique N-terminus (Holland et al., 1997). Moriguchi et al. cloned murine M K K 7 y and identified two M K K 7 isoforms using an antiserum raised against full length murine M K K 7 y (Moriguchi et al., 1997).  Based on  electrophoretic mobility, we believe that the larger isoform could represent the murine equivalent of our human MKK7(3 (Fig. 5.5). L u et al. have identified another isoform of human M K K 7 that has the same N-terminus and kinase domain as our M K K 7 isoforms but contains 70 amino acids that are not found in any of the M K K 7 isoforms reported to date (Lu et al., 1997). Based on sequence identities among the known M K K s (Fig. 5.7), we investigated the ability of M K K 7 to activate JNK or p38 M A P K and showed that in co-transfection assays M K K 7  140  acted upstream of J N K l (Fig. 5.11), but not p38 M A P K (Fig. 5.12). The identification of J N K l as an in vitro substrate for M K K 7 permitted us to investigate the ability of a range of stimuli to activate transiently expressed M K K 7 .  In HeLa cells, M K K 7 was strongly activated by  hyperosmotic shock, U V light, anisomycin, heat shock and to a lesser extent T N F a (Fig. 5.13). We observed no detectable activation of M K K 7 in HeLa cells treated with E G F (Fig. 5.13, top). These results are similar to that seen by Holland et al. with PDGF in NIH 3T3 cells (Holland et al., 1997). In Ba/F3 hematopoietic cells, hyperosmotic shock and U V light also activated M K K 7 (Fig. 5.14). Our observations that stimulation of Ba/F3 cells with IL-3 increased the activity of M K K 7 (Fig. 5.14 and 5.16) correlates with recent observations that J N K was activated by a range of hematopoietic growth factors including IL-3, GM-CSF, G-CSF, EPO or SLF (Foltz and Schrader, 1997; Nagata et al., 1997; Rausch and Marshall, 1997; Terada et al., 1997).  Moreover, our  observation that IL-4 failed to activate M K K 7 (Fig. 4c, 5c) correlates with the inability of IL-4 to activate the Ras, E R K , p38 or JNK M A P kinase pathways in hematopoietic cells (Duronio et al., 1992; Foltz et al., 1997; Foltz and Schrader, 1997; Satoh et al., 1991; Welham and Schrader, 1992). Co-expression of activated mutants of Ras, Rac or Cdc42 with M K K 7 in HeLa cells resulted in readily detectable activation of M K K 7 and M K K 4 (Fig. 5.19). This is consistent with previous work which demonstrated that the Ras and Rho family of small GTPases are capable of activating JNK (Bagrodia et al., 1995; Coso et al., 1995; Derijard et al., 1994; Hibi et al., 1993; Minden et al., 1995). Slightly different results were obtained in Ba/F3 cells where M K K 7 was activated by co-expression of activated Rac or Cdc42, but not Ras (Fig. 5.20).  The inability of  Ras to activate M K K 7 was not surprising as a constitutively active Ras was insufficient to activate JNK in Ba/F3 cells, although IL-3-induced J N K activation was blocked by expression of a dominant-negative mutant of Ras (Terada et al., 1997). Taken together, these results support the notion that Ras is necessary but not sufficient for JNK activation. Our evidence that IL-3 increased M K K 7 activity (Fig. 5.14 and 5.16), but that Ras alone failed to increase M K K 7 activity (Fig. 5.20), is also consistent with data on activation of JNK by G-CSF, which is structurally related to  141  IL-3 and acts through a similar receptor. These experiments showed that activation of JNK in cells stimulated with G-CSF depended upon both an intact Ras signalling pathway, as well as a specific tyrosine residue in the G-CSF receptor (Rausch and Marshall, 1997). Together these data suggest that activation of M K K 7 in response to IL-3 involves both activation of the Ras pathway and an as yet unknown signal. The receptor for the Fc fragment of Immunoglobulin G (FcR) has many important roles in the immune system (Takai, 1996).  These include the phagocytosis of Ig-coated particles by  macrophages and neutrophils, the antibody-dependent cell mediated cytotoxicity by N K cells, the down-regulation of signalling through the BCR, and the release of T N F a and other mediators by macrophages and mast cells. Our observation that ligation of FcR results in the activation of both endogenous M K K 4 and M K K 7 in MC/9 mast cells (Fig. 5.18) supports previous findings that signalling through the FcR activates J N K in bone-marrow derived macrophages (Rose et al., 1997). Recent studies have shown that signalling through the J N K pathway is required for the production of T N F a in MC/9 mast cells (Ishizuka et al., 1997). As signalling through the FcR activates JNK through M K K 4 and M K K 7 , it will be important to determine their individual roles in the production of TNFa in mast cells. The existence of multiple activators of JNK which are responsive to different stimuli was established by the fact that MKK4-deficient ES cells still exhibited activation of JNK in response to hyperosmolarity and U V light, but not heat shock or anisomycin (Derijard et al., 1995; Lin et al., 1995). In that M K K 7 is activated by all of the above stimuli (Fig. 5.13, 5.14 and 5.16), and M K K 7 mRNA is present in ES cells (Table 5.3), the failure of MKK4-deficient ES cells to activate JNK in response to heat shock or anisomycin is paradoxical. It is possible that, although M K K 7 is expressed in ES cells, it is not activated by heat or anisomycin because of cell-specific differences in upstream activators. This notion is supported by a recent report that in K B and U937 cells TNFa activates M K K 7 , but not M K K 4 (Lawler et al., 1997; Moriguchi et al., 1997), whereas in HeLa cells we observed that T N F a activates both M K K 7 and M K K 4 (Fig. 5.17), an observation recently reported by Wu et al. (Wu et al., 1997). M K K 4 has also been recently  142  reported to be activated in normal bone marrow-derived macrophages treated with T N F a , supporting the notion that these kinases will be regulated differently depending on their cellular context (Winston et al., 1997). Furthermore, Wu et al. have reported that ASK1 and G C K activate M K K 7 in preference to M K K 4 , whereas M E K K 1 and M E K K 2 activate both M K K 4 and M K K 7 to comparable levels (Wu et al., 1997). The functional significance of the activation of M K K 7 is unclear but its activation by physiological stimuli such as IL-3 (Fig. 5.14 and 5.16), by ligation of immunoregulators such as FcR (Fig. 5.18), CD40, BCR, CD3 (Foltz et al., 1998, M . Luckach and R. Salmon, unpublished observations), and by the GTPases Ras, Rac and Cdc42 (Fig. 5.19 and 5.20) suggests that the role of M K K 7 will not be confined to stress responses. The existence of an ortholog of M K K 7 in C. elegans and our failure to identify an ortholog in S. cerevisiae suggest that M K K 7 arose in evolution during the transition from single cellular to multicellular organisms. The notion that M K K 7 is involved in processes important for multicellular organisms, such as embryonic development, chemotaxis or apoptosis, is in keeping with evidence that mutations in dHep and Bsk, the Drosophila homologue of J N K l , result in a similar failure of epithelial cell movement and dorsal closure (Glise et al., 1995; Riesgo-Escovar et al., 1996; Sluss et al., 1996). Holland et al. have demonstrated that M K K 7 is able to partially complement a deficiency of dHep in Drosophila (Holland et al., 1997).  This demonstrates that M K K 7 is highly conserved functionally and  suggests that it may play a role in embryological development in mammals.  The embryonic  lethality resulting from disruption of the M K K 4 gene in mice also points to the importance of normal J N K signalling during embryonic development (Ganiatsas et al., 1998; Nishina et al., 1997; Yang et al., 1997), and indicates that M K K 4 and M K K 7 have discrete physiological functions. Future work detailing the function of individual activators of J N K is likely to reveal roles in multiple aspects of development and other physiological processes, hematopoiesis and immune responses.  143  including  C H A P T E R 6 - Conclusion  The stress-activated protein kinases, p38 M A P K and JNK, are activated by diverse stimuli acting on a cell. When I began working in this field, these kinases were known to be activated by cellular insults including hyperosmotic shock, U V irradiation, heat shock and the pro-inllammatory cytokines TNFa and IL-1.  In contrast, growth factors such as E G F and PDGF failed to activate  these enzymes to comparable levels. We had some biochemical evidence that a protein with similar isoelectric point and electrophoretic mobility as p38 M A P K was tyrosine phosphorylated by the hematopoietic growth factors, IL-3 and S L F (Welham and Schrader, 1992), and we hypothesized this unknown phosphoprotein was p38 M A P K .  I demonstrated that p38 M A P K was indeed  tyrosine phosphorylated and activated by these growth factors. Furthermore, we demonstrated that p38 M A P K activity was required for the activation of M A P K A P kinase-2 by these cytokines. Previous work in our laboratory indicated that IL-4 was unable to activate E R K M A P K or Ras (Duronio et al., 1992; Welham et al., 1992; Welham et al., 1994), and we were interested to examine the ability of IL-4 to activate p38 M A P K . I found that IL-4 was unable to activate p38 M A P K , or M A P K A P kinase-2, consistent with the notion that M A P K A P kinase-2 was a substrate of p38 M A P K in vivo. Growth factors are important not only for the proliferation, but also for the survival of hematopoietic cells. We hypothesized that p38 M A P K would regulate some aspect of cytokine action. Indeed, we demonstrated using the inhibitor, SB 203580, that the activity of p38 M A P K was required for D N A synthesis. However, an SB 203580-resistant mutant of p38a M A P K failed to restore D N A synthesis, suggesting that p38a M A P K was not sufficient for D N A synthesis. The activation of M A P K A P kinase-2 was completely restored by the SB 203580-resistant mutant of p38a M A P K after hyperosmotic shock, suggesting that M A P K A P kinase-2 activity was not required for D N A synthesis. However it is not clear if p38a M A P K activity was completely restored under normal cell culture conditions. Since SB 203580 also inhibits p38p M A P K , these data suggest a role for p38p M A P K in D N A synthesis, or perhaps another target of this inhibitor.  144  When I began working in the stress-activated protein kinase field, all stimuli that activated p38 M A P K also activated JNK. I hypothesized that the hematopoietic growth factors that activated p38 M A P K would also activate J N K . Indeed, I demonstrated that IL-3, G M - C S F or S L F activated both 45 and 55 kDa isoforms of JNK1 and JNK2. Consistent with the failure of IL-4 to activate p38 M A P K , I also hypothesized that IL-4 would fail to activate JNK. The inability of IL-4 to activate Ras also supported the notion that IL-4 would fail to activate J N K as growth factors such as E G F activated J N K in a Ras-dependent fashion. Indeed, I found that IL-4 failed to activate JNK in hematopoietic cells. Together, these results indicated that IL-4 was unique among cytokines that we examined in its failure to activate any M A P K family member, or the small GTPase Ras. These findings lead to many important biochemical questions, including what is the role of Ras for the activation of p38 M A P K , do these hematopoietic growth factors activate Rac or Cdc42, and what are the biological implications of IL-4 failing to activate Ras or any M A P kinases? The JNK family of protein kinases was known to be regulated by a single M A P K kinase, M K K 4 , at the time I was studying the activation of J N K by hematopoietic growth factors. Therefore, we presumed the activation of J N K by hematopoietic growth factors was through M K K 4 . Consistent with this hypothesis, we detected an increase in the threonine phosphorylation of M K K 4 after stimulation with G M - C S F or SLF.  However, the phosphorylation was greater  with SLF than with G M - C S F or NaCl despite the fact that these stimuli activate J N K to a similar extent. We hypothesized this finding reflected the existence of another unidentified J N K kinase in MC/9 mast cells that was activated in cells after treatment with G M - C S F or NaCl.  Several  independent reports supported the existence of another JNK kinase (Meier et al., 1996; Moriguchi et al., 1995; Nishina et al., 1997a; Yang et al., 1997b). We screened the expressed sequence tags database and identified a novel JNK kinase, M K K 7 , with a great degree of homology to M K K 4 . We demonstrated this enzyme was activated by NaCl and IL-3, a cytokine that signals through a receptor that is shared with G M - C S F . Furthermore, M K K 7 was expressed in MC/9 mast cells,  145  implicating M K K 7 as the molecule we hypothesized to be activated after cells were treated with hyperosmolarity or GM-CSF. My demonstration that J N K and p38 M A P K were activated by hematopoietic growth factors provided the first evidence that these enzymes were strongly activated by growth factors, in contrast to previous findings with EGF or PDGF.  The activation of both of these enzymes by  G M - C S F and S L F were on the same order of magnitude as that seen with hyperosmotic shock. Since then several physiologically relevant stimuli have been identified as activators of JNK and p38 M A P K including the hematopoietic growth factors Erythropoietin and Thrombopoietin, and receptors involved in the regulation of the immune system including the Fc receptors for IgG or IgE, the B cell antigen receptor and CD3, a component of the T cell antigen receptor.  These  findings are consistent with the notion that these enzymes have important physiological functions beyond simply a response to stress, and are supported by proposed roles for these kinases in cytokine production, apoptosis, proliferation, tumorigenesis, embryogenesis and organogenesis. However, the specific functions of these enzymes in mammalian cells, and indeed in the whole organism is poorly understood.  Most of our understanding of the function of these  enzymes are inferred through the use of over-expressed dominant negative mutants that act to sequester either upstream activators or downstream effectors of these kinases. This approach is useful as it can potentially tell us something about enzyme functions, but it has inherent problems as one never really knows if the dominant negative specifically inhibits the intended pathway. The ability to use both dominant negative proteins and a specific inhibitor has allowed a rapid determination of the functions of p38a M A P K and p38(3 M A P K . However, the inhibitor also has inherent problems as unknown targets for the compound might confound the interpretation of data. The production of new p38 M A P K mutants that are resistant to SB 203580, but retain their substrate specificity, will hopefully provide more plausible data on the biological function of these proteins and maybe differentiate between functions of p38a M A P K and p38|3 M A P K isoforms in vivo.  146  Genetic analysis of the function of these enzymes is certainly a major focus of the future research in this field.  Mice lacking M K K 4 provided the first evidence for the non-redundant  functions for M K K 4 and M K K 7 , as cells lacking M K K 4 were still able to activate JNBC (Nishina et al., 1997). However, these mice die in utero, and as such did not provide that much information on the role of JNK kinase activity in vivo. A better understanding of the role of M K K 4 in adult mice will likely require tissue specific or conditional disruption of this gene using the Cre recombinase. Mice lacking JNK and p38 M A P K are also being generated. As there are three J N K genes and four p38 M A P K genes, the determination of in vivo function may require the disruption of multiple genes. However, mice with the JNK1, the JNK2 or the JNK3 gene disrupted had phenotypes, indicating that these enzymes are not entirely redundant at least in some tissues (Yang et al., 1997a; Yang et al., 1998a, Dong et al., 1998). The field is moving rapidly, and as our understanding grows, better experiments are being devised to address these fundamental questions.  Future work may proceed to generate mice  expressing SB 203580-resistant isoforms of both p38cc M A P K and p38(3 M A P K  under  endogenous promoters. As the mechanism of action of this drug is very well established, these mice would address issues such as other targets of the compound. Most importantly, ES cells derived from these mice could then be used to introduce mutations into other kinases to confer sensitivity to SB 203580 (Gum et al., 1998). 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