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Cytokine regulation of glycogen synthase kinase-3 (GSK-3) Vilimek, Dino Alexander Henry 2001

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C Y T O K I N E R E G U L A T I O N O F G L Y C O G E N S Y N T H A S E KINASE-3 (GSK-3) by DINO A L E X A N D E R H E N R Y V I L I M E K B.Sc. (Hon.), University of British Columbia, 1998 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F SCIENCE in T H E F A C U L T Y O F G R A D U A T E STUDIES {t^r&meMa\. '^hMm^W^am^l. We accept this thesis as conforming to the required standard T H E UNIVERSITY O F BRITISH C O L U M B I A October 2001 © Dino Alexander Henry Vilimek, 2001 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that p e r m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g ain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada A B S T R A C T Cytokines are soluble growth factors that are essential for the continued survival of numerous hematopoietic cell lines. For example, in cells of the erythromyeloid lineage, interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor ( G M - C S F ) are powerful anti-apoptotic agents. Glycogen synthase kinase-3 (GSK-3) is an ubiquitous and yet enigmatic protein kinase implicated in various cellular functions, including glycogen metabolism, embryonic development, cell survival, proliferation, and protein translation. Numerous extracellular stimuli can promote the inactivation of G S K - 3 through serine phosphorylation of the amino-terminal region. Growth factors are among the agents capable of inactivating G S K - 3 , presumably to promote cell survival and proliferation. Interestingly, an extensive literature search has found no studies examining G S K - 3 in the context of cytokine-mediated signaling. This study is the first to demonstrate the elevation of G S K - 3 a and G S K - 3 R serine phosphorylation by IL-3, IL-4, and G M - C S F in several hematopoietic cell lines. IL-4 required PI3-K activity, and presumably P K B activity, for full G S K - 3 modification, while IL-3 and G M - C S F did not. Although M A P K and p 7 0 S 6 K did not regulate the control of G S K - 3 by IL-3, IL-4 or G M - C S F , P K C did demonstrate a strong effect. Inhibition of P K C activity, through the addition of pharmacological compounds, led to a dramatic abrogation of G S K - 3 serine phosphorylation induced by all three cytokines. However, the lack of specificity of these inhibitors made it difficult to identify the class of P K C or the individual isoforms responsible for G S K - 3 regulation. Furthermore, the inhibition of diacylglycerol production also impacted on the cytokines' ability to phosphorylate G S K - 3 . Diacylglycerol is a necessary component for the activation of numerous P K C isoforms. However, like the P K C inhibitors, some evidence suggests that these P L C inhibitors may be acting on non-specific targets. Interestingly, increased serine phosphorylation of GSK -3 did not appear to correlate with a decrease in catalytic activity, although the quality of the assay system is questionable. Regardless, this study is the first to examine GSK -3 in cytokine-mediated signaling, and to implicate PI3-K and P K C as mediators of this event. Further studies w i l l help develop a clear signaling model for the regulation of GSK -3 and determine GSK -3 ' s role in the cellular effects induced by cytokines. i i i T A B L E O F C O N T E N T S Page A B S T R A C T i i T A B L E O F C O N T E N T S iv LIST O F FIGURES vi LIST O F ABBREVIATIONS v i i i A C K N O W L E D G E M E N T S x DEDICATION xi C H A P T E R 1: INTRODUCTION 1.1. Hematopoiesis 1 1.2. Hematopoietic Growth Factors 4 1.2.1. Interleukin-3 4 1.2.2. Granulocyte-Macrophage Colony Stimulating Factor 5 1.2.3. Interleukin-4 6 1.3. Cytokine Receptors 7 1.4. Cytokine Signaling Pathways 10 1.4.1. J A K 10 1.4.2. M A P K 14 1.4.3. p90 r s k 19 1.4.4. Phosphatidylinositol 3'-kinase (PI3-K) 20 1.4.5. Protein kinase B 21 1.4.6. Protein kinase C 26 1.4.7. Protein kinase A 31 1.4.8. p 7 0 s 6 K •. 34 1.5. Glycogen synthase kinase-3 (GSK-3) 35 1.5.1. Isoforms 35 1.5.2. Regulation 36 1.5.3. Embryonic development 39 1.5.4. Substrates 40 iv C H A P T E R 2: M A T E R I A L S A N D M E T H O D S 2.1. Materials 44 2.1.1. Chemicals 44 2.1.2. Disposables 45 2.1.3. Proteins/Peptides 46 2.1.4. Antibodies 46 2.2. Methods 47 2.2.1. Ce l l culture 47 2.2.1.1. F D C - P l , M C / 9 a n d M C / 9 ( P K B - E R ) 47 2.2.1.2. TF-1 47 2.2.2. Cytokine stimulation 48 2.2.3. Protein Biochemistry 48 2.2.3.1. Immunoprecipitation 48 2.2.3.2. Immunoblotting 49 2.2.3.3. Stripping blots 50 2.2.4. Kinase Assays 50 2.2.4.1. G S K - 3 a 50 2.2.4.2. P K B a 51 2.2.5. L i p i d Analysis 51 2.2.5.1. Thin layer chromatography (TLC) 51 2.2.5.2. Densitometry analysis 52 C H A P T E R 3: R E S U L T S 3.1. Rationale and Hypothesis 53 3.2. Results 53 C H A P T E R 4: CONCLUSIONS 91 C H A P T E R 5: R E F E R E N C E S 105 v LIST O F FIGURES Page C H A P T E R 1: INTRODUCTION Figure 1.1. Hematopoiesis 3 Figure 1.2. Cytokine receptors 8 Figure 1.3. J A K / S T A T pathway 12 Figure 1.4. M A P K pathways 15 Figure 1.5. P I 3 - K / P K B pathway 22 Figure 1.6. Diacylglycerol formation 27 Figure 1.7. P K C isoforms 28 Figure 1.8. P K A activation 32 C H A P T E R 3: R E S U L T S Figure 3.1. Effect of cytokines on serine phosphorylation of GSK-3a/f3 56 Figure 3.2. Effect of cytokines on GSK-3a/p is post-translational 59 Figure 3.3. Role of PI3-K in G S K - 3 regulation by cytokines 61 Figure 3.4. P K B activity is sufficient for G S K - 3 serine phosphorylation 64 Figure 3.5. The role of M A P K in G S K - 3 regulation 67 Figure 3.6. The role of p 7 0 s 6 K in G S K - 3 regulation 69 Figure 3.7. Ce l l specific regulation of G S K - 3 by forskolin 71 Figure 3.8. Effect of phorbol esters on G S K - 3 a/p 73 Figure 3.9. G S K - 3 phosphorylation via P K C in M C / 9 76 Figure 3.10. G S K - 3 phosphorylation via P K C in TF-1 77 Figure 3.11. G S K - 3 phosphorylation via P K C in FDC-P1 78 vi Figure 3.12. PKC8 does not regulate GSK-3 phosphorylation by cytokines 80 Figure 3.13. Diacylglycerol formation upon cytokine treatment 83 Figure 3.14. Role of PLC in GSK-3 regulation by cytokines 85 Figure 3.15. Regulation of GSK-3a activity 88 Figure 3.16. Regulation of PKB activity 90 vi i LIST O F ABBREVIATIONS A T F - 1 activating transcription factor-1 A T F - 2 activating transcription factor-2 A T P adenosine triphosphate B C G F B-cel l growth factor c D N A complementary D N A C / E B P CCAAT/enhancer core binding protein C R E B c A M P response element binding D A G diacylglycerol D M S O dimethylsulfoxide D N A deoxyribonucleic acid D T T dithiothreitol eIF4E elongation initiation factor 4E E R K extracellular signal-related kinase G A D D 1 5 3 growth arrest and DNA-damage-inducible 153 G A S y-IFN activated sequences G M - C S F granulocyte-macrophage colony stimulating factor Grb2 growth factor receptor-bound protein 2 G S K - 3 glycogen synthase kinase-3 TL-3 interleukin-3 IL-4 interleukin-4 I L K integrin-linked kinase IRS insulin receptor substrate J A K Janus kinase J H J A K homology J N K Jun amino-terminal kinases kDa kilodalton M A P K mitogen-activated protein kinase M E F 2 C myocyte enhancer factor 2C M E K M A P K kinase M H C major histocompatibility complex m R N A messenger ribonucleic acid m T O R mammalian target of rapamycin N K natural killer cells N-linked amino-terminus O H hydroxide P A K p21-activated protein kinase P C phosphatidylcholine P D K 1 phosphoinositide-dependent kinase 1 P H pleckstrin homology PI3-K . phosphatidylinositol 3'-kinase P K A protein kinase A P K B protein kinase B P K C protein kinase C v i i i P L C phospholipase C P L D phospholipase D P M A phorbol myristic acid P M S F phenylmethylsulfonyl fluoride PS phosphatidylserine P T B phosphotyrosine binding R S K ribosomal S6 kinase S A P K stress-activated protein kinases SH2 sre homology 2 She SH2-containing Sos son of sevenless S R F serum response factor S T A T signal transducer and activator of transcription T L C thin layer chromatography ACKNOWLEDGEMENT Behind every individual achievement is the team of people who provided support. I would be hard-pressed to find a more kind and empathetic supervisor than Vincent Duronio. M y sincerest gratitude for letting me join your team. To Drs. Krystal, Salh, Brownsey, and Steinbrecher, I 'd like to say thanks for taking the time to review my work. To my colleagues who became my friends, you made it fun to come to work everyday. Thank you to Payman, Trish, Tina, Sarwat, Jen, Carmen, and Kathy. Remember to raise the roof and to eat chi l l i everyday. To Dad, M i k e , Jane and Taylor, I thank you for supporting me. Your encouragement is invaluable and never forgotten. To my best friend. Words fail to describe how much you mean to me...and thanks for helping me stuff tips, stimulate cells, and make gels. It was always fun with you there. I love you, Christiane. x To Mom xi C H A P T E R 1: I N T R O D U C T I O N 1.1. Hematopoiesis The numerous types of cells that comprise mammalian blood perform a variety of crucial physiological functions. The formation and development of these cells is termed hematopoiesis. The process originates with a population of pluripotent stem cells from which every cell type in the blood derives.1 To ensure continual and extensive cell production, stem cells have an unlimited capability for self-renewal and can differentiate into any one of the types of cells found 2 3 in the peripheral blood. ' Although stem cells reside primarily in the bone marrow, these cells are also able to circulate through the bloodstream. Unfortunately, research on stem cells has been complex due to their low numbers in the body and the difficulty of culturing them in vitro. However, recent advances are facilitating studies using stem cells. 4 ' 5 Pluripotent stem cells can exist in several states. A large portion of the population of stem cells are quiescent, halted at an intermitotic phase of the cell cycle which can be maintained for a long period of time. 6 This reserve pool of resting cells ensures a continual capacity for hematopoiesis. Meanwhile, a smaller population of stem cells can continue to divide without differentiating, allowing for a self-renewal capacity. 7 Finally, a very small group of stem cells can divide and differentiate into progeny cells of a specific type.8 Hematopoiesis is an on-going process beginning in the embryonic stage. Early on, hematopoiesis takes place in the embryonic yolk sac.9 In humans, between the third and seventh gestation months, stem cells migrate to the fetal liver and then later to the spleen. 9 ' 1 0 After seven months, and from then on, hematopoiesis occurs primarily in the bone marrow. 1 1 Most large bones participate in blood cell production, including the limb bones, pelvis, sternum, vertebra, 1 and ribs. In times of severe hematopoietic stress however, the liver and the spleen can once again lend themselves to blood cell production. 1 2 The developmental path a stem cell wi l l follow is dependent on the microenvironment, particularly the presence of soluble growth factors and matrix components. Blood cells can be categorized into two major lineage groups (Figure 1.1). The erythromyeloid lineage gives rise to neutrophils, macrophages, eosinophils, basophils, mast cells, erythrocytes, megakaryocytes and platelets. Neutrophils and macrophages provide protection against bacteria by engulfing foreign pathogens through phagocytosis. Eosinophils defend against parasitic infections and downregulate allergic responses, while basophils participate in inflammatory and allergic responses through IgE binding and granular release. Meanwhile, mast cells, which are morphologically and functionally similar to basophils, are found mainly in the tissues. Erythrocytes carry oxygen to the tissues through a hemoglobin carrier, while megakaryocytes produce platelets, which in turn promote healing of wounds to the circulatory system. The other major group is the lymphoid lineage, which includes B and T lymphocytes and natural killer cells. B cells secrete immunoglobulins against specific antigens, while T cells provide a cellular-based immunity. Natural killer cells, which are less specific in their targets, are also cytotoxic cells that act on microbes and tumour cel ls . 1 4 Hematopoiesis is an essential function for survival. Humans are estimated to produce some 3.7 X 10 1 1 blood cells a day. A s such, there exists a need for delicate balance between blood cell production/survival and cell death via apoptosis. This is often achieved through the presence or absence of necessary growth factors. If this process becomes unbalanced however, the physiological results can range from immune deficiencies to leukemia. 2 SONE MARROW THYMUS 2 * !L-3 GM-CSF I 1_ Monocyte Myeloid call Neutrophil § Stem celf. i ;sirc Eosinophil Basophil L @ • • * ®r Plateieis Bcelt precursor !«§;§ | .- lymphoid; , j A ' 7 ' - . celt , !-Pre-B 'Mature 8 cell< cat/:/- ri~~:^.-... .w&k MM Plasma cell 3L0C0/TISSUES Monocyte Macrophage Neutrophil eosinophil Basoohil/Mast Cell © © ,' Platelets ' Mature Activated ; B;lymphocyte 8 lymphocyte 1 m Annbbdies: PRECURSOR CELLS Pfe-T Mature-T: ceil celt COMMITTED PROLIFERATING/DIFFERENTIATING CELL LINE CELLS Mature Activated iyrphocyte Tfymphocy e EFFECTOR CELLS/MOLECULES Figure 1.1 - Hematopoiesis. The development of all blood cells begins with a pluripotent stem cell that is capable of differentiating into members of both the myeloid and lymphoid lineages. This process typically takes place in the bone marrow, with the effector cells later moving into the blood and surrounding tissues to perform their critical functions.3 1.2. Hematopoietic Growth Factors A n efficient means of communication between cells is necessary in any large multicellular organism. Neighbouring cells are able to communicate through direct cell-to-cell contact, while cells of the nervous system can propagate signals via neurotransmitters at synaptic connections. In contrast, cells of the blood are mostly regulated by the release of proteins within the circulatory system. These secreted peptides can travel through the bloodstream to target cells that are far away from the original source. Soluble hematopoietic growth factors called cytokines are glycosylated proteins that can regulate cell differentiation, survival, and proliferation of pluripotent and mature blood cell lines. Cytokines can also mediate direct functional responses in many blood cells. To date, a wide range of cytokines have been characterized, however, many of these exhibit redundant responses as well as functional pleiotropy. 1 5 In fact, there is ample evidence that various cytokines are evolutionarily related. For example, the genes for IL-3, EL-4, and G M - C S F are all clustered on chromosome 5 of the human genome. 1 6 1.2.1. Interleukin-3 (IL-3) Interleukin-3 (IL-3) is a hematopoietic growth factor responsible for the growth, survival and proliferation of pluripotent and committed cell types, particularly those of the erythromyeloid lineage. The c D N A encoding the murine IL-3 was first identified in 1984 and the human form two years later. 1 7 ' 1 8 The human protein is comprised of 152 amino acids (murine 166 amino acids) and contains two putative N-linked glycosylation sites and a single disulfide bond. While the disulfide linkage is necessary for cytokine activity, the glycosylation process only promotes 4 an increase in JL-3'S half life in vivo}9'20 Remarkably, the homology between the human and murine forms is only 29%, which is consistent with the high species specificity of TL-3. The cytokine is produced primarily by C D 4 + T helper cells, but other sources, such as thymic 21 23 epithelial cells, and stimulated mast cells and eosinophils can also contribute small amounts. JJL-3's effects are generally directed at cells of the hematopoietic system. A s Figure 1.1 illustrates, IL-3 can promote differentiation of hematopoietic cells in the bone marrow, particular those of the myeloid lineage. As well , IL-3 promotion of cell survival and proliferation in progenitor and terminally differentiated blood cell types is well documented. In fact, several myeloid and lymphoid cell lines depend on JL-3 to protect against apoptosis. For example, EL-3 is sufficient for the survival and proliferation of the M C / 9 mast cell line, the BaF/3 pro B-cel l line, the TF-1 myelomonocytic cell line and the hematopoietic myeloid progenitor FDC-P1 cell l ine . 2 4 " 2 7 IL-3 is also a potent regulator of the functional activity of mast cells, basophils, eosinophils, and macrophages. 2 8" 3 1 For example in mast cells, JJL-3 induces the synthesis of vasoactive histamines. 1.2.2. Granulocyte-macrophage colony stimulating factor (GM-CSF) Granulocyte-macrophage colony stimulating factor ( G M - C S F ) is a versatile cytokine that regulates specific sets of hematopoietic cells. Murine G M - C S F was first cloned in 1984, and the human gene a year later. 3 2 ' 3 3 The mature human G M - C S F , derived from a precursor with a secretory signal sequence, is a 127 amino acid protein sharing only 54% homology with the 124 amino acid murine form. G M - C S F contains two sites for N-linked glycosylation that enhance biological activity, and two disulfide bonds of which one is essential for cytokine act ivi ty. 3 4 ' 3 5 5 There exists a wide variety of cells capable of producing G M - C S F , including T cells, endothelial cells, macrophages, mast cells, and stromal cells in the bone marrow. 3 6" 4 0 Similar to IL-3, G M - C S F regulates differentiation of myeloid blood cells. A s its name implies, it is also an essential factor for the growth and differentiation of macrophages and granulocytes such as neutrophils and eosinophils. The factor-dependent cell lines M C / 9 , TF-1 , BaF/3, and F D C - P 1 all exhibit long-term proliferation in the presence of G M - C S F . 4 1 " 4 4 This cytokine is also a mediator of certain cell functions in neutrophils, macrophages, and eosinophils. 4 5" 4 7 For example, G M - C S F can enhance the phagocytic capabilities of neutrophils and the cytotoxicity of eosinophils. 4 5 ' 4 7 1.2.3. Interleukin-4 (IL-4) Interleukin-4 (IL-4) is a pleiotropic cytokine responsible for various cell functions. First identified as a B-cel l growth factor ( B C G F ) , both the murine and the human form of IL-4 were cloned in 1986. 4 8 ' 4 9 Mature human and murine IL-4 are derived from precursor proteins containing hydrophobic secretory signal sequences, which are cleaved to produce the 129 amino acid and 120 amino acid species respectively. IL-4 has three N-linked glycosylation sites and six cysteine residues involved in disulfide linkages, the latter being essential for cytokine activity. 5 0 ' 5 1 This cytokine is produced primarily by C D 4 + T cells, but also in smaller amounts by mast cells and basophils. 5 2" 5 4 IL-4 can elicit responses in a diverse subset of cells from many hematopoietic lineages. B cells and basophils differentiate in response to this cytokine, and IL-4 can direct differentiation of naive T cells into cells of the T H 2 subclass of T cel ls . 5 5 " 5 7 Unlike IL-3 and G M - C S F , interleukin-6 4 does not typically act as a growth factor on its own. However, EL-4 can synergize with co-stimulatory agents to support proliferation of various cell lines. For example, IL-4 in conjunction with JJL-IO can support proliferation of M C / 9 mast cells, however, neither cytokine alone can accomplish the same task. 5 8 F D C - P 1 and M C / 9 cells grown in EL-4 supplemented media display survival for only a few days with minimal proliferation. 5 9 ' 6 0 In fact, EL-4 can inhibit proliferation of various cell types, including FDC-P1 cells treated with EL-3 and G M - C S F . 6 1 On the other 62 hand, TF-1 cells can proliferate in response to EL-4 without co-stimulatory agents. Therefore EL-4's ability to upregulate proliferation is dependent on cell type. A s well , EL-4 can also trigger 63-67 functional responses in B cells, T cells, macrophages, mast cells, and endothelial cells. " For example, EL-4 induces the expression of M H C II on the cell surface of B cel ls . 6 3 A s well, this cytokine's role in the allergic response is of particular interest due to its unique ability to induce immunoglobulin class switching to IgE . 6 8 1.3. Cytokine receptors Soluble cytokines exert their effects through their respective receptor molecules found on the surface of various cell types. These receptors are biochemically quite similar, but are unique in comparison to other receptor families. The type I cytokine receptor family includes the receptors for EL-3, G M - C S F , EL-4 and many more. 6 9 They are single-spanning transmembrane glycoproteins lacking intrinsic kinase activity. Many cytokines utilize a dimeric receptor system comprised of a unique receptor for each ligand (a chain), and a common chain to assist in transduction of an intracellular signal (Figure 1.2).70 Both chains contain a 200 amino acid stretch in the extracellular domain with four conserved cysteine residues and a conserved region 7 IL-3/GM-CSF/IL-5 IL-4 Receptor Receptor XX GM-CSF/IL-3/IL-5 receptor a-chain domain p Haemopoietin domain i~l Box 1 or 2 I 2 | homology domain Transmembrane domain • SH2 homology domain a p. XX I 1 I . 5 IL-4-Ra Figure 1.2 - Cytokine receptors. Schematic display of the type I cytokine receptors for IL-3, G M -CSF, and IL-4. A l l three cytokines bind to a unique receptor (a chain), which associates with a shared receptor component (P or y chain). The intracellular portions of these receptor complexes contain domains essential for protein recruitment and propagation of an intracellular signal.6 8 8 containing a W S X W S sequence. A s well , some receptor subunits possess Box 1/Box 2 domains in the cytoplasmic region of the receptor that are important for the binding of associated J A K proteins. B o x 1 is a membrane proximal region rich in proline residues while Box 2 is less conserved, and typically contains several hydrophobic residues followed by negatively charged 71 -73 amino acids and ending in one or two positively charged residues. " Cytokine receptors may also contain other motifs to mediate protein-protein interactions, such as the putative SH2 domain found in the EL-4 receptor. 7 4 The binding of a cytokine by the a receptor chain is a low affinity interaction. Association of the a subunit with the non-ligand-binding common receptor subunit creates a high-affinity heterodimeric receptor complex that can better respond to the physiological concentrations of cytokines. 7 5 A signal is then propagated into the cell, a process that is primarily driven by the common chain, but which requires the intracellular portions of both receptor subunits in EL-3, G M - C S F , and EL-4 signalling. 7 6" 7 8 The 70 kDa EL-3 receptor a chain (EL-3Rcc) and the 45 kDa G M - C S F receptor a chain ( G M - C S F R a ) are both glycoproteins that conform to the standards of type I cytokine receptors. 7 9 , 8 0 Both bind their respective cytokines with low affinity. As such, the receptor complexes for EL-3, G M - C S F , and EL-5 all utilize the 130 kDa (3 common ((3C) chain for 81 increased binding affinity and the transmission of an intracellular signal. This subunit houses the Box 1 and Box 2 motifs which are essential for receptor function. 8 2 Interestingly, the murine system has a pV-3 chain as well as a (3C chain, with the former being found only in EL-3 receptors. In contrast, human cells do not possess this restricted p chain. 9 The E L - 4 R a is a 140 k D a transmembrane receptor that binds EL-4 wi th l o w affinity and houses the B o x 1 and B o x 2 motifs that mediate some o f the receptor's protein interactions w i t h i n the c e l l . 7 1 T o enhance phys io log ica l sensit ivity, the EL-4 receptor, a long w i t h the EL-2 , EL-7 , EL-9, EL-13 and EL-15 receptors, also contain the 65 k D a y c receptor subuni t . 7 4 The importance o f the y c protein is h ighl ighted i n the human disease X - l i n k e d severe combined immunodef ic iency ( X - S C E D ) , where a mutated y c chain results i n dysfunctional B cells and d imin i shed product ion o f T cel ls and natural k i l l e r ( N K ) cel ls . 1.4. Cytokine signaling pathways C y t o k i n e associat ion w i t h its respective receptor initiates a cascade o f various s ignal ing pathways w i t h i n the target c e l l . A diverse array o f protein kinases can mediate the plethora o f ce l lu lar act ivi ty that cytokines promote. 1.4.1. JAK Type I cytokine receptors contain no intr insic kinase act ivi ty, and as such require the recruitment o f other protein kinases. The Janus kinases ( J A K ' s ) are key components i n the early stages o f cytokine- induced intracellular s ignal l ing . M e m b e r s o f the J A K tyrosine kinase fami ly , J A K 1 , J A K 2 , J A K 3 , and T Y K 2 , contain seven conserved J A K homology (JH) regions (JH1-7) and two tyrosine kinase domains . The latter are loca l i zed at the carboxyl- terminus wi th a catalytic tyrosine kinase doma in (JH1) and a pseudo-kinase domain ( JH2) . " The pseudo-kinase domain regulates J A K act ivi ty by interacting w i t h the J H 1 domain and inh ib i t ing J A K activi ty i n 10 the absence of ligand stimulation. Meanwhile, the amino terminus houses the region responsible for binding to the cytokine receptor. 8 7 JAK-receptor interactions promote tyrosine phosphorylation of the receptor subunits and association of other adapter molecules at the receptor. In resting cells, J A K ' s are constitutively associated with the proline-rich receptor motifs Box 1 and Box 2. J A K 2 is the predominant isoform found at the (3C receptor, with minor J A K 1 and T Y K 2 presence, while the yc chain of the EL-4 receptor favors J A K 3 and J A K 1 . Cytokine-receptor binding promotes heterodimerization of the a and common receptor chains into a single receptor complex, followed by oligomerization of these dimeric units. As such, two J A K molecules, one at each receptor complex, come into close proximity. This permits transphosphorylation of each J A K by the other, resulting in the upregulation of J A K tyrosine kinase activity (Figure 1.3). The activated J A K ' s then target key tyrosine residues within the receptor chains themselves. These phosphorylations allow for binding of various SH2-containing proteins at the receptor. Extensive mapping of receptor 88 89 docking sites has been accomplished through truncation and amino acid substitution studies. ' Recruitment of the S T A T family of transcription factors to the receptors is a direct result of receptor phosphorylation by J A K ' s . There are seven known S T A T isoforms, all of which contain an amino-terminal oligomerization domain, a D N A binding domain, an SH2 domain, and a carboxyl-terminal transactivation domain. 9 0 EL-4 potently activates S T A T 6 and to a lesser extent STAT5a/b, while STAT5a/b plays a key role in EL-3 /GM-CSF signalling with some S T A T 1 , S T A T 3 , and S T A T 6 activation. 9 1 STAT's are normally latent in the cytoplasm, but cytokine stimulation promotes their nuclear translocation, D N A binding, and transactivation l l Ligand -courtesy ofpromega.com Figure 1.3 - JAK/STATpathway. Cytokine association with its respective receptor activates the J A K family of tyrosine kinases and the STAT family of transcription factors. Upon cytokine binding, the receptor-associated J A K ' s phosphorylate each other and the receptor chains. This promotes STAT recruitment to the receptor complex where it is phosphorylated by J A K , and becomes an active tran-scription factor. abilities. Upon receptor phosphorylation, the S T A T proteins bind to these sites via their SH2 domains. For example, tyrosine phosphorylation of the EL-4Ra at T y r 5 7 8 and T y r 6 0 6 permits recruitment of S T A T 6 to the receptor. 7 8 Once associated with the receptor, the J A K ' s then phosphorylate the STAT's directly at a conserved tyrosine residue which initiates SH2-mediated homo- or heterodimerization of various S T A T molecules (Figure 1.3). S T A T can then pass across the nuclear membrane and target the palindromic G A S (y-EFN activated sequences) element T T N C N N N A A found in various gene promoter regions. Other kinases can further modulate the transactivational and D N A binding abilities of the STAT's via phosphorylation of key serine residues. 9 3 Once bound to a gene's promoter sequence, S T A T ' s can either act alone, or in concert with other weak DNA-binding proteins, such as CJ3P/p300, to initiate gene 94 transcription. J A K ' s can play an important role in regulating both cell survival and cell proliferation in factor-dependent hematopoietic cells. Several transcription factors implicated in proliferative responses, including c-fos and c-myc, are themselves regulated by S T A T proteins. For example, promotion of c-fos transcription in GM-CSF-treated TF-1 cells is partially controlled by S T A T family members. 9 5 In BaF/3 cells, expression of a dominant negative J A K 2 blocks c-fos and c-myc promoter activation, and EL-3/GM-CSF-mediated cellular proliferation. 9 6 A s well , S T A T 5 repression can block EL-3-induced cell growth in BaF/3 cells and 32D myeloid cells, while STAT5a-deficient bone marrow-derived macrophages show impaired proliferative responses to G M - C S F . 9 7 ' 9 8 However, there is mounting evidence that J A K can also mediate these cellular effects without S T A T . For example, constitutive activation of J A K 2 in BaF/3 cells demonstrates no S T A T activity but can confer. EL-3-independent growth nevertheless.9 9 This apparent paradox 13 of STAT involvement in IL-3-mediated proliferation is still under scrutiny. It is plausible that JAK's utilize alternative pathways that bypass the need for STAT-mediated transcription. For example, EL-3-induced JAK activity can upregulate the expression of the anti-apoptotic B C 1 - X L protein independent of STAT in FDC-P1 cells.100 As well, JAK's can activate the Ras/MAPK cascade without STAT. 1 0 1 1.4.2. M A P K The mitogen-activated protein kinase (MAPK) family is a group of evolutionarily conserved kinases that are one of the most commonly used kinase pathways involved in eukaryotic cell regulation. Various MAPK isoforms have been isolated, with the three most predominant being the extracellular signal-related kinases (ERK1 and ERK2), the p38 family (p38 a, (3, y, 8), and the Jun amino-terminal kinases/stress-activated protein kinases (JNK1, 2, 3/SAPK Y, a, P)-102 The ERK's are stimulated by a variety of growth factors, while p38 and JNK isoforms are often activated by stress stimuli and have been implicated in the apoptotic 103 process. The mechanism of MAPK isoform activation is highly conserved and utilizes protein kinase modules and sequential phosphorylations. As Figure 1.4 illustrates, all three major MAPK modules utilize a GTP-binding protein, followed by activation of a MAPK kinase kinase (MEKK), a MAPK kinase (MEK), and finally the MAPK itself. ERK is largely activated by the monomeric GTPase Ras. Ras is a plasma membrane-localized protein, the result of post-translational isoprenylation.104 It acts as an adapter, targeting the Raf family of proteins (A-Raf, B-Raf, Raf-1) and recruiting these to the plasma membrane as 14 Extracellular Stimuli CELL MEMBRANE (Proliferation/Differentiation) (Stress Responses) ERK/MAPK JNK/SAPK p38/HOG Kinase Pathway Pathway Pathway SMALL GTP-BIND1NG PROTEINS MEKK MEK MARK •courtesy ofpromega.com Figure 1.4 - MAPKpathways. A cascade of protein kinase phosphorylations regulates all three families of M A P K isoforms, including E R K , J N K , and p38. Small GTP-binding proteins transduce a signal initiated by extracellular stimuli, promoting activation of M E K K , M E K , and finally the M A P K isoforms. 15 w e l l . 1 0 5 A l l Raf isoforms are serine/threonine kinases activated by association with Ras. Raf may also require prior phosphorylation at key residues by p21-activated protein kinase ( P A K ) and/or P K C to elicit full act ivat ion. 1 0 6 ' 1 0 7 A l l three Raf isoforms are regulated by similar means, although B-raf does not require phosphorylation to achieve activation. In turn, the Raf proteins can phosphorylate M E K 1 and M E K 2 within their respective activation loops, resulting in full kinase activity. M E K 1 and M E K 2 are dual specificity M A P K K that phosphorylate both the threonine and the tyrosine found in a Thr-Glu-Tyr motif in the activation loop at kinase subdomain VIJI within E R K 1 and E R K 2 (Thr 1 8 5 and T y r 1 8 7 of E R K 2 ) . 1 0 8 These modifications promote full E R K activity. Meanwhile, the p38 and J N K family of M A P K isoforms are also regulated by similar modules of protein kinases induced by a variety of pro-inflammatory and stress stimuli. The Rho family of GTP-binding proteins, including Rac and Cdc42, initiate a M A P K cascade similar to Ras in the E R K pathway. 1 0 9 These GTPases can then activate a wide range of M E K K proteins including T A K 1 , A S K 1 ( M E K K 5 ) , M E K K 1 , Tpl-2, M E K K 2 , M E K K 3 , M U K and T A O . 1 1 0 - 1 1 3 Each of these can then lead to propagation of the J N K pathway, the p38 pathway, or both. The numerous players at this stage create a complex protein kinase network. A s in E R K regulation, these M E K K ' s phosphorylate M E K ' s , which in turn promote p38 and J N K activation. p38 activity is upregulated by M E K 3 and M E K 6 through the dual phosphorylation of a Thr-Gly-Tyr sequence analogous to the crucial E R K motif. Likewise, the J N K pathway utilizes M E K 4 and M E K 7 to target a Thr-Pro-Tyr motif within the J N K activation loop . 1 0 8 A l l M A P K ' s phosphorylate substrates at a serine or threonine residue that precedes a proline site. A s such, M A P K ' s are commonly referred to as proline-directed protein kinases. A 16 common target of M A P K ' s are transcription factors. E R K can target E L K - 1 , c-fos, and c-jun transcription factors resulting in enhanced expression or transactivational a b i l i t y . 1 1 4 - 1 1 6 As well , E R K can regulate cell proliferation by enhancing the activity of carbamoyl phosphate synthetase, a rate-limiting enzyme involved in nucleotide synthesis. 1 1 7 J N K ' s can phosphorylate the transcription factors c-jun and A T F - 2 , while p38 members can target M E F 2 C , A T F - 1 , C / E B P p , G A D D 1 5 3 , and S R F transcription factors. 1 1 8" 1 2 4 The M A P K cascades are common intracellular pathways utilized by a variety of cytokines. Wi th EL-3 or G M - C S F stimulation, phosphorylation of the (3C receptor at T y r 5 7 7 by J A K 2 allows for the binding of the adapter protein She via its phosphotyrosine binding (PTB) domain. 8 9 She is then itself phosphorylated, whereupon another adapter protein, Grb2, binds to She. The guanine nucleotide exchange factor, Sos, can associate with Grb2 and catalyze the exchange of G D P with G T P , converting Ras to an active form. From there, the Ras-Raf -MEK-E R K pathway can proceed. Meanwhile, EL-4 is less consistent in the activation of M A P K compared to EL-3 and G M - C S F , with EL-4-mediated upregulation of M A P K being a cell type-specific response. 1 2 5 In some cases, the EL-4 receptor complex recruits the insulin receptor substrate molecules (IRS) to T y r 4 9 7 of the y c chain, where IRS is phosphorylated by Fes and/or J A K 2 . 1 2 6 , 1 2 7 From there, Grb2 can bind to IRS and exert similar effects on Ras as seen in EL-3 signalling. However, these general mechanisms do not account for alternative means that exist to promote E R K activity. 1 2 8 Meanwhile, IL-3 and G M - C S F can also activate the J N K / S A P K and p38 isoforms. 1 2 9 " 1 3 0 In a pattern similar to EL-4's ability to activate E R K in a cell specific 131 132 manner, enhanced p38 activity is also restricted to certain cell lines. ' However, how p38 and J N K isoforms are activated by cytokine treatment is less well understood. 17 M A P K isoforms have been shown to be important in cell proliferation and cell survival in factor-dependent cell lines. Expression of a Raf or a M E K 1 construct fused to the estrogen receptor permitted survival and proliferation of FDC-P1 and TF-1 cells in the presence of estrogen, despite a lack of EL-3 or G M - C S F . 1 3 3 Meanwhile, the expression of an active Ras blocked cell death in BaF/3 cells possessing a truncated (3C receptor chain. Furthermore, transfection of an oncogenic form of Raf suppressed apoptosis in EL-3-deprived BaF/3 and 32D ce l l s . 1 3 4 A s well , expression of a dominant negative M E K in BaF/3 blocked EL-3-induced 135 survival. However, Ras activity is not essential for BaF/3 proliferation in response to EL-3. This suggests an alternative mechanism for E R K activity is available, or that E R K is not essential for cell proliferation in all cases. J N K and p38 isoforms are primarily activated by stress and inflammatory cytokines and often mediate pro-apoptotic processes. For example, the removal of EL-3 leads to apoptosis in TF-1 cells, a process blocked by the co-presence of a p38 inhibitor. 1 2 9 In contrast, p38 inhibition cannot deter ceramide-induced apoptosis in the M C / 9 cell l i ne . 1 3 6 A s such, there exists a complex role for these M A P K isoforms. The activation of the J N K / S A P K and p38 families have 132 137 also been reported in the EL-3/GM-CSF-stimulation of various factor-dependent cell lines. ' Interestingly, J N K may in fact promote EL-3-induced proliferation. 1 3 7 Some have suggested that the pro-apoptotic effects of J N K and p38 may be offset by the activation of E R K during mitogen stimulation. 18 1.4.3. p90rsk Another downstream target of the E R K isotypes is the family of serine/threonine kinases termed p90 ribosomal S6 kinase (p90 r s k or M A P K A P - K 1 ) . This group is comprised of three isoforms, R S K 1 , R S K 2 , and R S K 3 , all encoded by distinct genes. 1 3 8 R S K , first isolated from the Xenopus laevis oocyte, is peculiar in that it has two catalytic domains. The amino-terminus domain is responsible for R S K ' s ability to phosphorylate a variety of cellular substrates involved in cell proliferation, protein synthesis, and gene transcription. Meanwhile, the carboxyl-terminus kinase domain plays a role in R S K activation. 1 3 9 R S K is usually activated when E R K is stimulated, and mitogens are typically strong activators of both E R K and R S K . The activation of R S K by E R K is a multi-step process involving not only E R K , but also other protein kinases as w e l l . 1 4 0 Cytokines such as I L - 3 and G M - C S F are mitogens, and as such can activate R S K in factor-dependent cell lines. For example, the TF-1 response to G M - C S F leads to R S K activation and FDC-P1 cells demonstrate IL-3-dependent R S K ac t iv i ty . 1 4 1 ' 1 4 2 Numerous proteins are targeted by R S K , including the transcription factors c A M P response element binding ( C R E B ) protein, c-fos, and I K B O / N F K B . 1 4 3 1 4 5 C R E B is phosphorylated by R S K 2 at Ser 1 3 3 , a site whose modification is essential for C R E B transactivational ability and possibly cell survival. Meanwhile, the expression of the c-fos gene can be upregulated by R S K proteins. As well, N F K B activation my also be upregulated by R S K ' s phosphorylation of IKBCX, allowing for the degradation of IKBCC, N F K B nuclear translocation, and transcription of important genes. Finally, the translation of a specific group of m R N A important for cell growth is upregulated by R S K via its phosphorylation of the 40S ribosomal subunit protein S 6 . 1 4 6 19 1.4.4. Phosphatidylinositol 3'-kinase (PI3-K) Activation of the P I 3 - K / P K B pathway is another common response to cytokine signaling. Three classes of PI3-K have been defined with respect to substrate specificity and structural motifs . 1 4 7 The Class I P I 3 - K isoforms, which are best understood, are all heterodimeric proteins comprised of a catalytic and a regulatory subunit. Members of this class preferentially phosphorylate phosphatidylinositol-4,5-biphosphate [PI(4,5)P2] at the 3 ' - O H position within the inositol ring to form PI(3,4,5)P 3, but can also target PI and PI(4)P to a lesser extent to generate PI(3)P and PI(3,4)P 2 respectively. 1 4 8 This class is further subdivided into two groups based on the type of adapter subunit associated with the catalytic unit. Class IA PI3-K's are comprised of a 110 kDa catalytic subunit and a regulatory protein. There are three Class IA catalytic isoforms termed p i 10a, P, and 8. p i 108 expression appears restricted to the hematopoietic lineage. 1 4 9 The common regulatory subunit is an 85 kDa protein housing SH2 and SH3 domains for protein interactions. The Class IB PI3-K isoform is p i 10y associated with a 101 kDa regulatory protein. This isoform is sensitive to the (3y subunits of the trimeric G-proteins and does not associate with the p85 regulatory protein. 1 5 0 The proposed regulation of Class IA PI3-K during E L - 3 / G M - C S F signaling involves multiple steps. Cytokine ligation to its respective receptor results in the phosphorylation of the Pc chain at T y r 6 1 2 , which acts as a docking site for the tyrosine phosphatase SHP2. A novel adapter protein plOO may act as an intermediary, linking to both SHP2 and p85 via their SH2 domains . 1 5 1 ' 1 5 2 In EL-4 signalling, EL4-Ra-associated IRS molecules bind the p85 subunit of PI3-K . 1 5 3 In both cases, the association of the p85 regulatory protein with the cytokine receptor 20 complex results in the activation of the p i 10 catalytic protein and membrane translocation. This permits proximity of the enzyme to the necessary l ipid substrates for PI3-K. 1.4.5. Protein kinase B (PKB) P K B was so named for its homology to the catalytic domain of P K A and P K C , and was identified as the cellular homolog of the viral oncogene Ak t (v-Akt) from a transforming retrovirus ( A K T 8 ) in spontaneous thymoma of the A K R mouse. 1 5 4 It is a serine-threonine protein kinase whose full activity requires membrane localization and phosphorylation at two sites conserved in the A G C kinase family. Three mammalian isoforms have been identified and cloned, PKBoc /Ak t l , PKBp7Akt2 , and P K B y / A k t 3 , with PKBoc being the more abundant species in most tissues. 1 5 5 A l l three isoforms are ubiquitously expressed, and share a high degree of homology and similar structural features. This includes an amino-terminus pleckstrin homology (PH) domain that binds to phospholipids and two conserved phosphorylation sites (Ser 4 7 3 and T h r 3 0 8 in P K B a). The activation of P K B is a complex multi-step process (Figure 1.5). 1 5 6 PI3-K activation generates elevated levels the of PI(3,4,5)P 3 and PI(3,4)P 2 lipids, which can promote recruitment of the constitutively active phosphoinositide-dependent kinase 1 (PDK1) to the membrane surface via the latter's pleckstrin homology (PH) domain. P D K 1 acts as a link between PI3-K and a variety of protein kinases implicated in mitogenic activation, including P K B . P D K 1 targets a conserved threonine/serine found in the T-loop between subdomains V E and V I E of a Inactive cytosolic P K B Figure 1.5 - PI3K-PKBpathway. Cytokine signaling often initiate PI3-K activation. The heterodimeric PI3-K produces a series of lipids, which promote P D K 1 and P K B membrane transloca-tion. P D K 1 can then phosphorylated P K B at Thr ' 0 8 , while an elusive P D K 2 can target Ser 4 7 3 . These phosphorylation promote activation of P K B . 1 5 6 22 variety of A G C protein kinases leading to catalytic activation, including PKBoc (Thr ), P K C ^ (Thr 4 1 0 ) , p 7 0 S 6 K (Thr 2 5 2 ) , Rsk2 (Ser 2 2 7 ) , and S G K 2 ( T h r 1 9 3 ) . 1 5 7 " 1 6 2 Meanwhile, PI3-K-generated lipids promote cell membrane localization of protein kinase B (PKB) . Initial studies on P K B regulation focused on the constitutive activity of its viral counterpart, v-Akt. A myristoylation signal in the gag domain targets v-Akt to the plasma membrane, suggesting that membrane localization was essential for P K B activation. 1 5 6 Subsequent studies confirmed the translocation of a pool of P K B to the plasma membrane upon mitogen stimulation, a process mediated by P K B ' s P H domain. In fact, point mutations in the P H domain that reduce l ipid binding affinity impairs P K B activation, while enhanced lipid binding hyper-activated the enzyme. 1 6 3 Both PI(3,4,5)P 3 and PI(3,4)P 2 bind to the P H domain of P K B , but the relative contributions of each in vivo is still a mystery. 1 6 4 However, studies on SHIP, a 5'-phosphatase that converts PI(3,4,5)P 3 to PI(3,4)P 2, showed that SHIP is an inhibitor of P K B activity in vivo.165 This data suggests PI(3,4,5)P 3 is primarily responsible for P K B translocation. 308 Upon membrane localization, P K B is phosphorylated at two residues. Thr is found within the T-loop of the kinase domain, and Se r 4 7 3 on a hydrophobic region of the carboxyl-terminus. Both sites are necessary for P K B activation as demonstrated through mutation of each to a non-phosphorylatable alanine. 1 6 6 The inhibition of PI3-K blocked phosphorylation of both these residues, demonstrating that the kinases responsible for these modifications are themselves 308 dependent on PI3-K-generated lipids. The kinase responsible for Thr phosphorylation is in fact P D K 1 . The phosphorylation of Ser 4 7 3 is more enigmatic. The integrin-linked kinase (ELK) has recently been suggested to regulate P K B activity through direct phosphorylation of Se r 4 7 3 in a 23 PI3-K-dependent manner. 1 6 7 Interestingly, P D K 1 can phosphorylate both T h r 3 0 8 and Ser 4 7 3 when P D K 1 associates with the carboxyl-terminal region of the PKC-related protein kinase P R K 2 . 1 6 8 Another theory on the table is that T h r 3 0 8 phosphorylation allows P K B to autophosphorylate at Ser 4 7 3 , possibly via close proximity to other P K B molecules in a multimeric complex. 1 6 9 Regardless, once Se r 4 7 3 is phosphorylated, P K B detaches from the membrane region, re-enters the cytosol and translocates to the nucleus. The evidence to date suggests that this is the means of activation for P K B a , P K B p \ and P K B y . There have been, however, reports of P K B activation independent of PI3-K. In particular, activation of the P K A pathway can activate P K B without the need for its P H domain or Ser 4 7 3 phosphorylation, although T h r 3 0 8 is still required. 1 7 0 Numerous potential P K B targets have been implicated in the possible control of cell survival and proliferation. Recently, P K B was identified as a regulator of the pro-apoptotic Bcl -2 family member, B A D , via phosphorylation at S e r 1 3 6 . 1 7 1 This leads to sequestration of the protein and aids in the inhibition of apoptosis. Unsequestered, B A D can heterodimerize with the anti-apoptotic Bcl -2 and B c l - x L and neutralize their pro-survival abilities. However, the regulation of B A D is still under scrutiny and its role in cytokine-mediated survival has been called into doubt. 1 7 2 " 1 7 3 Furthermore, the expression level of B A D is low and not ubiquitous, suggesting that any role P K B has in cell survival may be accomplished independently of B A D . Meanwhile, P K B may also phosphorylate and inactivate casapse-9, blocking execution of the apoptotic program. 1 7 4 However, the phosphorylation site targeted by P K B in caspase 9 is not conserved among the species. 1 7 5 As well , P K B can inhibit the forkhead family of transcription factors that primarily target pro-apoptotic genes, including the Fas l i gand . 1 7 6 ' 1 7 7 Phosphorylation of the forkhead family of transcription factors results in their sequestration in the cytoplasm, thus 24 preventing transcription of these destructive genes. It has been reported that the transcription factor C R E B may also be targeted by P K B , inducing expression of the pro-survival Bcl -2 family 178 179 member M c l - 1 . ' Protein synthesis, a necessary component for cell proliferation, might be under P K B control as well . During basal conditions, a regulator of m R N A translation, eEF4E, is 180 found to be inactive and in complex with a repressor of translation termed 4E-BP . 4 E - B P phosphorylation by P K B might allow 4 E - B P to dissociate from eEF4E and subsequently promote m R N A translation. 1 8 1 The P I 3 - K / P K B pathway is a potential regulator of cell survival, proliferation, and a variety of hematopoietic cell functions. Numerous factor-dependent hematopoietic cell lines have demonstrated enhanced PI3-K and P K B activity upon IL-3, EL-4, and G M - C S F s t imula t ion . 6 0 ' 1 7 3 ' 1 8 2 ' 1 8 3 This activation of P K B may be essential for cell survival and/or proliferation. A dominant-negative form of P K B has been noted to abrogate EL-3-induced proliferation in the 32D factor-dependent cell line, while expression of the constitutively active v-Akt protects these cells from apoptosis induced by EL-3 withdrawal. 1 8 4 However, the role of PI3-K and P K B in cytokine-mediated survival and proliferation has been a contentious issue. While constitutive P K B activity in BaF/3 is sufficient to protect against apoptosis induced by EL-3 withdrawal, P K B activity is not necessary for the EL-3-induced inhibition of apoptosis due to D N A damage. 1 8 5 It has also been shown that there is little correlation between P K B activity and cell survival in M C / 9 and other cell l ines. 6 0 BaF/3 proliferation in the presence of EL-3 is reduced with the expression of a dominant negative form of the p85 adapter protein (Ap85), but 186 187 there is no effect on survival. ' However, recent evidence points to significant communication between the PI3-K and E R K pathways. Some data suggests that the E R K 25 pathway is regulated by PI3-K, and that the abrogation of PI3-K also blocks IL-3 induced E R K activity and myeloid cell prol i ferat ion. 1 3 4 , 1 8 8 It is plausible that the M A P K and PI3-K pathways synergize to mediate cell survival and proliferation in many factor-dependent cells. 1.4.6. Protein kinase C (PKC) Diacylglycerol ( D A G ) is commonly produced by the cell receptor-mediated hydrolysis of phosphatidylinositol and phosphatidylcholine by phospholipase C (PI-PLC and P C - P L C ) and phospholipase D (PLD) isoforms (Figure 1.6). 1 8 9 A potent second messenger, D A G targets the P K C family of serine/threonine kinases that are ubiquitously expressed in a variety of species. 1 9 0 P K C is also activated through its role as an intracellular receptor for phorbol esters. 1 9 1 Among the eleven known mammalian isoforms of P K C , there are various reoccurring structural features. Four domains termed C1-C4 are found in some or all of the P K C isoforms (Figure 1.7). 1 9 2 The amino-terminus C I domain found in some P K C ' s contains two sets of zinc-finger motifs. D A G binds at this region, and is competitive with phorbol esters for the same binding domain. Beside the C I domain is a pseudosubstrate region that binds to the P K C catalytic region and suppresses P K C activity prior to effector binding. The C2 domain permits calcium and phosphatidylserine association. Some P K C ' s have a pseudo-C2 domain that lacks key aspartate residues needed for calcium binding. Finally, the C-terminus C3 and C4 domains comprise the catalytic region. As well , a hinge region between the regulatory and catalytic portions becomes proteolytically labile upon plasma membrane association of P K C . 1 9 3 26 Figure 1.6-Diacylglycerol formation. Cellular agonists often induce diacylglycerol formation through one of several mechanisms. The phospholipase C (PLC) enzymes, which can hydrolyze either phos-phatidylcholine (PC-PLC) or phosphatidylinositol (PI-PLC) to produce D A G , are commonly activated after agonist stimulation. Meanwhile, phospholipase D (PLD) also acts on phosphatidylcholine to produce phosphatidic acid, which can be further dephosphorylated to yield diacylglycerol. 27 Classical PKC's a, P, y Regulatory Hinge Catalytic C l C2 C3 C4 2Zn^ 2Zn + + A T P Substrate Novel PKC's 8, r|, e, 9 C2-like 2Zn + + 2Zn + Atypical PKC's 2Zn + PKC u 2Zn^ 2Zn + + P H Pseudosubstrate | | Cystein rich region: D A G binding Calcium binding D A G C a 2 + PS Phorbol esters Classical X X X X Novel X X x Atypical X PKC | i X X X Factor required = X Phorbol stimulates = X -adaptedfrom www. cmb. uab. edu/courses/Lectures/theibertlipid2.pdf Figure 1.7 - PKC isoforms. The three classes of P K C , while sharing certain structural features, have unique cofactor requirements. The classical or conventional P K C ' s (cPKC) include the a, Pi, Pn (an alternative splicing variant of PO and y subtypes and were the first to be cloned. This subset of P K C ' s is activated by a combination of calcium, phosphatidylserine (PS), and diacylglycerol, and is responsive to 192 phorbol esters. D A G binding via the C I region increases P K C ' s affinity for PS and calcium into the physiological range. The novel isoforms of P K C (nPKC), 8, e, 0, and T|, are calcium-independent, but still require diacylglycerol and phosphatidylserine for activation. The absence of calcium regulation is due to the lack of key residues in the C2 domain . 1 9 2 Like the classical P K C ' s , these proteins act as receptors for phorbol esters as well. The atypical subgroup, with £ and A, (mouse i) isoforms, are activated in a unique manner from the other two families. Without a C 2 domain and only one cysteine-rich fold in the C I region, neither diacylglycerol nor calcium regulate the function of the atypical members of P K C . 1 9 2 Meanwhile, the enigmatic P K C u / P K D is a unique family member, possessing a P H domain and an N-terminal hydrophobic region. 1 9 4 Furthermore, it lacks the canonical pseudosubstrate region familiar in other P K C ' s . 1 9 5 However, P K C u . is responsive to diacylglycerol and phorbol esters thanks to a C l - l i k e domain. Meanwhile, a group of PKC-related protein kinases or PRK's , have also been identified. 1 9 6 There are at least three members, including P R K 1 (PKN) , P R K 2 , and P R K 3 , which share similar properties with the atypical P K C ' s in that they are insensitive to calcium, D A G , and phorbol esters. Recently, a role for both the R h o A GTPase and P D K 1 in P R K activation has been i 197 suggested. The activation of P K C isoforms is a complex, multi-step process involving phosphorylation, membrane localization, and pseudosubstrate release. P K C is phosphorylated at 198 three key residues soon after protein translation. The activation loop of classical, novel, and 29 atypical P K C ' s are phosphorylated within a conserved residue (Thr of PKC(3n) by P D K 1 . Autophosphorylation within a turn motif in all P K C isoforms (Thr 6 4 1 of PKCfJn) and within a hydrophobic motif in classical and novel types (Ser 6 6 0 of PKC(3n), primes the kinase for activation. Atypical P K C ' s possess a glutamic acid residue at the site corresponding to Ser 6 6 0 , which mimics a phosphorylated residue. Later, agonist-induced production of D A G promotes translocation of P K C to the cell membrane. The actions of calcium, D A G , and PS promote membrane translocation, pseudosubstrate release and creation of a fully competent P K C enzyme. Remarkably, the role of phospholipases and P K C isoforms in the cytokine regulation of cell survival and proliferation has undergone minimal investigation. However, cytokine-mediated elevation of intracellular D A G has been observed. Phosphatidylcholine-specific phospholipase C (PC-PLC) activity has been detected in cells upon EL-3, G M - C S F , and EL-4 stimulation, with concurrent elevation of diacylglycerol levels . 1 9 9 " 2 0 1 Furthermore, phospholipase D activity has also been detected upon cell treatment with G M - C S F , but not with EL-4 treatment. " Since cytokines do not initiate a calcium spike, and since a byproduct of phosphatidylinositol-directed phospholipase C (PI-PLC) activity is E P 3 , an activator of transient calcium fluxes, P I -PLC is not considered to be involved in the intracellular cytokine response. Numerous investigators have reported cytokine-induced activation of P K C isoforms. M C / 9 , TF-1 , and FDC-P1 cells all display heightened P K C function upon EL-3 and G M - C S F stimulation. 2 0 4 " 2 0 8 Interestingly however, no information exists on EL-4-mediated P K C activity in these factor-dependent cells. Nevertheless, in EL-3 and G M - C S F signaling, P K C isoforms may play a critical role in cell survival. Avoiding apoptosis may require P K C for its ability to induce expression of the pro-survival bcl-2 gene and to phosphorylate the Bcl-2 protein. For example, 30 overexpression of P K C e in the TF-1 cell line exhibits elevated bcl-2 transcription and permits cell survival in the absence of I L - 3 . 2 0 9 Chronic phorbol ester exposure results in reduced P K C 210 activity and bcl-2 expression, as well as increased apoptosis even in the presence of IL-3. As 211 well, pharmacological intervention of EL-3-stimulated P K C activity decreases bcl-2 transcripts. Furthermore, inhibition of P C - P L C in TF-1 cells also abrogates IL-3-enhanced bcl-2 expression and promotes apoptosis even in the presence of I L - 3 . 1 9 9 However, some isoforms, including P K C 8 and 6, may in fact promote apoptosis in certain cell types. ' They are targeted and activated by caspase cleavage during apoptosis, and over-expression of these in hematopoietic cells can further drive cell death. 1.4.7. Protein kinase A The elevation of adenosine 3',5' cyclic monophosphate ( c A M P ) by extracellular signals is a powerful and ubiquitous intracellular second messenger. The classical mechanism of c A M P production is mediated by receptor activation of GTP-binding proteins (G-proteins). The G -protein subunit, G a s , activates adenylyl cyclase and enhances conversion of A T P to c A M P . 2 1 4 215 Increased levels of c A M P can then promote protein kinase A ( P K A ) activation. The inactive P K A holoenzyme tetramer is comprised of two regulatory (R) and two catalytic (C) subunits (Figure 1.8). 2 1 6 The R mammalian isoforms (RIoc, RIp\ R l l a , RII(3) derive from distinct genes and vary in tissue expression, although the physiological importance of these varieties is unresolved. Meanwhile, C a , C(3, and C y are the three known mammalian genes encoding the catalytic subunits, with three additional splice variants of Cp1 (C(3l, C(32, C(33). Each C isoform also differs in tissue expression profile and catalytic properties. During basal r -courtesy of promega, com Figure 1.8 - PKA activation. Agonist stimulation o f cells can in some cases stimulate P K A activation. The canonical pathway involves activation of G proteins, followed by elevated levels of c A M P induced by adenylyl cyclase activation. c A M P is then able to free P K A from the inhibition brought on by its regulatory subunits. 32 stages, the C subunits are inactive due to the inhibition conferred by the inhibitory pseudosubstrate domain of the R subunits. Wi th increased intracellular c A M P , each R subunit is capable of binding two molecules of c A M P . Wi th this event, their affinity for the C subunit is decreased, permitting dissociation of the R and C complex and activation of P K A catalytic activity. Negative feedback control is achieved through PKA-mediated phosphorylation and activation of cyclic nucleotide phosphodiesterases, which degrade intracellular c A M P and thus reduce P K A activity. 2 1 7 P K A is also involved in negative feedback of c A M P production via phosphorylation of membrane receptors, leading to heterologous desensitization whereby receptors exhibit decreased affinity for their respective ligands. 2 1 8 P K A was first noted for its role in the hormonal regulation of glycogen metabolism. It is a serine/threonine protein kinase that targets a consensus sequence containing two basic adjacent residues ( R R X S / T X ) . P K A can regulate gene expression through a variety of transcription factors, including A T F - 1 and the cAMP-responsive element binding protein ( C R E B ) . The latter is phosphorylated by P K A at Ser 1 3 3 , which enhances C R E B ' s transactivational ability. In this manner, P K A and c A M P can regulate various gene transcription events via promoter regions containing a cAMP-responsive element (CRE) . Although G-protein activity has not been routinely detected during cytokine signalling, several tyrosine kinase-based receptors have been shown to mediate marked c A M P accumulation, possibly via inhibition of phosphodiesterase isoforms. In B cells, IL-4 can induce sustained elevated c A M P levels and increased P K A activity. 2 1 9 Meanwhile, EL-3-induced phosphorylation and inactivation of B A D at S e r " 2 exhibits P K A dependency. 2 2 0 However, G M -C S F stimulation of TF-1 cells does not induce a c A M P spike nor P K A activity. 2 2 1 Equally 33 confusing is P K A ' s role in cell survival and cell proliferation. In B cells, forskolin-induced elevation of c A M P results in enhanced apoptosis, while microinjection of c A M P into the myeloid cell line EPC-81 displays a similar e f fec t . 2 2 2 , 2 2 3 In contrast, c A M P can abrogate apoptotic events upstream of caspase 3 in human neutrophils. 2 2 4 A s such, it appears that the effect of c A M P and P K A in cell survival is potentially cell type- and stimulus-specific. 1.4.8. p70S 6 K Targeting the S6 protein of 40S (small) ribosomal subunit, p70/p85 s 6 K are splice variants 225 S6IC of a serine/threonine kinase implicated in various mitogenic signaling pathways. The p85 isoform has an amino-terminal 23-amino acid nuclear localization signal that p70 lacks. The S6K functional significance of these two isoforms is still being unraveled, and the term p70 is still often used to describe both species. The regulation of p70 is a complex process requiring multiple upstream inputs. Activation of p 7 0 s 6 K involves serial phosphorylations within the regulatory domain, transactivation loop, and autoinhibitory domain. Activation via these critical phosphorylations is controlled by the mammalian target of rapamycin (mTOR/FRAP) and P D K 1 , both of which are 226 227 252 essential for protein activation. ' The latter targets a conserved residue (Thr ) that is homologous to T h r 3 0 8 in P K B and other residues throughout the A G C family of protein kinases. 1 6 0 The cytokine activation of p 7 0 s 6 K has been documented in a variety of cases, including treatments with EL-3, G M - C S F , and I L - 4 . 1 2 7 , 2 2 8 - 2 3 1 The kinase may mediate cell proliferation through the phosphorylation of S6 ribosomal proteins. This enhances protein translation of a class of m R N A containing tract of oligopyriomidines (TOP) at the 5' terminus. Many of these m R N A encode for proteins involved in cell cycle control, and p 7 0 s 6 K activity is in most cases essential for progression past the G l phase and thus cell proliferation. However, p 7 0 s 6 K may not be essential for cell proliferation in all cases . 2 3 0 ' 2 3 2 1.5. Glycogen synthase kinase-3 (GSK-3) In the 1980's, biochemists were keen to characterize the enzymes responsible for metabolic reactions. During this search, G S K - 3 , a serine/threonine protein kinase, was identified as an enzyme that phosphorylates and inactivates glycogen synthase, a key checkpoint in the regulation of glycogen synthesis. ' ' In particular, insulin upregulation of glycogen production was found to be a result of G S K - 3 inhibition and subsequent glycogen synthase activation. Since that discovery, the manner in which G S K - 3 isoforms are regulated has been under scrutiny. Interestingly, since its initial characterization, the level of G S K - 3 activity has been implicated in regulating a variety of cell functions including embryonic development, cell proliferation and 235 240 survival, protein translation, cell cycle progression, and protein degradation. " However, there has been no published data into G S K - 3 and its role in cytokine stimulation of factor-dependent hematopoietic cells. 1.5.1. Isoforms G S K - 3 , although originally purified from skeletal muscle, is ubiquitously expressed among cell types and evolutionarily conserved among diverse species such as yeast, plants, and mammals . 2 3 9 ' 2 4 1 ' 2 4 2 In lower organisms, a large family of G S K - 3 homologs have been 35 characterized. 2 4 3 In mammals, genetic screening has identified two distinct isoforms of G S K -3 . 2 4 4 The 51 kDa G S K - 3 a and the 47 k D a GSK-3(3 share substantial sequence homology throughout the key regulatory and catalytic regions. Although most studies have focused on either G S K - 3 a or GSK-3p \ little has been published with respect to the differences between the two. However, there is some evidence of dissimilarity in the regulation, function, and substrate specificity of each mammalian i s o f o r m . 2 3 7 ' 2 4 5 ' 2 4 6 1.5.2. Regulation of GSK-3 Extracellular stimuli typically lead to the inactivation of the constitutively active G S K - 3 isoforms. The best-studied model of G S K - 3 inactivation involves insulin stimulation of glycogen production. Further studies has found that a variety of stimulants can inhibit G S K - 3 activity, including growth factors, immunological events, cardiac hypertrophy, and hypox ia . 2 3 6 ' 2 4 7 " 2 4 9 These external signals can promote G S K - 3 inactivation through the phosphorylation of an amino-terminus serine residue found in all G S K - 3 isoforms studied. The modification of the Ser 2 1 and Ser 9 residues of GSK-3oc and GSK-3P respectively, promote inactivation of G S K - 3 protein kinase activity. Conversion of this critical serine residue to a non-phosphorylatable alanine results in a constitutively active G S K - 3 enzyme that is non-responsive to extracellular i 236,250 signals. ' A variety of protein kinases have been shown to phosphorylate these serine residues in vitro including P K B , p90 r s k , P K A , ELK, and some P K C isoforms. If and how each of these kinases modulate G S K - 3 activity is still under investigation in various model systems. The 36 classical model of insulin-mediated G S K - 3 inactivation was found to involve the PI3-K and P K B pathways. The presence of a dominant negative P K B can abrogate insulin's effect on G S K - 3 , thus demonstrating the essential need for P K B activity in the serine phosphorylation of G S K -3 . 2 5 1 In fact, various other stimuli utilize the P I 3 - K / P K B pathway to inactivate G S K - 3 . In cultured neurons, insulin growth factor-1 (IGF-1) blocks G S K - 3 activity, a process that is sensitive to PI3-K inhibitors. 2 5 2 Furthermore, hypoxic and hypertrophic stimuli require the PI3-K / P K B pathway to mediate their inhibitory effects on G S K - 3 . 2 4 8 ' 2 4 9 However, there are several studies that have demonstrated PI3-K/PKB-independent regulation of both isoforms of G S K -2 253,254 Several other growth factors mediate inhibition of G S K - 3 via the M A P K / R S K pathway. In P C 12 cells, epidermal growth factor (EGF) and nerve growth factor (NGF) inhibition of G S K -3 is blocked by the M E K inhibitor, PD98059 . 2 5 5 In vitro, GSK-3oc was phosphorylated at Ser 2 1 and inactivated by p 9 0 r s k . 2 5 6 Furthermore, R S K has been implicated in mediation of G S K - 3 257 activity in Xenopus. As well , a role for some protein kinase C (PKC) isoforms in G S K - 3 inhibition has also been demonstrated. 2 4 5 In embryonic development, P K C mediates G S K - 3 inactivation, despite an insu l in /PKB/GSK-3 pathway in the same cell l i ne . 2 5 8 Over-expression of PKCpn in intestinal epithelium cells leads to deceased G S K - 3 activity. 2 5 9 As well , phorbol esters have been demonstrated to lead to phosphorylation and inactivation of G S K - 3 in a variety of cellular models. 2 5 8 However, there is evidence that each isoform of G S K - 3 shows varying susceptibility to P K C ' s actions. 2 4 5 In particular, GSK-3(3 is more responsive to many P K C isoforms than is G S K - 3 a . 37 Even the cAMP-dependent protein kinase ( P K A ) can associate and phosphorylate G S K -3 a and p at the inhibitory serine residues both in vitro and in vivo.260'261 In cerebellar granule neurons, elevated c A M P promotes Ser 9 phosphorylation of G S K - 3 and subsequent enzyme inhibi t ion. 2 6 1 Meanwhile, in EGF-treated human myoblasts, there is a decrease in G S K - 3 activity and upregulation of glycogen synthesis. The increase in glycogen production is blocked by the m T O R inhibitor, rapamycin, which suggests a possible link between G S K - 3 regulation and p 7 0S6K 262 I n f a c t ^ G S K a c a n b e i n a c t i v a t e d through phosphorylation of Ser 2 1 by p 7 0 S 6 K . 2 6 3 Finally, another protein kinase implicated in G S K - 3 control is the integrin-linked kinase (ILK). I L K is a serine/threonine protein kinase that associates with integrin subunits and whose activity is regulated by cell interaction with the extracellular matrix. I L K can directly phosphorylate and inactivate G S K - 3 at the key serine residue. 2 6 4" 2 6 6 Transfected I L K promotes G S K - 3 inactivation while those transfected with a kinase-dead version of I L K demonstrate enhanced G S K - 3 activity. 2 6 6 Furthermore, I L K has been described as the elusive P D K 2 , a PI3-K-dependent kinase that phosphorylates Se r 4 7 3 of P K B , thus regulating P K B activation. As such, it remains to be determined whether or not ILK's primary effect on G S K - 3 in vivo is direct or via the enhanced P K B activity. In addition to the inhibitory serine phosphorylation sites, G S K - 3 can also be phosphorylated on key tyrosine residues. These conserved residues ( T y r 2 7 9 in G S K - 3 a and T y r 2 1 6 in G S K - 3 p ) are phosphorylated during the resting state, and are necessary for full G S K - 3 activity. ' In fact, mutation of this site to phenylalanine results in a highly inactive G S K - 3 possessing only a small level of residual catalytic activity. The importance of tyrosine 38 phosphorylation in regulating G S K - 3 activity is demonstrated with G S K - 3 constructs lacking the amino-terminus region and thus the critical serine residue. In these mutants, the enzyme is still inactivated through dephosphorylation of the tyrosine. 2 6 7 The kinase responsible for tyrosine phosphorylation of G S K - 3 is a novel study, although recent evidence suggests that P Y K 2 may serve that ro le . 2 6 9 Although it appears these tyrosine residues are crucial for G S K - 3 regulation, they might not be essential for G S K - 3 activity in all systems. 2 5 2 For example, in H E K - 2 9 3 cells, neither IGF-1 nor insulin treatment, which lead to inactivation of G S K - 3 , induced tyrosine dephosphorylation of GSK-3(3. A s such, the relationship between tyrosine and serine phosphorylation of G S K - 3 in growth factor-mediated inhibition remains unsolved. Furthermore, 270 there is evidence that G S K - 3 can autophosphorylate itself at other amino acid residues. Therefore, this complex regulation of G S K - 3 by phosphorylation is a continuing study. 1.5.3. GSK-3 in embryonic development The study of G S K - 3 has been conducted extensively in various embryonic development models. G S K - 3 first garnered attention in Drosophila melanogaster where it was implicated in the Wnt /Wg (wingless) signaling pathway. The Wnt ligand binds to serpentine receptors of the 271 Frizzled family, leading to activation of the protein Dishevelled (Dsh). Dsh, through an unknown mechanism, promotes inactivation of the shaggy/zeste-white 3 protein, a homolog of the mammalian G S K - 3 . 2 7 2 ' 2 7 3 This inactivation leads to increased stability and nuclear accumulation of the (3-catenin protein, a promoter of the lymphoid enhancer factor/T cell factor (Lef/Tcf) family of transcription factors. The resulting gene products are involved in the 39 formation of the dorsal axis during Drosophila development. In contrast, G S K - 3 activity permits phosphorylation of [3-catenin, ubiquitination, and subsequent proteasome-mediated degradation. Meanwhile, in Xenopus, G S K - 3 also modulates embryo polarity and development. Rotation of the outer cytoplasm relative to the inner cytoplasm upon fertilization results in 273 274 rotation of the dorsalizing activity to the side opposite to the site of sperm contact. ' This promotes G S K - 3 inactivation and nuclear accumulation of (3-catenin. Meanwhile, the ventral side of the embryo exhibits active G S K - 3 and limited [3-catenin levels. As such, a polarity is established within the embryo. Finally, in Dictyostelium, cell polarity is also mediated by G S K - 3 activity. During starvation conditions, a buildup in c A M P levels results in the activation of the tyrosine kinase, Z A K 1 . This kinase is able to phosphorylate G S K - 3 at the key tyrosine residues and promote 275 276 G S K - 3 activation. In turn, this regulates genes that direct cell determination. ' Although much of the G S K - 3 developmental studies have emphasized primitive organisms, embryo development and tumorigenesis in higher organisms appears to also utilize the canonical Wnt /Wg pathway involving G S K - 3 . 2 7 7 ' 2 7 8 1.5.4. Substrates of GSK-3 Since the initial discovery of G S K - 3 in glycogen metabolism, a number of other G S K - 3 targets and effects have been noted. For the most part, active G S K - 3 appears to be an antagonist of events leading to cell proliferation. In fact, G S K - 3 has the potential to mediate a variety of cell functions through a number of cellular targets, including cellular metabolism, cell 40 proliferation, cell survival, and protein translation. " ' Furthermore, G S K - 3 ' s role in the pathogenesis of Alzheimer's disease is of recent interest. 2 5 4 Many substrates of G S K - 3 require pre-phosphorylation at a serine residue in the +4 position relative to the G S K - 3 target site. In skeletal muscle, G S K - 3 wi l l phosphorylate and activate eEF-2B at Ser 5 3 5 of its epsilon subunit, but only with a priming phosphorylation at S e r 5 3 9 . 2 7 9 Mutation of Ser 5 3 9 to a non-phosphorylatable alanine results in abrogation of G S K - 3 phosphorylation at Ser 5 3 5 . However, G S K - 3 can also target peptide regions containing no prior phosphorylation, as in the case of (3-catenin. Interestingly, it is this variation in substrate recognition that allows G S K - 3 to mediate certain downstream cellular effects without eliciting others. For example, insulin stimulation abrogated G S K - 3 phosphorylation of the pre-phosphorylated glycogen synthase, but had no effect on GSK-3-mediated stability of (3-catenin, a protein requiring no prior phosphorylation. 2 8 0 Therefore, in a cellular context, G S K - 3 can sort out its multiple effector pathways through differences in substrate modification. G S K - 3 ' s founding role was in regulating cell metabolism, particularly the regulation of glycogen synthesis. Glycogen synthase is the rate-limiting enzymatic process in glycogen biosynthesis. A t the carboxyl-terminus, G S K - 3 can phosphorylate four serine residues (Ser 6 4 0 , Ser 6 4 4 , Ser 6 4 8 , Ser 6 5 2 ) , which are known as sites 3a, 3b, 3c, and 4 respectively. 2 8 1 Phosphorylation of each site is dependent on prior phosphorylation at the +4 residue, beginning with casein kinase 2 (CK2) phosphorylation of Ser 6 5 6 . This sequential phosphorylation leads to inactivation of glycogen synthase activity. Ce l l proliferation and cell survival may also be regulated by G S K - 3 . For example, specific inhibitors of G S K - 3 protect peripheral and central nervous system neurons from 41 261 282 283 apoptosis caused by growth factor withdrawal and PI3-K inhibition. ' ' A s well , lithium 236 284 inhibition of G S K - 3 can prolong cell proliferation in various cell lines. Furthermore, a dominant negative G S K - 3 transfectant can also prevent apoptosis. In contrast, constitutively active G S K - 3 reduces cell proliferation, while overexpression of active G S K - 3 can cause significant increase in cell dea th . 2 3 6 ' 2 8 3 ' 2 6 1 ' 2 8 5 However, active G S K - 3 may not be essential for all forms of apoptosis. 2 8 3 ' 2 8 6 In fact, murine fibroblasts lacking G S K - 3 R demonstrated a greater impairment in cell survival. G S K - 3 ' s pro-survival effects may be mediated in part by its interaction with various transcription factors. For example, phosphorylation of c-jun, a component of the AP-1 transcription factor, by G S K - 3 in vitro can inhibit A P - l ' s D N A binding capacity. 2 8 7 Likewise, 58 288 the degradation of c-myc is mediated by phosphorylation at Thr , a G S K - 3 target site. G S K - 3 can also regulate the nuclear accumulation of the calcium-regulated transcription factor N F - A T . Calcineurin, a calcium-dependent phosphatase, dephosphorylates key serine sites on N F - A T , thus promoting nuclear entry. However, G S K - 3 can oppose this action through phosphorylation of 236 289 these same serine residues, thereby supporting nuclear export of N F - A T . ' G S K - 3 has also been implicated in modulating various other transcription factors including C R E B , heat shock factor-1 (HSF-1) and N F - K B . 2 9 0 " 2 9 2 Cel l cycle progression and protein translation may also be altered by GSK-3 's actions. G S K - 3 may promote cyclin D I expression indirectly through R-catenin since the Lef/Tcf group of transcription factors regulates cyclin D I transcription. 2 9 3 Cycl in D I phosphorylates and inactivates the retinoblastoma (RB) tumour suppressor protein allowing the cell to pass the G l / S phase restriction point. Furthermore, G S K - 3 can also directly influence cyclin D I through 42 phosphorylation of Thr . This modification promotes increased proteasomal degradation of the c y c l i n . 2 9 4 Fumonisin B , a well-known rat hepatocarcinogen, leads to inhibition of GSK-3(3 and overexpression of cyclin D I . 2 9 5 A s such, G S K - 3 may be a crucial factor in tumorigenesis. Meanwhile, the debate continues on G S K - 3 ' s role in regulating eEF-2B. Protein translation is mediated by this multimeric eukaryotic initiation factor, which can be downregulated through phosphorylation of the e subunit at Se r 5 3 5 by G S K - 3 . 2 3 8 Indirect data shows a correlation 247 between IGF-1-induced G S K - 3 inactivation in cultured neurons, and activation of eEF-2B. A s well , insulin treatment of skeletal muscle results in GSK-3-mediated phosphorylation and activation of eEF-2B via the P I 3 - K / P K B pathway. 2 9 6 In contrast, when EGF-induced inactivation 255 of G S K - 3 is blocked, there is no change in eEF-2Be phosphorylation. Finally, GSK-3 's role in the pathology of neurodegenerative diseases is also a hot topic. In Alzheimer's disease, there is an accumulation of neurofibrillary fibers, which many believe to be a causative factor of this devastating disease. These fibers contain large amounts of hyperphosphorylated tau, and recent work has suggested that G S K - 3 can directly phosphorylate t au . 2 9 7 ' 2 9 8 Furthermore, inhibition of G S K - 3 can block hyperphosphorylation of tau both in vitro and in vivo.299 In fact, transgenic mice expressing the GSK-3P gene within the brain demonstrate advanced tau phosphorylation and high rates of neurodegeneration. 3 0 0 As such, G S K - 3 represents an interesting focus in the on-going study of Alzheimer's disease. 43 C H A P T E R 2: M A T E R I A L S AND M E T H O D S 2.1. Materials 2.1.1. Chemicals H 3 [ 3 2 P ] 0 4 I C N 4-hydroxytamoxifen Sigma Acetic acid Fisher Scientific Acrylamide BioRad Acrylamide:bis BioRad Ammonium persulphate BioRad Adenosine 5'-triphosphate Sigma P -mercaptoethanol Fisher Scientific Bis BioRad Bovine serum albumin Fisher Scientific Bromophenol blue Fisher Scientific Butanol B D H Calphostin C, Cladosporium cladosporioides Calbiochem Chloroform Fisher Scientific D609 Calbiochem Diethylether Fisher Scientific D M E M Gibco D M S O Fisher Scientific Dithiothreitol Sigma Enhanced chemiluminescence reagents (ECL) Amersham Ethanol Commercial Alcohols E D T A B D H E G T A B D H Ficoll-Paque Amersham Forskolin, Coleus forskohlii Sigma y - [ 3 2 P ] A T P I C N Glycine BioRad Go 6976 Calbiochem Go 6983 Calbiochem Glycerol Fisher Scientific H E P E S Sigma Hydrochloric A c i d Fisher Scientific L-glutamine Gibco Leupeptin Sigma LY294002 Calbiochem Magnesium chloride Fisher Scientific Methanol Fisher Scientific Microcystin Calbiochem 44 Penicill in/ streptomycin Gibco Pepstatin A Sigma Petroleum ether Sigma Phorbol myristic acetate ( P M A ) Sigma Phosphatidic acid Sigma Phosphoric acid Fisher Scientific P M S F Sigma Ponceau S concentrate Sigma Potassium oxalate B D H Rapamycin V W R Ro-31-8220/bisindolylmaleimide IX Calbiochem Ro-31-8425/bisindolylmaleimide X Calbiochem Rottlerin I C N Sk im milk Safeway Canada Sodium azide B D H Sodium chloride Fisher Scientific Sodium dodecyl sulphate (SDS) Fisher Scientific Sodium fluoride Fisher Scientific Sodium molybdate Sigma Sodium pyruvate Sigma Sodium orthovanadate Sigma Soybean trypsin inhibitor Boehringer Mann. T E M E D BioRad Tris BioRad Triton X-100 Boehringer Mann. Tween-20 B D H U0126 Promega U73122 Calbiochem Urea Fisher Scientific Wortmannin/KY 12420 Calbiochem .2. Disposables Aluminum-backed silica plates E M Science Autoradiography fi lm Kodak Conical tubes Falcon Disposable pipettes V W R Eppendorfs V W R Filter paper V W R Gel loading tips V W R Glass pipettes V W R Kimwipes V W R Latex gloves V W R Liquid nitrogen Membrane Filters Nitrocellulose P81 phosphocellulose Petri plates Pipette tips Scintillation fluid Scintillation vials Praxair Nalgene BioRad Whatman V W R V W R Pharmacia V W R 2.1.3. Proteins/peptides C G M I Crosstide Fetal bovine serum Human recombinant G M - C S F Murine recombinant G M - C S F Murine recombinant interleukin-3 Murine synthetic interleukin-4 Phospho-glycogen synthase peptide-2 Protein G-Agarose RPMI-1640 WEHI-3 (from WEHI-3 cell line) Chris Brown, Calgary Upstate Biotech Gibco R & D R & D R & D James Wieler, U B C Upstate Biotech Gibco Gibco A T C C 2.1.4. Antibodies A k t l / P K B a polyclonal 1 ug/ml G S K - 3 a polyclonal GSK-3oc polyclonal 1:1000 GSK-3 (3 polyclonal 1:1000 Phospho-Akt (Ser 4 7 3 ) polyclonal 1:1000 Phospho-CREB (Ser 1 3 3 ) polyclonal 1:1000 Phospho-GSK-3o/(3 (Ser 2 1 / 9 ) polyclonal 1:1000 Phospho-GSK-3(3 (Ser9) polyclonal 1:1000 Phospho-p44/p42 M A P K (Thr 2 0 2 /Tyr 2 0 4 ) E10 monoclonal 1:1000 Phospho-p70 s 6 K (Thr 4 2 1 /Ser 4 2 4 ) polyclonal 1:1000 PI3-K (p85) polyclonal 1:2000 p 7 0 S 6 K polyclonal 1:1000 R s k / M A P K A P - K l polyclonal 1:1000 Upstate Biotech Upstate Biotech Santa Cruz Stressgen Cel l Signaling Ce l l Signaling Ce l l Signaling Cel l Signaling Ce l l Signaling Cel l Signaling Upstate Biotech Ce l l Signaling Upstate Biotech 46 2.2. Methods 2.2.1. C e l l culture 2.2.1.1. F D C -P1 , M C / 9 and M C / 9 ( P K B - E R ) The murine mast cell line M C / 9 and the murine myeloid progenitor cell line FDC-P1 (American Type Culture Collection, Manassas, V A ) were grown in RPMI-1640 growth medium, supplemented with 10% heat-inactivated F B S , 5-10% WEHI-3 conditioned medium containing murine recombinant EL-3, 2 m M L-glutamine, 1 m M sodium pyruvate, 50 n M (3-mercaptoethanol, and penicillin/streptomycin. Starvations were performed for 4 to 6 hours in the absence of any W E H I - 3 . A l l incubations were carried out in a humidified incubator at 5% C 0 2 . M C / 9 ( P K B - E R ) cells are M C / 9 clones expressing P K B fused with the estrogen receptor. This line was cultured and treated in the same manner as M C / 9 . 2.2.1.2. TF -1 The human erythroblast cell line, TF-1 (American Type Culture Collection, Manassas, V A ) , was grown in RPMI-1640 growth medium, supplemented with 10% F B S , 1% C G M I conditioned media containing human recombinant G M - C S F , 2 m M L-glutamine, 1 m M sodium pyruvate, 50 n M (3-mercaptoethanol, and penicillin/streptomycin. Starvations were performed for 12 to 16 hours in the absence of any C G M I . A l l incubations were carried out in a humidified incubator at 5% C 0 2 . 47 2.2.2. Cytokine stimulation Starved cells were collected, spun down (1500 rpm, 5 minutes), and resuspended in warm RPMI-1640 with 20 m M H E P E S to 5 X 10 6 cells/ml. Immediately before and during stimulations, the cells were kept in a 37°C water bath and routinely vortexed to keep treatments homogenous. Upon completion of a stimulation time point, cells were spun down at 20,000 x g for 1 minute. The supernatant was aspirated, and the cell pellet resuspended in 16 ul of ice-cold lysis buffer (50 m M Tr is -Cl , p H 7.7, 1% Triton X-100, 10% glycerol, 100 m M N a C l , 2.5 m M E D T A , 10 m M NaF, 0.2 m M N a 3 V 0 4 , 1 m M N a M o 0 4 , 40 ug/ml P M S F , 0.5 ug/ml leupeptin, 10 ug/ml soybean trypsin inhibitor, 1 u M pepstatin). Ce l l lysates were left on ice for 10 minutes after which they were again spun at 20,000 x g for 1 minute. The supernatant was extracted and either used for immunoprecipitation or immunoblotting. 2.2.3. Protein Biochemistry 2.2.3.1. Immunoprecipitation The lysates of approximately 8 mill ion cells were reconstituted in 500 ul lysis buffer. Either 2 | ig of an t i -PKBa or 2 pig of ant i -GSK-3a were added, and the samples were then placed at 4°C on a rotator for 1 hour. Protein G-Agarose was added (20 | i l ) and samples were again rotated for 1 hour. Afterwards, the agarose beads were isolated through centrifugation at 10,000 rpm for 1 minute and then washed as needed. 48 2.2.3.2. Immunoblotting The lysates of approximately 1 mill ion cells (16 ul) were added to 4 u4 5 X S D S - P A G E loading buffer (14.81 ml 20% SDS, 9.26 ml 1 M Tris, p H 6.8, 14.81 ml glycerol, 3.70 ml 0.1% bromophenol blue, 7.41 ml (3-mercaptoethanol) and boiled for 10 minutes. Samples were used immediately or frozen down at -20°C. For immunoblotting, a 12.5% low-bis (120:1 acrylamide:bis) polyacrylamide gel (6.64 ml 30% acrylamide, 3.52 ml water, 1.68 ml 1% bis, 4 ml 1.5 M Tris, p H 8.8, 160 ul 10% SDS, 112 ul 10% A P S , 11.2 ul T E M E D ) and a 5% stacking gel (453 ul 1.5 M Tris, p H 6.8, 50 ul 10% SDS, 835 ul 37.5:1 acrylamide:bis, 3.66 ml water, 16.7 pil 10% A P S , 5 ul T E M E D ) were used for protein separation. Gels were run at 200 V in reservoir buffer (3 g. Tris, 14.4 g. glycine, 1 g. SDS; final volume of 1 L) for approximately 45 minutes, or until the dye front had bled off. The gel contents were then transferred to a nitrocellulose membrane at 40 m A for 1 hour 15 minutes in a semi-dry transfer apparatus. Transfer buffer consisted of 5.81 g. Tris, 2.93 g. glycine, 0.0375 g. SDS, and 200 ml methanol, in a final volume of 1 L . The nitrocellulose was soaked briefly in Ponceau S stain to visualize successful protein transfer, and then blocked in 3% skim milk in T B S for 1 hour at room temperature. Afterwards, blots were washed 3 times in T B S with 0.05% Tween-20 added (TBST) for 10 minutes each wash. Overnight incubation of the nitrocellulose in primary antibody followed. Antibody dilutions were in T B S T as noted in section 2.1.4. The blots were again washed 3 times with T B S T for 10 minutes each. Afterwards, the appropriate secondary antibody conjugated to the horseradish peroxidase enzyme was added at 1:5000 dilution for 1 hour in T B S T . Three T B S T washes followed, with a final wash in E C L for 1 minute. Proteins 49 were visualized through fi lm exposure of the blot. Pre-stained molecular weight markers were used and their positions were noted on the developed film. 2.2.3.3. Stripping blots Nitrocellulose blots were re-probed by stripping off the previous antibodies. Blots were kept moist in T B S , and then soaked in stripping buffer (60 m M Tr i s -HCl , p H 6.7, 100 m M (3-mercaptoethanol, 2% SDS) for 30 minutes at 50°C with frequent agitation. The nitrocellulose was then washed with copious amounts of T B S T , and then re-blocked in 3% skim milk. The immunoblotting procedure was continued from this point. 2.2.4. Kinase activity assays 2.2.4.1. GSK-3<x Anti-GSK-3oc (2 ug) was added to cell lysates for one hour, followed by an hour with 20 ul of Protein G-Agarose, all undergoing rotation at 4°C. The beads were then washed 3 times with lysis buffer and once with kinase buffer (5 m M H E P E S , p H 7.4, 0.5 m M E D T A , 0.2 m M Na3V04). They were resuspended in 30 ul kinase buffer with 75 J I M phospho-glycogen synthase peptide-2 substrate added. The assay proceeded with the addition of 10 (i.1 containing 5 ] iCi Y-[ 3 2 P]ATP , 500 u M A T P , and 75 m M M g C l 2 in kinase buffer, with the reaction continuing for 15 minutes at 30°C. Immediately after, the contents were briefly spun down, and the phosphorylated products were isolated by spotting 25 ul of the reaction supernatant onto P81 phosphocellulose paper. The paper was allowed to dry for a few minutes, and was then washed six times in 1% phosphoric acid for 4 minutes each. Quantification was achieved through scintillation counting of each P81 square. Background controls were done simultaneously in samples containing no cell lysate. 2.2.4.2. P K B Cel l lysates were immunoprecipitated with 2 ug of an t i -PKB-a antibody for one hour under rotation at 4°C, followed by an addition of 20 (ll of Protein G-Agarose for one hour. Upon completion, the beads were washed 3 times with lysis buffer and once with kinase buffer (20 m M H E P E S , p H 7.4, 25 m M ^-glycerophosphate, 5 m M E G T A , 0.2 m M N a 3 V 0 4 , 1 m M D T T , 1 u M microcystin, 1 m M MgCl2, 40 Ug /ml P M S F , 0.5 Ug /ml leupeptin, 10 ug/ml soybean trypsin inhibitor). The beads were then resuspended in 10 u.1 kinase buffer and 10 | i l of 100 u M Crosstide substrate. The assay was initiated with the addition of 10 u.1 containing 10 u.Ci y-[ 3 2 P]ATP , 500 u M A T P , and 75 m M M g C l 2 in kinase buffer. The reaction continued for 18 minutes at 30°C after which the contents were briefly spun down. The phosphorylated products were isolated by spotting 20 u.1 of the reaction supernatant onto P81 phosphocellulose paper. The paper was allowed to dry for a few minutes, and was then washed six times in 1% phosphoric acid for 4 minutes each. Quantification was achieved through scintillation counting of each P81 square. Background controls were done simultaneously in samples lacking cell lysate. 2.2.5. L i p i d analysis 2.2.5.1. T h i n layer chromatography ( T L C ) Cells were washed three times and starved for the final 2 hours in phosphate-free R P M I -1640 with 20 m M H E P E S , and labeled with 0.25 mCi /ml [ 3 2 P]P0 3 " 4 at approximately 5 X 1 0 6 51 cells/ml. After 2 hours, the cells were spun down and resuspended in the same volume of R P M I -1640 with 20 m M H E P E S in polypropylene tubes. Stimulation proceeded as in section 2.2.2. To terminate the reaction, 1.6 ml of 2:1 chloroform:methanol was added, and the contents were shaken vigorously. Another 1 ml of chloroform was then added to aid in separation of the organic and aqueous phases. The samples were spun at 3000 rpm for 3 minutes and the lower organic phase was removed and kept. Lipids were dried down using a speed-vac centrifuge, and resuspended in 70 (ll chloroform. Meanwhile, aluminum-backed silica plates were soaked in 1% potassium oxalate for 1 minute and dried overnight. The plate was then baked at 80-100°C for 60 minutes. To the plate, 7 ul of each l ipid sample was spotted and 5 u,g of unlabelled phosphatidic acid standard was included on each side. The plate was placed in a solvent system containing 9:1:1 (v/v) chloroform:methanol:acetic acid, and the solvent front was allowed to climb to 10.5 centimetres from the origin. After a 10-15 minute drying period, the plate was soaked in a second solvent system (60:40:1 petroleum ethendiethylether:acetic acid) with this solvent front reaching the top of the plate. The silica plate was finally dried for 4 hours, and the unlabelled standards were visualized through iodine staining. The labeled lipids were detected through overnight exposure of a phosphoimaging plate. 2.2.5.2. Densitometry analysis Quantification of l ipid levels was achieved through phosphoimaging development and analysis using BioRad's Quantity One densitometry program. C H A P T E R 3: C Y T O K I N E R E G U L A T I O N O F GSK-3 3.1. Rationale and Hypothesis Regulation of cell survival and proliferation is a vital factor within the hematopoietic system. Since these cellular events are modulated by cytokines in many cases, the signaling cascades propagated by these growth factors deserve scrutiny. With mounting evidence of G S K -3's role in cell proliferation and survival, an investigation into the effects of cytokines on G S K - 3 seems warranted. ' Since a diverse array of extracellular signals can target G S K - 3 , it would be of no surprise i f cytokines also show the same effect . 2 3 6 ' 2 4 7 " 2 4 9 In particular, serine phosphorylation of G S K - 3 isoforms upon cytokine stimulation would be anticipated. Whether or not the P I 3 - K / P K B pathway is sufficient and/or essential to any G S K - 3 phosphorylation event is difficult to postulate considering the diverse upstream kinases that have demonstrated an effect on G S K -3 245,251,255,260,263 A g w e l , ^ i t w o u l d b e o f i n t e r e s t t 0 determine whether or not the GSK-3cc and GSK-3(3 isoforms share similar regulatory features in these model systems. 3.2. Results Effects of cytokine stimulation on serine phosphorylation of GSK-3 - The murine M C / 9 mast cell line is able to proliferate in IL-3- or GM-CSF-supplemented media. In contrast, media containing IL-4 only permits prolonged cell survival of M C / 9 cells, but no proliferative response. A n approximate dosage range and an appropriate incubation time for each cytokine was established from previous w o r k . 1 3 6 ' 1 7 3 Using these values as a reference point, M C / 9 cells were treated with a dose response of each cytokine. The phosphorylation status of Ser 9 and Ser 2 1 of 53 GSK-3 (3 and GSK-3oc respectively was analyzed using an equal mix of the phospho-GSK-3rj(/(3 antibody and the phospho-GSK-3(3 antibody (Figure 3.1 A ) . The two antibodies are required since the phospho-GSK-3ot/|3 antibody alone displays a weak affinity for phospho-GSK-3(3 (data not shown). Wi th EL-3 stimulation, both G S K - 3 isoforms exhibited increased serine phosphorylation after 10 minutes, correlating with the increase in cytokine dosage. Even with the presence of both antibodies, the signal was strongest with the G S K - 3 a isoform. While the level of G S K - 3 a phosphorylation was saturated across all dosages of EL-3 used, phosphorylation of GSK-3 (3 reached a maximal level with 1 u.g/ml EL-3 treatment. A s such, all further experiments performed with EL-3 were conducted at that dosage. Likewise, both EL-4 and G M -C S F induce elevated levels of serine phosphorylation of G S K - 3 a and (3. Maximum levels of G S K - 3 phosphorylation were reached at 10 | lg/ml EL-4 and 25 ng/ml G M - C S F at 10 minutes and 5 minutes respectively. These doses and time points were also used in all subsequent experiments. The human TF-1 cell line also proliferates in the presence of G M - C S F . Appropriate 301 doses of G M - C S F and an ideal incubation time have also been previously established. A dose response ranging from 0.1 ng/ml to 10 ng/ml demonstrated increased serine phosphorylation of G S K - 3 at a maximum induced by 10 ng/ml G M - C S F after 5 minutes (Figure 3.1 B) . Another factor-dependent cell line of murine origin is F D C - P 1 . Like M C / 9 , this cell line wi l l proliferate in response to EL-3 and G M - C S F , but not EL-4. The cytokine doses and treatment times used were equivalent to those established for maximal serine phosphorylation of G S K - 3 in M C / 9 . Despite a high background level of phospho-GSK-3a in the unstimulated sample, all three cytokines promoted a recognizable increase in the phosphorylation of G S K - 3 a (Figure 3.1 54 C). Likewise, GSK-3(3 also showed an increased phosphorylation signal with each cytokine. Once again, these treatment dosages were maintained in all further experiments utilizing F D C - P 1 cells. Interestingly, there is a third unidentified band of about 40 kDa in size that was consistently observed in all phosphoserine-GSK-3 immunoblots utilizing FDC-P1 cell lysates. L ike G S K - 3 a and G S K - 3 p \ this band was found to increase in intensity with the presence of each cytokine. Whether this is an example of non-specific binding to a completely different protein, or proof of proteolytic cleavage of G S K - 3 , remains to be determined. However, there is no previous mention of a physiological G S K - 3 cleavage event in the literature. 55 A IL-3 M C / 9 ^ r s/ — - • P - G S K - 3 a •P - G S K - 3 (3 IL-4 •P-GSK-3oc •P - GSK-3(3 G M - C S F <r *y V V • P - G S K - 3 a •P - GSK-3(3 B G M - C S F TF-1 O f ** S -P - GSK-3oc •P - G S K - 3 p Figure 3.1. Effect of cytokines on serine phosphorylation of GSK-3a/p. A . M C / 9 cells were cytokine-starved and stimulated with either IL-3 (10'), IL-4 (10'), G M - C S F (5') or the diluent alone ( D M S O - 10'). B . TF-1 cells were cytokine-starved and stimulated with either G M - C S F (5 ' )ordi luenta lone(DMSO-5 ' ) . C . FDC-P1 cells were cytokine-starved and stimulated with either IL-3 (10'), IL-4 (10'), G M - C S F (5') or the diluent alone ( D M S O -10 ') . S D S - P A G E was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of GSK-3 . These results are typical of at least 4 experiments. IL-3 FDC-P1 IL-4 — P - G S K - 3 P —? •P - G S K - 3 a G M - C S F P - G S K - 3 a P - GSK-3p Cytokines do not alter expression ofthe GSK-3 proteins - Although the short cytokine incubation times utilized in Figure 3.1 make it unlikely that the effects seen on G S K - 3 are the result of increased protein levels, it was prudent to establish that the apparent increase in G S K - 3 phosphorylation was indeed due to post-translational modification of the protein. In Figure 3.2 A , M C / 9 cells were stimulated with each of the three cytokines at the established doses and incubation times. A s before, EL-3, EL-4, and G M - C S F all induced increased serine phosphorylation of both G S K - 3 a and GSK-3p\ After stripping the blot of this antibody mix, they were re-probed with antibodies specific for G S K - 3 a and GSK-3(3, regardless of phosphorylation state. A s the figure illustrates, there was no change in the protein levels of either isoform of G S K - 3 . As such, the changes demonstrated with the phospho-GSK-3 antibodies were solely due to increases in phosphate content at the amino-terminal serine residue of G S K - 3 . The use of the p85 antibody was to simply insure equal total protein content throughout the experimental samples. Likewise, Figure 3.2 B established the consistency of G S K - 3 protein content between GM-CSF-stimulated TF-1 cells and unstimulated ones. 58 Figure 3.2. Effect of cytokines on GSK-3a/p* is post-translational. A. MC/9 cells were cytokine-starved and stimulated with either IL -3 (10'), I L - 4 (10'), GM-CSF (5') or the diluent alone (DMSO -10 ' ) . B. TF-1 cells were cytokine-starved and stimulated with either GM-CSF (5') or diluent alone (DMSO - 5'). SDS-PAGE was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of GSK-3, GSK-3cc, GSK-3p\ and p85. These results are typical of 2 experiments. Role ofPI3-K in cytokine-induced GSK-3 inactivation - The first reports focusing on the regulation of G S K - 3 involved the insulin-mediated inactivation of G S K - 3 . 2 3 3 ' 2 3 4 This inhibition was shown to require the phosphatidylinositol-3 kinase (PI3-K)/Protein kinase B (PKB) 251 pathway. In previous studies, pharmacological abrogation of PI3-K activity eliminated 252 phosphorylation of G S K - 3 at the crucial amino-terminus serine residues. To assess the role of this pathway in the phosphorylation of G S K - 3 in factor-dependent hematopoietic cell lines, two structurally distinct inhibitors of PI3-K were used. Both LY294002 ( IC 5 0 = 1.4 u M ) and wortmannin (IC50 = 3 nM) are potent catalytic site inhibitors of P I 3 - K . 3 0 2 ' 3 0 3 Cells were pre-treated with either inhibitor for 15 minutes prior to stimulation, at dose ranges well-established for in vivo PI3-K inhibi t ion. 3 0 4 EL-3- and GM-CSF-stimulated phosphorylation of both G S K - 3 isoforms was unchanged with either 100 u,M LY294002 or 100 n M wortmannin present (Figure 3.3 A ) . Assessment of P K B phosphorylation at Ser 4 7 3 , which is known to be phosphorylated downstream of PI3-K activation, allowed for assurances of total PI3-K inhibition. However, despite a total loss of Se r 4 7 3 phosphorylation at 10 u M of either inhibitor, G S K - 3 phosphorylation was unchanged with EL-3 and G M - C S F treatment. In contrast, EL-4 regulation of G S K - 3 was completely impaired by the presence of either wortmannin or LY294002. Meanwhile, TF-1 cells pretreated with either PI3-K inhibitor showed some reduction in GM-CSF-stimulated G S K - 3 phosphorylation (Figure 3.3 B) . However, despite total abrogation of P K B Ser 4 7 3 phosphorylation at 100 u M LY294002 and 100 n M wortmannin, the G S K - 3 phosphorylation signal was still greater then that found in the unstimulated samples. 60 A + L Y 2 9 4 0 0 2 ( u M ) 1 + 10 100 + Wortmanriiri (nM) 1 10 100 + + + + IL -3 P - GSK-3cx P - G S K - 3 p P - P K B (Ser 4 7 3) L Y 2 9 4 0 0 2 ( u M ) 1 + 10 + 100 + Woitmannin (nM) 1 10 100 + + + + I L - 4 P - G S K - 3 c c P - G S K - 3 P + L Y 2 9 4 0 0 2 ( u M ) 1 + 10 + 100 + Woitmannin (nM) 1 10 100 + G M - C S F P - G S K - 3 c c P - G S K - 3 p P - P K B (Ser 4 7 3 ) Figure 33. Role of P13-K in GSK-3 regulation by cytokines. A . M C / 9 cells were cytokine-starved, pre-treated wi th L Y 2 9 4 0 0 2 or woi tmannin for 15 minutes, and stimulated wi th either IL-3 (10 ' ) , I L - 4 ( 1 0 ' ) , G M - C S F (5 ' ) or the diluent alone ( D M S O - 1 0 ' ) . B . T F - 1 cel ls were cytokine-starved, pre-treated wi th L Y 2 9 4 0 0 2 or woi tmannin for 15 minutes, and stimulated wi th either G M -C S F (5') or diluent alone ( D M S O - 5 ' ) . S D S - P A G E was performed on whole ce l l lysates and the resulting blot probed for serine phosphorylation o f G S K - 3 , phosphoserine 4 7 3 P K B , and p85. These results are typical o f 3 experiments. LY294002(uM) + 1 + 10 + 100 + Wortmannin (nM) 1 10 100 + + + + G M - C S F P - G S K - 3 a P - GSK-3p P - P K B (Ser 4 7 3) PKB activity is sufficient for induction of GSK-3 serine phosphorylation - Since PI3-K inhibition did have dramatic effects with EL-4 signaling for G S K - 3 phosphorylation, and lesser effects with other cytokines, it would be of interest to see i f P K B can in fact mediate phosphorylation of G S K - 3 . Unfortunately, there are numerous downstream targets of PI3-K, making it difficult to differentiate between the effects of P K B on G S K - 3 versus other kinases. To examine P K B ' s ability to mediate G S K - 3 phosphorylation required a system whereby P K B could be stimulated without changes in PI3-K activity. As such, we utilized an M C / 9 cell line stably transfected with a P K B protein fused to a receptor for estrogen ( P K B - E R ) . The addition of an estrogen derivative, 4-hydroxytamoxifen (4-HT), provides a steroid group for the P K B - E R protein, promoting activation of the P K B construct. In fact, treatment with 4-HT alone led to increased G S K - 3 a/(3 phosphorylation in a dose-responsive manner with maximal stimulation at 10 u M (Figure 3.4 A ) . A s well , this dosage of 4-HT produced levels of G S K - 3 phosphorylation comparable to those found with the standard panel of cytokines (Figure 3.4 B) . While cytokine treatments promote P K B 4 7 3 phosphorylation, they have no effect on the P K B - E R construct. In contrast, the 4-HT has no effect on endogenous P K B , but does promote phosphorylation of the transfected P K B mutant. Regardless, this system forcefully activates P K B and is not a fair representation of P K B activation in vivo during cytokine stimulation. However, in this experiment, P K B can promote phosphorylation of G S K - 3 independent of PI3-K. 63 Figure 3.4. PKB activity is sufficient for GSK-3 serine phosphorylation. A . M C / 9 ( P K B - E R ) cells were cytokine-starved and stimulated with varying dosages of 4-hydroxytamoxifen ( 1 0 ' ) . B . M C / 9 (PKB-ER) cells were cytokine-starved and stimulated with either 1 0 | i M 4-HT (10'), 1 Ug/ml I L - 3 ( 1 0 ' ) , 1 0 ug/ml I L - 4 ( 1 0 ' ) , 25 ng/ml G M - C S F (5'), or the diluent alone ( D M S O - 1 0 ' ) . SDS-P A G E was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of G S K - 3 , phosphoserine4 7 3 P K B , and p85. These results are typical of at least 3 experiments. ERK/RSK do not regulate GSK-3 phosphorylation - Several studies have suggested a role for the E R K / R S K pathway in the phosphorylation of the G S K - 3 isoforms. 2 5 5 " 2 5 7 To examine this, we used the M E K 1 ( I C 5 0 = 72 nM) and M E K 2 ( IC 5 0 = 58 nM)-specific inhibitor U0126 to examine the role of these M E K isoforms, and their downstream target, E R K , on G S K - 3 regulation during cytokine treatment. 3 0 5 As is shown in Figure 3.5 A , both EL-3 and G M - C S F stimulated phosphorylation of the E R K 1 and E R K 2 proteins in the M C / 9 cell line. This was assessed with an antibody specific for the dual phosphorylation of the threonine and tyrosine found in the T E Y motif in p42 /p44 M A P K . These MEK-mediated phosphorylations are essential for E R K activation. Unlike EL-3 and G M - C S F however, EL-4 did not demonstrate any upregulation of E R K phosphorylation above the control sample. 3 0 6 Concomitantly, there was little to no change in G S K - 3 serine phosphorylation by EL-3, EL-4, or G M - C S F with 2 5 u M U0126 pretreatment in comparison to samples with no inhibitor. Likewise in the TF-1 cell line, GM-CSF-stimulated phosphorylation of G S K - 3 was unhindered by the presence of U0126 in the media (Figure 3.5 B) . Since E R K is upstream of R S K , we examined whether U0126 pretreatment also blocked this kinase. R S K is activated by numerous phosphorylation events via E R K , as well as through autophosphorylation upon activation by E R K . 1 4 0 In many cases, the change in phosphorylation state can be detected through the resulting change in molecular weight. For example, an antibody specific for p90 r s k demonstrates a clear bandshift to a higher molecular weight with cytokine treatment, indicating enhanced phosphorylation and thus reduced electrophoretic mobility (Figure 3.5). This same bandshift can be abrogated, along with E R K activity, with U0126 pretreatment, demonstrating the inhibition of p90 r s k phosphorylation and activity. Wi th the 65 abrogation of R S K activity and no change in cytokine-induced serine phosphorylation of GSK -3 , RSK' s role in GSK -3 regulation must be minimal. 66 B op (4 <V te # # -P - GSK-3a •P - GSK-3(3 p . p44/p42 MAPK p 9 0 r s k p85 Figure 3.5. The role of MAPK in GSK-3 regulation. A. MC/9 cells were cytokine-starved, pretreated with either 25 uM UO126 or diluent for 15 minutes, and stimulated with either IL-3 (10'), IL-4 (10'), GM-CSF (5') or the diluent alone (DMSO -10 ' ) . B . TF-1 cells were cytokine-starved, pretreated with either 25 |J.M UO 126 or diluent for 15 minutes, and stimulated with either GM-CSF (5') or diluent alone (DMSO - 5'). SDS-PAGE was performed on whole cell lysates and the resulting blots probed for serine phosphorylation of GSK-3, phospho-p44/p42 MAPK, p90 r s k and p85. These results are typical of 3 experiments. 67 p7(f6K does not modulate GSK-3 phosphorylation upon cytokine stimulation - Another kinase that potentially phosphorylates G S K - 3 isoforms is p 7 0 s 6 K . 2 6 3 To determine i f p 7 0 S 6 K does in fact regulate G S K - 3 phosphorylation, M C / 9 cells were pretreated with rapamycin, an inhibitor of p 7 0 s 6 K activity ( I C 5 0 = 0.05 nM). After 15 minutes of inhibitor treatment, cell samples were stimulated with the various cytokines to observe the effects on G S K - 3 phosphoserine content. In M C / 9 cells, EL-3 and G M - C S F induced elevated phosphorylation of p 7 0 S 6 K , with LL-4 producing less of an effect. The antibody for p 7 0 s 6 K phosphorylation recognizes the modification of both T h r 4 2 1 and Ser 4 2 4 . These residues must be phosphorylated to release pseudosubstrate inhibition of p 7 0 S 6 K and to thereby promote full catalytic activity. A s well , analysis using an antibody specific for the general p 7 0 S 6 K protein also noted a corresponding upward bandshift with cytokine treatment. Wi th all three cytokines, 100 ng/ml of rapamycin was able to reduce the SfSfC S6K phosphorylation of p70 and the resulting bandshift. However despite the inhibition of p70 activity, there was no change in GSK-3oc or GSK-3(3 phosphorylation in rapamycin-treated versus untreated cells (Figure 3.6 A ) . EL-3, EL-4 and G M - C S F upregulation of G S K - 3 serine phosphorylation was not blocked by rapamycin. Furthermore, G M - C S F induction of G S K - 3 S6K. phosphorylation in TF-1 cells was also uninterrupted by the cancellation of p70 activity (Figure 3.6 B) . 68 Figure 3.6. The role ofp7 n s*K in GSK-3 regulation. A . M C / 9 cells were cytokine-starved, pretreated with either 100 ng/ml rapamycin or diluent for 15 minutes, and stimulated with either IL-3 (10'), IL-4 (10'), G M - C S F (5') or the diluent alone ( D M S O -10') . B . TF-1 cells were cytokine-starved, pretreated with either 100 ng/ml rapamycin or diluent for 15 minutes, and stimulated with either G M - C S F (5') or diluent alone ( D M S O - 5'). S D S - P A G E was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of G S K - 3 , phospho-p70S 6 K, p70 S 6 K and p85. These results are typical of 3 experiments. 69 PKA activity may upregulate GSK-3 phosphorylation - Yet another protein kinase implicated in the regulation of G S K - 3 serine phosphorylation is the cAMP-dependent protein kinase ( P K A ) . 2 6 0 , 2 6 1 To further examine whether or not P K A can in fact phosphorylate G S K - 3 in vivo, forskolin, an activator of adenylyl cyclase, was used to induce P K A activation. The direct effect of this pharmacological treatment is an elevation in the levels of c A M P , which in turn can promote P K A activity. Wi th previously established doses and incubation times for forskolin therapy, the effects of P K A activation were assessed in M C / 9 , TF-1 , and F D C - P 1 cell l ines. 2 6 1 While increased forskolin dosages elevated G S K - 3 serine phosphorylation in TF-1 , there was little to no change in M C / 9 and FDC-P1 cells (Figure 3.7). The effect of forskolin on G S K - 3 in TF-1 cells was primarily directed at the GSK-3oc isoform, although a slight increase in phosphorylation was also detected in GSK-3R. The level of phosphorylation signal was highest at 40 u M of forskolin, but since this was the largest dosage tested, higher concentrations of forskolin may further promote G S K - 3 phosphorylation. To assess whether P K A was in fact activated through forskolin treatment, the phosphorylation of C R E B , a direct substrate of P K A , was also studied using antibodies specific for the phosphorylated residues targeted by P K A . While both TF-1 and F D C - P 1 cells demonstrate elevated increases in C R E B phosphorylation correlating with increasing forskolin dosage, M C / 9 cells did not appear to respond to forskolin therapy since modification of the C R E B protein was unchanged. Whether this is due to the cell line's inability to respond to forskolin through P K A activation, or P K A ' s inability to target C R E B , remains undetermined. However, other work has clearly demonstrated C R E B phosphorylation upon forskolin treatment in M C / 9 ce l l s . 1 3 6 70 Forskolin Forskolin Forskolin # # ^ i 5 ^ ^ j £ ° ^ # ^ - P - G S K - 3 c t -P - GSK-3P P - C R E B p85 1 3 3 M C / 9 TF-1 FDC-P1 Figure 3.7. Cell specific regulation of GSK-3 by forskolin. M C / 9 , TF-1 a n d F D C - P l cells were cytokine-starved and stimulated with either forskolin (4,10,40 | i M ) , or the diluent alone (DMSO) for 10 minutes. SDS-PAGE was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of G S K - 3 , phospho-CREB (Ser 1 3 3), and p85. These results are typical of 2 experi-ments. Phorbol ester stimulation of GSK-3 serine phosphorylation - Of further interest is the possibility that protein kinase C (PKC) may also play a role in the regulation of G S K - 3 i soforms. 2 4 5 ' 2 5 8 Wi th that in mind, members of the classical and novel subsets of the P K C family were activated through the use of the phorbol ester, phorbol myristic acetate ( P M A ) . P M A has a structure that resembles diacylglycerol, and is thus able to substitute for D A G in the activation of P K C . Phorbol esters increase the binding ability of P K C isoforms for C a 2 + , resulting in the enzyme's full activation without the need for calcium mobilization. In both the M C / 9 (Figure 3.8 A ) and the TF-1 (Figure 3.8 B) cell lines, 100 n M of P M A did promote G S K - 3 a and G S K - 3 p serine phosphorylation to levels well above those found in samples treated with the delivery vehicle alone ( D M S O ) . 72 A JMt B P M A - P - G S K - 3 a ~ P - G S K - 3 P -P - P K B (Ser 4 7 3) p85 Figure 3.8. Effect of phorbol esters on GSK-3a/p\ A . M C / 9 cells were cytokine-starved and stimulated with either P M A (100 nM) or the diluent alone (DMSO) for lOminutes. B . TF-1 cells were cytokine-starved and stimulated with either P M A (100 nM) or diluent alone (DMSO) for 10 minutes. SDS-PAGE was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of G S K - 3 , phospho-PKB (Ser 4 7 3), p85. These results are typical of >5 experiments. PKC regulates GSK-3 phosphorylation - Unfortunately, it is well established that 307 phorbol esters can activate a variety of protein kinases in addition to P K C / As such, it became essential to utilize other methods to evaluate P K C ' s effects on G S K - 3 during cytokine stimulation. To that end, a panel of P K C inhibitors was selected to pre-treat each cell line, and observe the capacity to which each cytokine could serine phosphorylate G S K - 3 under these conditions. Two of these inhibitors included the staurosporine analogs, R6-31-8220 and R6-31-8425, which target P K C at the C3 domain of the catalytic region as a competitive inhibitor of A T p 308-310 B o m o f t h e s e c o m p o u n ( j s are strong inhibitors of the classical P K C ' s ( I C 5 0 = 5-24 nM) and less so for P K C e ( I C 5 0 = 24-39 nM) . However, there is evidence that these two drugs 311 312 are capable of inhibiting a wide range of members from all three P K C families. ' Two other staurosporine-based analogs also used were the Go 6976 and Go 6983 compounds. The classical P K C ' s are strongly inhibited by both of these drugs (IC 5o = 2-10 nM) , although their effects on the novel and atypical isoforms at higher concentrations remain unsolved. 3 1 3 " 3 1 6 A s well , these two inhibitors can be helpful in discerning between the effects of various P K C isoforms. For example, Go 6976 also blocks P K C L I but not P K C ^ ( I C 5 0 > 20 uM), while Go 6983 blocks P K C C ( IC 5 0 = 60 nM) but not P K C ^ i . 3 1 7 " 3 1 8 With this arsenal of P K C inhibitors, studies were performed to determine the ability of each to block cytokine-induced G S K - 3 serine phosphorylation. Concentration and pre-incubation times were identified from dose response studies performed earlier (data not shown). In M C / 9 cells, we can see the minor effect of PI3-K inhibitors on IL-3 and GM-CSF-responses again (Figure 3.9). Likewise, PI3-K inhibition totally blocked DL-4-mediated G S K - 3 phosphorylation. As for the P K C inhibitors, Ro-31-8220, Ro-31-8425, and Go 6976 all blocked 74 phosphorylation of G S K - 3 , irrespective of the cytokine used. This was true for both G S K - 3 a and G S K - 3 p. Interestingly, Go 6983 consistently had a lesser effect on blocking G S K - 3 phosphorylation. Meanwhile in TF-1 cells, the same three P K C inhibitors also blocked G S K - 3 phosphorylation, although Ro-31-8220 was less effective in this case (Figure 3.10). Once again, Go 6983 had little to no effect. Finally, a similar pattern was observed in F D C - P 1 cells, although many of the inhibitors showed a weaker effect (Figure 3.11). Ideally, these P K C inhibitors would be most useful to help distinguish between the role of P K C and P K B in G S K - 3 regulation. Unfortunately, some of these P K C inhibitors produce an effect on P K B phosphorylation at Ser 4 7 3 . Ro-31-8220 did not block cytokine-mediated P K B phosphorylation in M C / 9 but it did have some effect in TF-1 and FDC-P1 cells. Ro-31-8425 also blocked P K B phosphorylation in M C / 9 and TF-1 , but had a variably effect in F D C - P 1 . Meanwhile, Go 6976 consistently blocked P K B phosphorylation in all cases. Finally, Go 6983 had no effect on P K B phosphorylation in any of these systems. 75 cP <# ^ <# J p A b ^ P - G S K - 3 o c •P - GSK-3p < # c # < # Ab oft ^ v # & <gr 6 ° ' 6° ' 1 1 4 ? , / njS* ^ b^ b^ P - G S K - 3 a P -GSK-3F3 P - P K B (Ser 4 7 3) P - G S K - 3 a P - G S K - 3 P P - P K B (Ser 4 7 3) Figure 3.9. GSK-3 phosphorylation via PKC. M C / 9 cells were cytokine-starved and pretreated with either LY294002 (100 U.M), wortmannin (100 nM), Ro-31 -8220 (10 uM) , Ro-31 -8425 (10 UM), Go-6976 (5 ( iM), or Go-6983 (5 | i M ) for 15 minutes. Samples were then stimulated with either IL-3 (10'), IL-4 (10'), G M - C S F (5') or the diluent alone ( D M S O -10 ' ) . S D S - P A G E was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of G S K - 3 , phospho-PKB (Ser 4 7 3), and p85. These results are typical of 3 experiments. 76 Figure 3.10. GSK-3 phosphorylation via PKC. TF-1 cells were cytokine-starved, pretreated with either Ro-31-8220 (10 uM) , Ro-31-8425 (10 j i M ) , Go-6976 (5 \M), or Go-6983 (5 u M ) for 15 minutes. Samples were then stimulated with either G M - C S F (5') or diluent alone ( D M S O - 5'). SDS-P A G E was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of G S K - 3 , phospho-PKB (Ser 4 7 3), and p85. These results are typical of 3 experiments. 4 4? 6 ° 4 ? 6 ° P - G S K - 3 a • P - G S K - 3 R b 6 ° P - G S K - 3 a P - G S K - 3 ( 3 P - P K B (Ser 4 7 3) p85 <^ cj? 4? 4? c>0' 6 b ' P - G S K - 3 a P - G S K - 3 (3 Figure 3.11. GSK-3 phosphorylation via PKC. FDC-P1 cells were cytokine-starved and pretreated with either LY294002 (100 uM) , wortmannin (100 nM) , Ro-31 -8220 (10 uM) , Ro-31 -8425 (10 | i M ) , Go-6976 (5 uM) , or Go-6983 (5 u M ) for 15 minutes. Samples were then stimulated with either IL-3 (10'), IL-4 (10'), G M - C S F (5') orthe diluent alone ( D M S O - 1 0 ' ) . SDS-PAGEwas performed on whole cell lysates and the resulting blot probed for serine phosphorylation of GSK-3 , phospho-PKB (Ser 4 7 3), and p85. These results are typical of 2 experiments. 78 GSK-3 is not a target ofPKCS- The unconfirmed specificity of many P K C inhibitors, with respect to each P K C isoform, makes any study of P K C difficult. One drug that showed 319 promising isoform specificity is rottlerin with strong effects on P K C 8 (IC50 = 3-6 uM). However, publications have also noted rottlerin's effects on some classical P K C ' s (IC50 = 30-42 uM) and some novel and atypical P K C isoforms ( I C 5 0 = 80-100 uM). However, the effect of rottlerin pre-incubation in M C / 9 and TF-1 cells with respect to cytokine-mediated G S K - 3 phosphorylation was still examined (Figure 3.12). A s illustrated, 5 u M of rottlerin had no effect on G S K - 3 phosphorylation in any case. A s such, it appears that P K C 8 is not an important regulator of G S K - 3 serine phosphorylation. 79 A Figure 3.12. PKC8 does not regulate GSK-3 phosphorylation by cytokines. A . M C / 9 cells were cytokine-starved and pretreated with either 5 u M rottlerin or diluent only (DMSO) for 15 minutes. Samples were then stimulated with either IL-3 (10'), IL-4 (10'), G M - C S F (5') or diluent alone ( D M S O -10') . B . TF-1 cells were cytokine-starved and pretreated with either 5 u M rottlerin or diluent only (DMSO) for 15 minutes. Samples were then stimulated with either G M - C S F (5') or diluent alone ( D M S O -10') . S D S - P A G E was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of GSK-3 and p85. These results are typical of 3 experiments. Cytokines increase diacylglycerol levels - Considering the prominent role of P K C in the regulation of G S K - 3 serine phosphorylation by cytokines, the regulation of P K C itself is an interesting focus. Presumably the inhibition of factors mediating P K C activation should also inhibit G S K - 3 phosphorylation. Both classical and novel P K C isoforms, as well as P K C p , require diacylglycerol production for the upregulation of P K C catalytic activity. Cytokines have been noted to increase diacylglycerol levels in other cell systems. 1 9 9 - 2 0 1 To examine whether these cytokines promote D A G elevation in M C / 9 cells, phosphate-free media was supplemented with H 3 [ 3 2 P]04, and the cells pre-incubated and treated with cytokines in this solution. Thin layer chromatography permitted the separation of various l ipid products, and those radioactively labeled were visualized by exposure to X-ray fi lm. However, diacylglycerol does not contain phosphate. A s such, a metabolic byproduct of D A G was examined instead. Phosphatidic acid is formed from the actions of diacylglycerol kinase and changes in its intracellular level are intimately linked to diacylglycerol amounts. Quantification of radioactivity within each sample's phosphatidic acid pool was measured through densitometry analysis. Both EL-3 and G M - C S F stimulated at least a two-fold increase in phosphatidic acid levels (Figure 3.13). However, EL-4's effects were minimal in this study. To determine the effectiveness of certain diacylglycerol inhibitors, three candidate drugs were also selected for pretreatment in the cell samples prior to cytokine treatment. The D A G kinase inhibitor El (R59949) can block the transformation of diacylglycerol to phosphatidic acid, while the phospholipase inhibitors U73122 and D609 were also examined. 3 2 0 U73122 is a broad phospholipase C inhibitor with a higher affinity for phosphatidylinositol-specific P L C (PI-PLC) while D609 is more selective for P C - P L C . 3 2 1 ' 3 2 2 81 Effective dosages for these drugs were established through literature sources and previous dose response studies (Figure 3.14). Pretreatment of M C / 9 cells with D A G kinase inhibitor LI, D609, or U73122 reduced EL-3-and GM-CSF-induced phosphatidic acid accumulation (Figure 3.13). Meanwhile, these inhibitors produced little difference in the already minimal phosphatidic acid change induced by EL-4. Interestingly, P M A also produced elevated phosphatidic acid levels but these perturbations were not significantly affected by any of these three inhibitors. 82 Figure 3.13. Diacylglycerol formation upon cytokine treatment M C / 9 cells were cytokine-starved and labelled with [ 3 2P]-P0 4~ 3. The cells were then pretreated with either D A G kinase inhibitor II (10 u M ) , U73122 (50 uM) , D609 (100 u M ) or diluent alone for 15 minutes. Samples were then stimulated with either IL-3 (10'), IL-4 (10'), G M - C S F (5'), P M A (10') or the diluent alone ( D M S O -10'). Samples were then stimulated with either G M - C S F (5') or diluent alone ( D M S O - 5'). Lipids were extracted using the Bligh-Dyer method and run on silica plates. Results of autoradiography were quantified and graphed. Diacylglycerol production and GSK-3/PKB phosphorylation - To further examine the effects of the phospholipase C inhibitors on G S K - 3 phosphorylation during cytokine signaling, cells were pretreated with either U73122 or D609 in the dosages indicated (Figure 3.14A). In the M C / 9 cell line, fifteen minute pretreatment with U73122 at dosages as low as 10 u M led to abrogation of both cytokine-induced P K B Ser 4 7 3 phosphorylation and G S K - 3 a / G S K - 3 P Ser 2 1 /Ser 9 phosphorylation. Treatment with 50 u M U73122 further enhanced this effect in all three cases. Meanwhile, the D609 compound demonstrated no change in cytokine-mediated G S K - 3 or P K B phosphorylation. A similar response was noted in the TF-1 cell line, although the effect on G S K - 3 and P K B phosphorylation was first noted at 50 u M of the U73122 compound. While inhibition with U73122 implicates PI-PLC-mediated D A G production as having some function in the signalling pathway to G S K - 3 , this is unlikely since cytokines are not known to activate P I -PLC. Thus it is possible that the inhibitor acts non-specifically on some kinases such as PI3-K or P D K 1 . These possibilities w i l l have to be addressed by testing the effect ofthe inhibitors on the various possible kinases. 84 A 4 U73122(uM) 1 D609 (\LM) 10 + 50 + 1 + 10 + 100 + IL-3 • P - G S K - 3 c c •P-GSK-3F3 P - P K B (Ser 4 7 3) p85 + U73122 (uM) 10 50 + + D609(uM) 1 10 + 100 + IL-4 P - G S K - 3 a P-GSK-3P P - P K B (Ser 4 7 3) p85 U73122 (U.M) D609 (uM) 1 10 50 1 10 100 + + + + + + + G M - C S F Figure 3.14. Role of P L C in GSK -3 regulation by cytokines. A . M C / 9 cells were cytokine-starved, pre-treated with U73122, D609, or diluent (DMSO) for 15 minutes, and stimulated with either IL-3 (10'), IL-4 (10'), G M - C S F (5') or the diluent alone ( D M S O -10 ' ) . B . TF-1 cells were cytokine-starved, pre-treated with U73122, D609, or diluent (DMSO) for 15 minutes, and stimulated with either G M - C S F (5') or the diluent alone ( D M S O -10') . S D S - P A G E was performed on whole cell lysates and the resulting blot probed for serine phosphorylation of GSK-3 , phosphoserine473 P K B , and p85. These results are typical of 3 experiments. 85 86 Correlation of GSK-3 activity with serine phosphorylation - Although the above results demonstrate a pattern in the regulation of G S K - 3 phosphorylation in cytokine-treated cells, the catalytic activity of G S K - 3 needs to be directly examined to verify that the phosphorylation events are having their expected effects. Although the serine phosphorylation of G S K - 3 has been intimately linked to the inactivation of this kinase, there is also evidence that other modifications of G S K - 3 also play a crucial and maybe even a more important role. ' ' ' Using a pre-phosphorylated peptide substrate derived from the glycogen synthase protein, G S K - 3 a activity was examined in vitro. Interestingly, the pattern of G S K - 3 activity did not correlate with the expected results established in the phospho-serine studies (Figure 3.15). In TF-1 cells, treatment with G M - C S F resulted in no significant change in G S K - 3 activity, although Western blot analyses suggest that the serine residue of G S K - 3 a in this treatment shows elevated phosphorylation. Likewise, treatment of cells with LY294002 or U73122 also showed no conclusive change with respect to cytokine-stimulated T F - l ' s and those treated with the vehicle alone (DMSO) . However, the one pattern that did emerge was the lowered catalytic activity induced by the four P K C inhibitors. Ro-31-8220, R6-31-8425, Go 6976 and even Go 6983 pretreatment reduced G S K - 3 catalytic activity to levels between 50-70% of the control samples. 87 Figure 3.15. Regulation of GSK-3a activity. TF-1 cells were cytokine-starved and stimulated with either 10 ng/ml G M - C S F (5 min.) or diluent alone ( D M S O - 5 min.), in some cases'after pre-incuba-tion (15 min.) with either LY294002 (100 u M ) , R6-31-8220 (10 u M ) , R6-31-8425 (10 uM) , Go 6976 (5 u M ) , Go 6983 (5 u M ) , or U73122 (100 uM) . G S K - 3 a was immunoprecipitated and its phosphotranferase activity assayed with the phosphoglycogen synthase substrate. The substrate was isolated on P81 phosphocellulose paper and scintillation analysis performed. The results are shown as a mean of three experiments ± standard error. 88 PKC inhibitors and PKB activity - Likewise, the direct analysis of P K B activity can also support, or refute, the ability of some of these P K C inhibitors to inhibit cytokine-induced P K B activity. To that effect, P K B a was immunoprecipitated and its catalytic activity assessed in vitro with a peptide substrate. Figure 3.16 clearly demonstrates a 6 to 10 times increase in the activity of P K B as compared to the control samples. Not surprisingly, the well-established PI3-K inhibitor, LY294002, reduced GM-CSF-stimulated P K B activity substantially. O f the four P K C inhibitors and the P L C inhibitor U73122, only Go 6976 and U73122 reduced P K B activity by statistically significant amounts. In fact, these two downregulated P K B activity to levels found in cell pretreated with LY294002. 89 15-14-13-Control GM-CSF GM-CSF GM-CSF GM-CSF GM-CSF GM-CSF GM-CSF + + + + + + LY294002 R6-31-8220 R6-31-8425 Go 6976 Go 6983 U73122 Figure 3.16. Regulation of PKB activity. TF-1 cells were cytokine-starved and stimulated with either 10 ng/ml G M - C S F (5 min.) or diluent alone ( D M S O - 5 min.), in some cases after pre-incuba-tion (15 min.) with either LY294002 (100 uM) , R6-31-8220 (10 uM) , R6-31-8425 (10 uM) , Go 6976 (5 u M ) , Go 6983 (5 u M ) , or U73122 (100 u M ) . P K B a was immunoprecipitated and its phosphotranferase activity assayed with the phosphoglycogen synthase substrate. The substrate was isolated on P81 phosphocellulose paper and scintillation analysis performed. The results are shown as a mean of three experiments ± standard error. 90 CHAPTER 4: CONCLUSIONS A variety of studies have closely examined the enigmatic enzyme, glycogen synthase kinase-3 (GSK-3) . Many of these publications have noted G S K - 3 to be an anti-proliferative and an anti-survival kinase. Several growth factors and other extracellular stimuli promote inactivation of G S K - 3 , presumably to allow for cell survival. 2 3 6 , 2 4 7 " 2 4 9 This is supported through experiments where inactivation of G S K - 3 protected cells from entering an apoptotic phase . 2 3 6 ' 2 6 1 , 2 8 2 " 2 8 5 Likewise, cells over expressing G S K - 3 or expressing constitutively active G S K - 3 demonstrated impaired cell survival and proliferation. However, no clear and consistent pattern has emerged as to whether G S K - 3 is sufficient or even essential for cell survival in certain cell types. 2 3 7 Surprisingly, there are no published reports on the effects of cytokines on G S K - 3 . Cytokines are soluble growth factors that target primarily hematopoietic cells, and promote enhanced cell survival and/or increased proliferation. In fact, a variety of cells require these cytokines for survival in culture and are thus called factor-dependent cell lines, such as M C / 9 , TF-1 and F D C - P 1 . EL-3 and G M - C S F are examples of cytokines that strongly promote cell growth and differentiation of the myeloid lineage of hematopoietic c e l l s . 2 4 " 2 7 ' 4 1 " 4 4 ' 5 8 Others, such as EL-4 are less potent in their proliferative effects, but still demonstrate potential as a growth factor, particularly with other co-stimulatory agents. Despite these powerful anti-apoptotic effects, a thorough search of the literature reveals that there is no data available on the role of cytokines in G S K - 3 regulation. In this study, EL-3, EL-4 and G M - C S F induced a dramatic increase in the serine phosphorylation of both G S K - 3 a and G S K - 3 P within five to ten minutes of initial stimulation in M C / 9 , TF-1 and the FDC-P1 cell lines. Although the results obtained from the kinase assays are 91 preliminary, and w i l l have to be verified, it appears that the lack of inhibition of G S K - 3 activity, despite its serine phosphorylation, contradicts numerous studies where the serine phosphorylation of G S K - 3 was shown to be essential and necessary for G S K - 3 inactivation. ' " In considering possible explanations for this result, it is known that numerous other residues within G S K - 3 are also targets for post-translational modification. In particular, much focus has been directed on the conserved tyrosine residues found in both G S K - 3 mammalian isoforms ( T y r 2 7 9 in GSK-3a) . Some research has demonstrated that this tyrosine is also an active player in the regulation of G S K - 3 ac t i v i t y . 2 6 7 ' 2 6 8 In particular, phosphorylation at this site promotes G S K - 3 activity in cells during basal stages. It is plausible that, despite G S K - 3 serine phosphorylation, the phosphorylation of the tyrosine residue continues to promote G S K - 3 catalytic activity to levels comparable to that of untreated resting cells. A s well , i f G S K - 3 does indeed mediate cytokine-induced growth and survival, the effects of G S K - 3 inactivation would need to be observed over a longer time frame. In these studies, G S K - 3 was examined only five to ten minutes after the initial addition of cytokines. Perhaps a longer cytokine exposure does promote a decline in G S K -3 catalytic activity to mediate long-term cellular effects, although insulin-mediated abrogation of G S K - 3 occurs as quickly as five minutes. 2 5 1 Furthermore, the possibility that the in vitro kinase assay itself may have been flawed or misleading cannot be ruled out. The examination of G S K - 3 323 activity is notoriously difficult due to the kinase's high level of basal activity. As well, no clear positive control was established to confirm the accuracy of this assay system. In particular, comparison of G S K - 3 activity from the lysates of untreated and IgM-treated A31 B-cells demonstrated no observable differences (data not shown). In other studies, I g M treatment of this 324 particular cell line has been noted to reduce G S K - 3 a activity in comparison to untreated cells. 92 Numerous attempts to replicate this data using the same assay system and protocols have failed. It is possible that some of the reagents used in these assays, or the techniques employed by the examiner, were flawed. Alternatively, the substrate peptide utilized in these in vitro assays permits analysis of only one facet of G S K - 3 catalytic activity. The substrate was based on a sequence from glycogen synthase, a pre-phosphorylated protein targeted by G S K - 3 . The peptide substrate is also pre-phosphorylated at the +4 residue to the site of G S K - 3 ' s target, as is the case with glycogen synthase in vivo. A s such, this assay examined G S K - 3 ' s ability to phosphorylate this particular substrate, and is presumably indicative of G S K - 3 ' s affinity for pre-phosphorylated targets. However, G S K - 3 can also target various non-phosphorylated substrates. One example is the transcription factor B-catenin, which can mediate the enhanced transcription of genes implicated in cell survival and cell proliferation. Interestingly, G S K - 3 can be modulated to target one class of substrates but not the other. 2 8 0 Thus, this assay only focused on the catalytic activity of G S K - 3 a towards pre-phosphorylated substrates, as opposed to those containing no prior modification. Despite the apparent setback in correlating G S K - 3 serine phosphorylation with G S K - 3 activity, the pathways regulating G S K - 3 serine phosphorylation were still crucial targets for examination. Since IL-3, IL-4 and G M - C S F all promote cell survival in M C / 9 and TF-1 cells to some extent, the concomitant serine phosphorylation of G S K - 3 could suggest a correlation between G S K - 3 regulation and cell survival. The P I 3 - K / P D K / P K B pathway has long been of interest in the study of how cytokines manifest their anti-apoptotic effects in hematopoietic cells. A l l three cytokines used in this study are well-known to activate both PI3-K and P K B . 6 0 , 1 7 3 ' 1 8 2 ' 1 8 3 The question remains as to the need 93 for this pathway in mediating cell survival. In some cell systems, P K B presence is essential for cell survival and cell proliferation, while in other cell lines, P K B activity does not correlate with cell su rv iva l . 6 0 ' 1 8 4 " 1 8 7 Regardless, the PI3-K pathway has been demonstrated to be a key regulator of G S K - 3 serine phosphorylation in other model systems. As well , P K B can phosphorylate G S K - 3 at these critical serine residues in vitro. Examination of the PI3-K pathway in vivo often involves use of the two structurally distinct inhibitors, wortmannin and LY294002. Wortmannin is a fungal metabolite that covalently associates with the p i 10 subunit of PI3-K with high affinity (IC50 = 3 nM), resulting in the abrogation of both the phosphatidylinositol kinase and the serine kinase activities of PI3-K . 3 2 5 This drug is a potent inhibitor of PI3K, and at lower concentrations it is also selective for PI3-K. LY294002, an analog of the bioflavinoid quercetin, is also a potent inhibitor of PI3-K (IC50 = 1.4 u M ) . 3 0 2 It inhibits PI3-K through a different mechanism from wortmannin by acting as a competitive inhibitor of A T P binding. In fact, LY294002 is considered to be a more reliable inhibitor of PI3-K than wortmannin. In M C / 9 cells, the PI3-K pathway is clearly an important player in the regulation of G S K -3 serine phosphorylation during IL-4 stimulation. However, the function of PI3-K in the inhibition of G S K - 3 is less certain with IL-3 and G M - C S F treatment of M C / 9 and TF-1 cells. This pattern between IL-4 and DL - 3 / G M - C S F regulation of G S K - 3 is probably due to the differences in the cytokine receptor complexes and the signaling pathways promoted by each. The EL-4 receptor is comprised of the a chain specific for the cytokine itself, but also includes the y common chain found in receptors for IL-4, IL-2, IL-7, IL-9, IL-13, and IL-15 . 7 4 Meanwhile, the IL-3 and G M - C S F receptors contain the P common subunit. The primary role of the common 94 subunit chain is as an intracellular signal transducer of cytokine stimulation. In fact, the P and y chains can activate different pathways from each other. As such, the similar cellular effects induced by EL-3 and G M - C S F are most probably due to the shared p c receptor subunit. Both EL-3 and G M - C S F activate E R K , promote cell survival and proliferation in many hematopoietic cells, and exhibit continued G S K - 3 phosphorylation in the absence of PI3-K activity. Meanwhile, EL-4, with a yc receptor chain instead, does not activate E R K , does not induce cell proliferation in many cell lines, and demonstrates PI3-K-dependent G S K - 3 regulation. As such, while PI3-K plays a predominant role in EL-4-mediated G S K - 3 phosphorylation, EL-3 and G M - C S F may use other pathways to mediate G S K - 3 phosphorylation, or utilize redundant pathways to circumvent the blockage of PI3-K activity. However, even in EL-3 and G M - C S F signaling, PI3-K activity still plays a minor role in G S K - 3 phosphorylation, suggesting the existence of backup kinases to target G S K - 3 in the event of PI3-K shutdown. Numerous studies have suggested that PI3-K and P K B are required for cytokine-mediated survival . 1 8 4 If this is true, the inability of PI3-K inhibitors to block G S K - 3 serine phosphorylation in some cases would suggest that G S K - 3 is not a key player in cytokine-mediated survival. However, studies have noted that G M - C S F and EL-3 can promote cell survival independent of PI3-K in M C / 9 cells, although this is not true in TF-1 and F D C - P 1 ce l l s . 1 7 3 As G S K - 3 is not greatly affected by PI3-K inhibitors with these two cytokines in M C / 9 , this lends credence to the theory that G S K - 3 may play a role in M C / 9 cell survival. On the other hand, the minor role played by PI3-K during G S K - 3 regulation by G M - C S F in TF-1 cells suggests that G S K - 3 might not be an important factor in cell survival in all cytokine-responsive cell types. Clearly, further investigation of G S K - 3 in cytokine-mediated cell survival is warranted. 95 The use of LY294002 and wortmannin establishes an important role for PI3-K in EL-4-induced G S K - 3 phosphorylation, and a minor function in the EL-3 and G M - C S F stimulation of G S K - 3 serine phosphorylation. It is, however, difficult to correlate this data with P K B as a component in G S K - 3 signaling. PI3-K promotes elevated levels of 3'-phosphorylated phosphoinositides, which in turn can mediate the activities of many protein kinases. For example, P D K 1 becomes membrane localized and promotes phosphorylation of various substrates upon agonist-mediated increase of PI3-K lipid products. These substrates include P K B a , PKC£, p 7 0 S 6 K , R S K 2 , and serum glucocorticoid kinase 2 ( S G K 2 ) . 1 5 7 - 1 6 2 A s well , PI3-K displays some protein serine kinase activity and can phosphorylate its p85 subunit and the ERS-1 protein. As such, inhibition of PI3-K activity could conceivably alter the activity of numerous other kinases other then P K B . Although several studies have targeted P K B activity directly to demonstrate changes in the serine phosphorylation of G S K - 3 , other studies have also pointed to a possible link between G S K - 3 and kinases such as P K C , p70 , and R S K 2 . As such, pharmacological inhibition of PI3-K in the study of G S K - 3 phosphorylation is only an initial step in elucidating the upstream kinases responsible. Since no effective and/or specific inhibitor for P K B is commercially available, it is difficult to focus exclusively on P K B ' s effects on G S K - 3 through chemical intervention. However, it is clear that P K B can promote the serine phosphorylation of G S K - 3 in some instances. The M C / 9 cell line expressing P K B fused to a portion of the estrogen receptor is one example of a system demonstrating the role of P K B in G S K - 3 modification independent of PI3-K. Treatment of this altered M C / 9 cell line with the estrogen derivative 4-hydroxytamoxifen (4-HT), clearly demonstrates that P K B can mediate phosphorylation of G S K - 3 independent of PI3-K. However, the question remains as to whether 96 P K B is an in vivo regulator of G S K - 3 phosphorylation during cytokine signaling, particularly upon LL-4 treatment. Another putative regulator of G S K - 3 serine phosphorylation was the M A P K / R S K pathway. In particular, R S K has been demonstrated to phosphorylate the crucial serine residues in G S K - 3 in vitro. It is also plausible that some of the effects noted in the PI3-K inhibition studies could result from the inability of P D K 1 to target R S K . However, R S K also requires phosphorylation by M A P K at key residues in order to be fully activated. IL-3 and G M - C S F can stimulate M A P K phosphorylation and activity, while IL-4 demonstrates no such response. Wi th this in mind, R S K was inhibited through the abrogation of M E K activity. M E K is the dual-specific kinase that phosphorylates the threonine and tyrosine residues within the catalytic loop of M A P K , thus promoting full activity of the latter. As such, inhibition of M E K should abrogate R S K activity as well . The U0126 compound was utilized as a potent inhibitor of both M E K 1 ( I C 5 0 = 72 nM) and M E K 2 ( I C 5 0 = 58 n M ) . 3 0 5 U0126 is a specific non-competitive inhibitor of M E K with respect to both A T P and E R K , and does not effect the activities of P K C , A b l , Raf, M E K K , E R K , J N K , M K K 3 , M K K 6 , Cdk2, or C d k 4 . 3 2 9 Pretreatment of M C / 9 and TF-1 cells with this cell permeable inhibitor led to reduced IL-3- and GM-CSF-mediated M A P K phosphorylation. A s well , abrogation of R S K phosphorylation was confirmed through bandshift analysis. However, there was no effect on the phosphorylation of either G S K - 3 a or G S K - 3 P by any of the cytokines in these cell models. As such, it is clear that neither M E K , E R K , nor R S K are responsible for the regulation of G S K - 3 by IL-3, IL-4 or G M - C S F . Although M A P K is important for IL-3- and GM-CSF-mediated cell survival of some factor-dependent cell lines, many of these effects are cell-type spec i f ic . 1 3 3 ' 1 3 4 A s well, there is on-going debate over the 97 extent to which the M A P K and PI3-K pathways crosstalk. 1 3 4 , 1 8 8 As such, it is difficult to ascertain whether the lack of M A P K input into G S K - 3 serine phosphorylation indicates a minimal role for G S K - 3 in cell survival. Closer scrutiny of the p 7 0 S 6 K pathway also demonstrated its insignificance in the regulation of G S K - 3 . The mitogenic activation of p 7 0 s 6 K is regulated by phosphorylation events that release the protein from pseudosubstrate inhibition and promote full catalytic activity. To examine this kinase more thoroughly in its effects of G S K - 3 , the macrolide antibiotic, rapamycin, derived from the filamentous bacterium Streptomyces hygroscopicus, was utilized. This immunosuppressive drug associates with the F K B P protein, creating a complex which in turn binds to m T O R . 3 2 9 Although the exact biochemical nature of m T O R remains elusive, m T O R is a putative protein kinase that lies upstream of the p 7 0 s 6 K regulatory pathway. Treatment of cells with rapamycin can block p 7 0 S 6 K activity in various cell systems. However, rapamycin is highly specific and has demonstrated no effects on PI3-K, Raf, M A P K , or R S K . 3 3 0 , 3 3 1 Clearly, IL-3 and G M - C S F can induce phosphorylation of p 7 0 s 6 K in M C / 9 and TF-1 cells, while IL-4 cannot. Rapamycin can totally abrogate this effect, thus blocking p 7 0 s 6 K activity. EL-4's inability to activate p 7 0 S 6 K , combined with the absence of any inhibitory effect on G S K - 3 serine phosphorylation with rapamycin treatment, suggests that p 7 0 s 6 K is not a player in the mediation of either G S K - 3 a or G S K - 3 (3 serine phosphorylation. The role of p 7 0 s 6 K i n cellular proliferation 228 232 • is well-established, although these effects appear to be cell-type specific. " There is little S6K S6K information on p70 and cell survival. A s such, the fact that G S K - 3 is not a target for p70 still supports a putative role for G S K - 3 in cell survival. 98 Recent evidence also illustrates enhanced G S K - 3 phosphorylation in response to induced c A M P production. In fact, the cAMP-responsive P K A can phosphorylate G S K - 3 at the crucial serine residues in vitro. The production of c A M P can be accelerated in vivo via treatment with various pharmacological compounds, including forskol in . 2 6 0 ' 2 6 1 This compound is a naturally occurring diterpene that directly activates adenylyl cyclase to promote elevated intracellular c A M P concentration. 3 3 2 The E C 5 o range for forskolin has been noted to be between 1 u M to 20 u M for cells and tissues. This compound generally induces cAMP-dependent effects, although the drug has also been noted to target various membrane transport proteins and receptors. 3 3 3 ' 3 3 4 The elevation of c A M P induced by forskolin typically promotes P K A activation. One common substrate target for P K A is the transcription factor C R E B . Forskolin clearly promoted C R E B phosphorylation in TF-1 and F D C - P 1 , although M C / 9 did not appear to respond to forskolin. Other groups have reported models in which forskolin is unable to promote increased c A M P product ion. 3 3 5 ' 3 3 6 However, previous work from this laboratory has established the role of forskolin in M C / 9 cells as an activator of adenylyl cyclase, and further demonstrated increases in C R E B phosphorylation. 1 3 6 As such, the lack of forskolin-based responses in M C / 9 remains a mystery. However, even with an expected forskolin response, only TF-1 cells exhibited increased G S K - 3 serine phosphorylation. In particular, G S K - 3 a phosphorylation appeared much more targeted by forskolin treatment then GSK-3(3. This would support the previous notion that P K A can target G S K - 3 for phosphorylation in some cases. However, whether or not P K A repeats this role in cytokine signaling cannot be discerned from these results. While IL-4 can stimulate P K A activation in B cells, G M - C S F does not produce a c A M P spike in TF-1 99 c e l l s . 2 1 9 , 2 2 1 A s such, it is improbable that cytokines regulate G S K - 3 phosphorylation through P K A . The P K C inhibitors dramatically affect cytokine-induced G S K - 3 phosphorylation. The most efficient and consistent inhibitor for G S K - 3 serine phosphorylation was the staurosporine analog, Go 6976. Both P K C a and P K C (3i have been shown to be inhibited by this compound ( I C 5 0 = 2-6 nM) , while P K C 8, 8, r| and £ are not ( I C 5 0 = 700-20,000 n M ) . 3 1 6 Indirect evidence also suggests that this inhibitor blocks P K C y activation. 3 1 3 Furthermore, the unique P K C u. is also a target for Go 69.76 inhibition ( I C 5 0 = 20 n M ) . 3 1 7 , 3 3 7 A search of the literature found no evidence of P K C 9, or XA inhibition by Go 6976. However, despite the actions of Go 6976, it is unlikely that the classical P K C isoforms are involved in G S K - 3 phosphorylation during cytokine signaling, since classical P K C ' s are typically activated in conjunction with an increase in intracellular calcium. Cytokines do not promote such calcium mobilization. On the other hand, in vitro activation of classical P K C requires very small amounts of calcium to be present. A s such, it is plausible that the residual levels of intracellular calcium found in these hematopoietic cells may still be sufficient for activation of classical P K C ' s . However, it is more likely that the effects of Go 6976 are due either to the inhibition of P K C p or to a non-specific target of the inhibitor. Some of the non-specific targets of Go 6976 include M A P K A P - K l b , M S K 1 , and P D K 1 . 3 3 8 Interestingly, this inhibitor consistently blocked phosphorylation of P K B at Ser 4 7 3 , although other studies have suggested that this inhibitor has minimal effects on P K B a activity in vitro.33S Regardless, the abrogation of both P K B phosphorylation and activity by Go 6976 in this study makes it difficult to determine i f P K C or P K B are indeed involved in the regulation of G S K - 3 phosphorylation. However, since IL-3- and GM-CSF-induced G S K - 3 phosphorylation is 100 not heavily dependent on PI3-K activity, it is more likely that Go 6976's effects on G S K - 3 during IL-3 and G M - C S F stimulation is via inhibition of P K C [i. The inability of Go 6983 to dramatically inhibit G S K - 3 phosphorylation also suggests a role for P K C p since Go 6983 targets the classical P K C ' s ( I C 5 0 = 6-7 nM) , but not P K C p ( I C 5 0 = 20,000 n M ) . 3 1 7 ' 3 3 7 However, Go 6983 does not inhibit cytokine-induced P K B phosphorylation or activation either. Furthermore, phorbol ester treatment of M C / 9 and TF-1 cells demonstrated elevated levels of G S K - 3 serine phosphorylation. Phorbol esters are capable of regulating the classical and novel P K C family members, as well as P K C p, further suggesting that P K C u. might be involved in G S K - 3 regulation by some cytokines. Wi th concomitant loss of P K B activity during inhibition of G S K - 3 phosphorylation by Go 6976, it is difficult to determine i f the inhibitor's effect is due to P K B or P K C inhibition. However, Ro-31-8220, another P K C inhibitor, was also able to dramatically inhibit G S K - 3 phosphorylation in M C / 9 cells without affecting serine phosphorylation of P K B in some cell types. This would suggest that the actions of this P K C inhibitor on G S K - 3 are independent of P K B activity. Ro-31-8220 has been noted to inhibit the activities of all classical P K C ' s (IC50 = 5-27 nM), as well as P K C s (IC50 = 24 nM). However, this same inhibitor can target a plethora of other protein kinases, including M A P K A P - K l b , M S K 1 , S G K , p 7 0 S 6 K , and even GSK-3p 338 itself. This makes any conclusions drawn from studies with R6-31-8220, or any other P K C inhibitor, tenuous at best. While it is clear that P K C plays a predominant role in G S K - 3 signaling by cytokines, the isoform(s) responsible remain unknown. Although the inhibition of G S K - 3 serine phosphorylation should promote elevated G S K -3 catalytic activity, the in vitro kinase assays does not appear to corroborate this theory. Once 101 again, this may be due to procedural flaws in the assay itself (see above). In previous studies, acute stimulation of cells with phorbol esters have been noted to decrease tyrosine phosphorylation and concurrent G S K - 3 activity. 3 3 9 However in the current study, inhibition of P K C led to further reduction in G S K - 3 activity, and is thus the opposite effect of what previous groups have reported. Therefore, the data produced from these in vitro assays do not corroborate with the existing literature or signaling pathway mechanics. As such, the results of these assays must be viewed cautiously. In an effort to further support the theory that P K C regulates cytokine-mediated G S K - 3 serine phosphorylation, this study examined the production of diacylglycerol. D A G is an essential component for the activation of classical and novel P K C ' s . Furthermore, these D A G -dependent P K C isoforms have also been implicated as promoters of P K C p activity, while P K C p can also bind directly to diacylglycerol as w e l l . 3 4 0 EL-3 and G M - C S F both induce the production of phosphatidic acid, a byproduct of diacylglycerol metabolism. This process is susceptible to both the P I -PLC inhibitor U73122 and the P C - P L C inhibitor D609. However, PI-P L C activity has not been implicated in cytokine stimulation, primarily due to a lack of calcium mobilization that should result from the phosphoinositide hydrolysis product, EP3. Meanwhile, P C - P L C activity has been detected with EL-3, G M - C S F , and EL-4 stimulation. 1 9 9 " 2 0 1 Interestingly, P L D hydrolysis of phosphatidylcholine to produce D A G via phosphatidic acid has also been detected with G M - C S F stimulation, but not E L - 4 . 2 0 2 - 2 0 4 However, neither U73122 nor D609 is known to inhibit P L D activity. The specificities of the two phospholipase inhibitors used in this study are also suspect. Both U73122 and D609 inhibit phosphatidic acid production to some extent. However, the role 102 of P I -PLC in this production should be minimal, which cannot be corroborated with the apparent actions of U73122. Likewise, U73122 also blocks G S K - 3 and P K B serine phosphorylation. Although U73122 has been shown to inhibit agonist-stimulated P K B serine phosphorylation with thrombin, thrombin is also known to promote P I - P L C activity and calcium mobil izat ion. 3 4 1 This is unlike cytokines where no P I -PLC activity and no calcium signal are recorded. It is therefore more likely that the non-specific nature of U73122 is the underlying cause for many of the results obtained. The current study is the first to demonstrate that cytokines do promote G S K - 3 serine phosphorylation in various factor-dependent hematopoietic cell lines. The pathways regulating this phosphorylation event are primarily the PI3-K and P K C pathways. IL-4 requires PI3-K activity in order to manifest its effects on G S K - 3 . In contrast, neither IL-3 nor G M - C S F stimulation of G S K - 3 phosphorylation is dramatically altered with the presence of PI3-K inhibitors. While the M A P K / R S K and p 7 0 s 6 K pathways show no evidence of playing a role in G S K - 3 regulation, the use of P K C inhibitors suggests a strong function for this family of protein kinases. Unfortunately, the nature of the P K C pharmacological inhibitors makes it impossible to identify the specific class of P K C ' s , or the specific P K C isoform responsible for the effects seen on G S K - 3 . A s well , the effects of these P K C inhibitors on P K B make it difficult to distinguish the role of the P I 3 - K / P K B pathway from that of the P K C pathway. Likewise, studies of diacylglycerol production are also hampered by the questionable specificity of the inhibitors that are in common laboratory use. As such, it is difficult to establish which mitogenic pathways are essential for G S K - 3 serine phosphorylation and cell survival by only using pharmacological methods. 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