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Regulation of the c-myc gene by cytokines and growth factors Wieler, James Scott 1999

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R E G U L A T I O N O F T H E c-myc G E N E B Y C Y T O K I N E S A N D G R O W T H F A C T O R S by J A M E S S C O T T W I E L E R B.Sc. (Honors), Simon Fraser University, 1991 A THESIS S U B M I T T E D I N 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 D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Medicine) (Division of Experimental Medicine) U N I V E R S I T Y O F B R I T I S H C O L U M B I A M A Y 1999 © James Scott Wieler, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of f~ yy^^i^^^^/ JrfjL^fUtriJ> The University of British Columbia Vancouver, Canada Date / C DE-6 (2/88) 11 ABSTRACT The proto-oncogene transcription factor c-myc has long been associated with cell growth as this gene is frequently found in many tumors to have undergone amplification or rearrangements with other genes. These alterations result in unregulated expression of an otherwise normal gene product. Whi le c-myc is required for growth and differentiation the mechanism as to how c-myc contributes to these processes is not understood. Treatment of a variety of cells with a growth factor results in the accumulation of c-myc m R N A and M y c protein although the proteins involved in regulating this process are unknown. Growth factor stimulation also results in increased Phosphatidylinositol 3-Kinase (PI 3-Kinase) activity, which is also required for growth, however the function of PI 3-Kinase in mitogenesis is not well understood although it has been shown to prevent apoptosis. During the course of this thesis several lines of evidence suggested that PI 3-Kinase might be involved in the regulation of c-myc gene expression. I show here that increased levels of c-myc m R N A induced by growth factor treatment is dependent on PI 3-Kinase activity. Two structurally and mechanistically distinct inhibitors of PI 3-Kinase, the synthetic molecule L Y 2 9 4 0 0 2 or the fungal metabolite wortmannin, blocked the increases in c-myc m R N A induced by platelet derived growth factor (PDGF) treatment of NIH-3T3 fibroblasts, or mast cells stimulated with interleukin-3 (IL-3), I L -4 or Steel locus factor (SLF). Another means of blocking PI 3-Kinase activity is the expression of a dominant interfering mutant of the regulatory subunit of PI 3-Kinase called Ap85. This mutant possesses the domains required for recruitment to activated receptors, and other intermediate proteins, but lacks the domain required for binding to the catalytic subunit of PI 3-Kinase. Transiently over-expressing this mutant protein prevents activation of the endogenous I l l PI 3-Kinase by competing with the endogenous regulatory subunit for binding to these activated receptors and intermediate proteins. In these transient systems c-myc gene expression can not be monitored so a reporter plasmid containing the c-myc promoter fused to the firefly luciferase gene was also transiently introduced. Luciferase activity obtained by treatment of cells with IL-3 or IL-4 was blocked by the co-expression of the Ap85 mutant. These results show that PI 3-Kinase activity is required for expression of the c-myc gene and also suggest that the signaling pathway to the c-myc gene lies downstream of PI 3-Kinase. This suggests a novel function for PI 3-Kinase, in addition to its anti-apoptotic role, and possibly explains the requirement of PI 3-Kinase activity for mitogenesis. The role of molecules activated by PI 3-Kinase, in the regulation of the c-myc gene, was also investigated. One of these molecules is the protein serine/threonine kinase called mammalian Target of Rapamycin (mTOR) which is inhibited by the drug rapamycin but also by wortmannin or LY294002 . Rapamycin failed to block the increased levels of c-myc m R N A induced by treatment with IL-3 , IL-4 or S L F in hemopoietic cells or P D G F in NIH-3T3 fibroblasts. Thus c-myc induction correlated with PI 3-Kinase activity but not m T O R activity suggesting the signal transduction pathway, regulating the c-myc gene, lies downstream of PI 3-Kinase but does not involve mTOR. iv T A B L E O F C O N T E N T S Abstract i i Table of Contents iv List of Tables v i i i List of Figures ix List of Abbreviations x i Acknowledgements xv Chapter One Introduction 1.1 General Overview 1 1.2 Regulatory Transcription Factors 3 1.3 c-myc 6 1.3.1 Phosphorylation 7 1.3.2 Functional Domains 8 1.3.3 Family Members 9 1.3.4 Transcriptional Repression 14 1.3.5 M y c Proliferation 16 1.4 Signal Transduction Pathways Regulating c-myc Induction V 1.4.1 Tyrosine Kinases 17 1.4.2 R a s / M A P Kinase Pathway 17 1.4.3 v-aW 18 1.4.4 Vi ra l Induction of c-myc 18 1.5 Regulation of c-myc during Development 19 1.6 Induction of Apoptosis by M y c 19 1.7 Phosphatidylinositol 3-Kinase 21 1.7.1 Class I PI 3-Kinases 22 1.7.2 Class II PI 3-Kinases 25 1.7.3 Class III PI 3-Kinases 26 1.7.4 P K B and P D K 26 1.7.5 S6-Kinase 31 1.8 Ras and MAP-fami ly Kinases 33 1.9 Signal Transduction of Cytokines 1.9.1 c-kit Signaling 34 1.9.2 IL-3 Signaling 35 1.9.3 IL-4 Signaling 36 1.10 Induction of Nuclear Proto-oncogenes 3 8 Chapter Two Materials and Methods 2.1 Cloning of Steel Locus Factor 40 2.2 Bacterial Expression of Steel Locus Factor 40 2.3 Ce l l Stimulation Conditions 41 2.4 Kinase inhibitors 42 2.5 S D S - P A G E Immunoblots 42 vi 2.6 P K B Kinase Assays 43 2.7 P K B Immune-blots 44 2.8 J N K Kinase Assays 44 2.9 J N K Immunoblots 45 2.10 p70 s 6 K Gel Shift Assay 45 2.11 Transient Transfection of Baf/3 Cells 46 2.12 Isolation and Culture of Primary Bone Marrow Derived Mast Cells 46 2.13 Northern Blots 46 2.14 RNase Protection Assays 49 Chapter Three Regulation of c-myc by Cytokines and Growth Factors 3.1 c-myc Expression Correlates with Proliferation 53 3.2 Role of PI 3-Kinase in c-myc Induction in Hemopoietic cells 57 3.3 Role of m T O R in c-myc induction 58 3.4 Role of PI 3-Kinase in c-myc Induction in Non-hemopoietic cells 61 3.5 Specificity of PI 3-Kinase Inhibitors 62 3.6 Inhibition of P K B 63 3.7 Inhibition of p70 S 6 K 64 3.8 Role of S T A T - 6 in c-myc induction by IL-4 65 3.9 Dominant negative blocks c-myc induction 67 3.10 Role of PI 3-Kinase in constitutive expression of c-myc 68 3.11 Role of the Raf/Erk signal cascade in c-myc V l l induction 69 3.12 Role of PI 3-Kinase in MAP-family kinase activation 70 Chapter Four Conclusions and Future Work 72 Bibliography 79 V l l l L I S T O F T A B L E S Table 1.1 Genes positively regulated by M y c 13 Table 1.2 Genes negatively regulated by M y c 15 ix L I S T O F F I G U R E S Chapter 1 Figure 1.1 Functional regions of M y c 10 Figure 1.2 Functional regions of PI 3-Kinase 22 Chapter 3 Figure 3.1 c-myc m R N A expression is constitutive in a variety of cell lines 54 Figure 3.2 Expression of c-myc in parental and serum free R 6 X mast cells 55 Figure 3.3 Induction of c-myc, c-fos and c-jun by IL-3 or IL-4 56 Figure 3.4 PI 3-Kinase activity is required for the induction of c-myc in response to IL-4 58 Figure 3.5 LY294002 or wortmannin block c-myc induction in response to IL-3 in a dose dependent manner 59 Figure 3.6 PI 3-Kinase but not m T O R is required for c-myc induction in response to cytokines 60 Figure 3.7 PI 3-Kinase but not mTOR is required for c-myc induction in NIH-3T3 fibroblasts 61 Figure 3.8 Effect of LY294002, wortmannin or rapamycin on the growth factor induced activation of P K B 63 Figure 3.9 Effect of LY294002, wortmannin or rapamycin on the growth factor induced activation of p 7 0 s 6 K 64 Figure 3.10 S T A T - 6 is not required for increased levels of c-myc m R N A stimulated by IL-4 or S L F 66 Figure 3.11 Dominant inhibitory mutant of the p85 subunit of PI 3-Kinase X (Ap85) blocks the activation of a c-myc luciferase reporter 67 Figure 3.12 Constitutive expression of c-myc in HeLa cells is not PI 3-Kinase dependent Figure 3.13 Raf/Erk M A P kinase signaling path is not sufficient for c-myc Induction Figure 3.14 PI 3-Kinase activity is not required for Jnk activation in response t o I L - 3 o r S L F 71 68 69 xi L I S T O F A B B R E V I A T I O N S A P C Adenomatous Polyposis Co i i AP-1 Activator Protein-1 b Basic C / E B P C A A T T box/Enhancer Binding Protein C R E B c A M P Responsive Element B i n d i n g protein C S F Colony Stimulating Factor CSF-1 Colony Stimulating Factor-1 dbl double domain G N E F s Guanine Nucleotide Exchange Factors H L H Helix-Loop-Helix H T H Helix-turn-helix JEN Interferons IL Interleukin I N R Initiator element IRS 1/2 Insulin Receptor Substrate-1 and 2 K L c-kit Ligand L I F Leukemia Inhibitory Factor L Z Leucine Zipper M C 2 9 Myelocytomatosis retrovirus 29 M E K 1 M A P kinase kinase 1 M E K K s M A P kinase kinase kinases Xll M G F Mast cell Growth Miz -1 M y c interacting zinc finger protein M K K s M A P kinase kinases mT Polyoma middle T N L S Nuclear Localization Signal p70/p85 S 6 K Ribosomal protein S6 kinase P A H Paired Amphipathic a-Helixes P D G F Platelet-Derived Growth Factor P D K 1 / 2 3-phosphoinositide Dependent Kinase 1 and 2 P H Plekstrin Homology domain PI Phosphatidylinositol PI3,4,5P 3 Phosphatidylinositol-3,4,5 trisphosphate PI3,4P 2 Phosphatidylinositol-3,4 bisphosphate PI3P Phosphatidylinositol-3 phosphate PIC Pre-Initiation Complex P I K PI Kinase P K A Protein Kinase A P K B Protein Kinase B P K C Protein Kinase C P T B Phosphotyrosine-Binding R A C - P K Related to A and C Protein Kinases R N A Pol II R N A polymerase II S C F Stem Cel l Factor x i i i Src Homology-2 SH-2 containing Inositol-5 Phosphatase Steel Locus Factor Specificity protein-1 Serum Response Element Serum Response Factor Signal Transducers and Act ivators of Transcription T B P T A T A box Binding Protein Tcf-4 T-cell factor-4 T N F Tumor Necrosis Factor T P A 12-O-Tetradecanoylphorbol 13-Acetate Tpo Thrombopoietin Y Y 1 Yin-Yang-1 SH-2 SHIP S L F Sp-1 S R E S R F S T A T xiv A C K N O W L E D G E M E N T S I would l ike to thank Sidney Brenner whose pioneering work in m R N A production and processing made this thesis possible. I could not have completed this thesis without the support of my parents, close friends and my three lab comrades Dr. Ian Foltz, Dr . Megan Levings and Dr. Ruth Salmon. I would also like to thank Dr. Vince Duronio who gave me my first exposure to the world of signal transduction and was one of the best teachers I have ever had. Finally, I would like to thank my supervisor Dr. John Schrader who gave me complete academic freedom to explore any project of my choosing. This freedom has taught me much about the art of doing good science. 1 C H A P T E R 1. I N T R O D U C T I O N 1.1 Genera l Overview Growth factors, when bound to their appropriate receptors, initiate a cascade of molecular events, which eventually results in cell division. The identity of the intracellular molecules recruited by the various receptor systems and the function of these molecules in growth, survival and development has been the focus of much research. The hemopoietic growth factors belong to a family of cytokines and hormones that share a common four oc-helix bundle structure and regulate the growth, differentiation and survival of many different cell types including those of the hemopoietic compartment. The cytokine family consists of the interleukins (IL), colony stimulating factors (CSF), erythropoietin (Epo), thrombopoietin (Tpo), leukemia inhibitory factor (LIF), prolactin, growth hormone, interferons (JEN), and tumor necrosis factor (TNF). Other molecules that share the same three dimensional structure are the hemopoietic growth factors such as Steel locus factor (SLF) also known as stem cell factor (SCF), mast cell growth factor (MGF) or c-kit ligand (KL) and colony stimulating factor-1 (CSF-1). Despite their different biological effects, the intracellular cascades stimulated by many of these ligands are surprising similar. However, there are a few examples where the biological activities of the cytokines can be accounted for based on the specific intracellular signals generated. One such example, discussed below, is the comparison of the biological and biochemical events stimulated by IL-3, IL-4 and SLF. The binding of growth factors, in general, results in the dimerization, or possibly oligomerization, of their appropriate receptors. IL-3 and IL-4, like many of the hemopoietic growth factors, engage heterodimeric receptors that lack intrinsic tyrosine kinase activity and instead recruit cytoplasmic tyrosine kinases to initiate their biological actions. SLF binds a 2 homodimeric receptor, also known as c-kit, similar in structure to the receptors for platelet-derived growth factor (PDGF) , or C S F - 1 . These possess a split tyrosine kinase domain on the intracellular portion of the receptors. Despite the general similarities between IL-3 and IL-4 receptors, they do activate very different intracellular signaling pathways although a few are shared. For example both of these cytokines lead to increased Phosphatidylinositol 3-Kinase (PI 3-Kinase) activity (Rottapel et al., 1991) (Izuhara et al., 1994), (Gold et al., 1994) and activation of members of the Janus tyrosine kinases and the Signal Transducers and Activators of Transcription ( J A K / S T A T ) pathway. However IL-3 stimulation, but not IL-4 , leads to the activation of the Ras/Mitogen Activated Protein ( M A P ) kinase pathway (Duronio et al., 1992). This difference is probably the reason that IL-4 is not a true growth factor in that it can not support the proliferation of any normal cell type (Levings et al., 1999). Instead IL-4 seems to act as a cofactor for other stimuli or can act as a survival factor by itself. The receptor for S L F possesses no similarity to the receptor for IL-3; however, when activated it also has the ability to activate the R a s / M A P kinase pathway and increase PI 3-kinase activity, however it fails to activate the J A K / S T A T pathway (Duronio et al., 1992), (Rottapel et al., 1991). When I initiated this thesis, the molecular events and mechanisms regulated by IL-3 , IL-4 or S L F were largely unknown. Observing their similarities and differences provided an important clue to the mechanism for the up-regulation of the transcription factor M y c . This clue identified a novel mechanism for the regulation of this gene that was shared by multiple growth factors in several cell types and shed light on the poorly understood process of c-myc gene regulation. 3 1.2 Regulatory Transcription Factors Whi le the synthesis of R N A is carried out by R N A polymerases and the basal transcription factors, the process of regulating the transcription of the desired gene(s) in a specific and timely fashion are controlled by the regulatory or activator type transcription factors. These transcription factors bind to specific palindromic elements, found in the promoter region of certain genes, and stimulate the basal transcription machinery described above. These regulatory type transcription factors and how they are themselves regulated by various extracellular stimuli is the focus of this thesis. Most eukaryotic regulatory transcription factors contain multiple domains, each of which perform a specific function that allows a transcription factor to bind D N A and facilitate transcription of a particular gene. These domains include the transcriptional activation, D N A binding, dimerization, and some contain hormone-binding domains. The transcriptional activation domains are typically classified based on the most abundant amino acid present such as acidic, glutamine, proline and serine/threonine rich domains. However this classification system can be misleading as residues other than the most abundant appear to be critical, possibly for structural reasons. One of the best-characterized activation domains is the acidic domain from the yeast G A L 4 protein. Two models have been proposed for its mechanism of action. One model suggests this region adopts an amphipathic cc-helix in which the acidic residues on one face of the cc-helix interact with basal transcription factors (Giniger and Ptashne, 1987). The other suggests it has no structure and instead forms "acid blobs" that interact with basal transcription factors (Sigler, 1988). Whi le N M R and crystallography have not been able to provide any structural information, circular dichroism studies have shown this region can form p-strands (Van Hoy et al., 1993), (Leuther et al., 1993). It is possible that the structure of these 4 domains is more important rather than the obvious most prevalent amino acid currently used to distinguish these classes. However glutamine or proline repeats do form functional activation motifs when fused to the DNA-binding domain of GAL4 (Gerber et al., 1994). Transcription factors use several different types of DNA binding domains, each of which facilitate binding to DNA in the major groove in a sequence specific manner. Many transcription factors use zinc to stabilize their DNA-binding domains. The zinc finger and the zinc cluster are two different classes of such domains. SP-1 contains multiple repeats of the two-cysteine and two-histidine type zinc finger which is a loop of 12 amino acids containing a conserved serine, phenylalanine and leucine residues at positions two, four and ten respectively as well as several basic amino acids which interact directly with the acidic DNA (Fairall et al., 1993). The base of the loop is held together by the cysteine and histidine residues that bind a zinc atom in a tetrahedral fashion. GAL4 contains a Cys6-Zn2 cluster that is connected by a linker to an oc-helix which forms a coiled-coil with the other monomer (Marmorstein et al., 1992). The homeotic genes involved in development such as the Drosophila melanogaster proteins engrailed, antennapedia and bicoid all contain a region of approximately 60 amino acids known as the helix-turn-helix (HTH) domain (Kissinger et al., 1990). This region typically contains three a-helixes with the second and third helixes comprising the HTH motif. The turn consists of three to four amino acids, and the two helixes make an angle of 120°. This structure is similar to the DNA-binding motif found in bacteriophage proteins such as the lambda Cro protein (Jordan and Pabo, 1988). X-ray crystallographic studies have shown that the third helix fits into the major groove where it can makes contact with the DNA. This sequence specific binding is carried out by what is called the recognition helix. As a result a single amino acid substitution in helix III of antennapedia with that from bicoid, produced an antennapedia protein with the DNA binding specificity of bicoid (Hanes and Brent, 1989). 5 Several helix-loop-helix (HLH) structures have been solved, the first being from the eukaryotic transcription factor Max (Ferre-D'Amare et al., 1993). Max, a basic H L H leucine zipper (LZ) protein, contains a basic DNA-binding region, a H L H protein motif and a heptad repeat of leucine residues known as the leucine zipper protein dimerization domain. Max forms a dimer with Myc and together they form a parallel four-helix bundle, with two helixes provided by each monomer. The basic region, located at the amino-terminal end, is an extension of the first helix, and binds the major groove of D N A . The leucine zipper is an extension of the carboxy-terminus helix and forms a coiled-coil with the leucine zipper of Myc. This structure contains leucine residues that repeat every seventh amino acid forming a unique right-handed helix with the leucine residues lining up every second turn on the same face. This region was referred to as a zipper based on an original proposal that the side chains of these leucine residues would interdigitate with the reciprocal parallel leucine zipper of another transcription factor to effect dimerization. However subsequent work demonstrated that these leucine residues do not interdigitate but instead line up in a head to head fashion. The first and second residues form the hydrophobic face while the charged residues in the fifth and seventh position control specificity and affinity. Examples of proteins that contain the leucine zipper are the transcription factors C A A T T box/enhancer binding protein (C7EBP), and the proto-oncogene products Fos and Jun. While the leucine zipper is not a D N A binding domain, it does facilitate binding by orienting the adjacent basic domain contained in these proteins. Thus the leucine zipper brings two basic domains into close proximity such that each basic region binds in the major groove to its half of the D N A palindrome on either side of the D N A helix. 6 1.3 MYC A l l cells from prokaryotes to mammals respond to various environmental treatments by inducing or repressing the expression of particular genes. These genes are controlled by regulatory or activator type transcription factors that recognize specific palindromic D N A sequences found in the promoter region of the gene to be regulated. Some of these activators play key roles in controlling proliferation, differentiation and cell death and are thus targets for mutations that subvert the normal coordinated activities of intracellular signaling. These proteins encode a growing list of proto-oncogenes that are positioned within the cell from the plasma membrane to the nucleus, and are important players in these intracellular signal transduction pathways. The first nuclear oncogene discovered was c-myc, isolated as the cellular homologue of the transforming gene from the myelocytomatosis retrovirus 29 (MC29) responsible for a chicken leukemia (Sheiness and Bishop, 1979) (Roussel et al., 1979) (Sheiness et al., 1980). The myc family of proto-oncogenes includes c-myc, N-myc and h-myc (this thesis will use the term myc when referring to the gene and Myc for the protein). These genes encode nuclear phosphoproteins that bind specifically the canonical D N A sequence C A C G T G (Blackwell et al., 1990), or the non-canonical sequences C A T G T G or C A T G C G (Blackwell et al., 1993), referred to as the c-myc E-box. Expression of c-myc mRNA is barely detectable in quiescent cells, but is induced in following the stimulation of resting cells with growth factors. Production of c-myc mRNA reaches maximal levels within two to three hours and gradually returns to a basal level that is maintained in continuously growing cells. Levels of c-myc mRNA correlate with the proliferative potential of cells by increasing following treatment with mitogens and decreasing following treatments that induce growth arrest or stimulate differentiation. Moreover, forced expression of Myc blocks differentiation of some cell types, such as erythroblasts. Since the 7 half-life of M y c protein is 20 to 30 minutes, production of c-myc m R N A seems to be the primary means of regulating the level of M y c , and thus functional activity. 1.3.1 Phosphorylat ion of M y c Posttranslational modifications such as glycosylation and phosphorylation have been reported and might have effects in some in vitro assays (Chou et al., 1995). O f key interest are two proline-directed potential phosphorylation sites, Thr 58 and Ser 62. Two-dimensional phosphopeptide mapping have revealed that a peptide containing Thr 58 and Ser 62 was phosphorylated in vivo after stimulation of fibroblasts with serum and this event occurred in the cytoplasm (Lutterbach and Hann, 1994). Although p42 Erk M A P kinase and glycogen synthase kinase-3cc (GSK-3oc) can phosphorylate Ser 62 and Thr 58 in vitro respectively, over-expression of M A P kinase, or an activated p21 r a s , in fibroblast cells did not enhance phosphorylation of M y c in vivo. Additionally there is disagreement as to whether mutation of these sites to alanine, leucine or acidic residues has an effect on the transactivation potential of M y c . Mutation of these residues to alanine or leucine has been reported to decrease the ability of M y c to transactivate a reporter, and increase if the substitutions are to glutamic acid (Gupta et al., 1993). Lutterbach et. al. have shown that similar substitutions have no effect with a similar reporter (Lutterbach and Hann, 1994). While these biochemical effects may vary, the biological evidence from these two groups is in agreement. Both groups report that any kind of mutation at either of these sites has no effect on the ability of c-myc to cooperate with ras in the transformation of primary fibroblasts. It is noteworthy that chromosomal rearrangements, placing the coding region of c-myc downstream of an immunoglobulin promoter, frequently results in the amino terminal deletions up to and including Thr 58. Whether these deletions produce a weaker allele of c-myc is unknown; however, these sites are clearly not required for transformation. While the 8 role of amino terminal phosphorylation remains unclear, the accepted view is that gene amplification or chromosomal translocation resulting in the over-expression of an otherwise normal protein is the mechanism by which M y c perturbs cell cycle control (Spencer and Groudine, 1991). 1.3.2 Functional Domains Functional studies have revealed that the M y c protein can be divided into several domains (Figure 1.2). Fusing different regions of M y c to the D N A binding portion of the G A L 4 protein identified that the amino terminus of M y c contains the transcriptional activation (TA) domain (Kato et al., 1990). Sequence comparison of this region from several eukaryotic species identified two highly conserved amino terminal motifs, referred to as M y c box I (amino acids 47-63 in human M y c ) and M y c box II (amino acids 121-143 in human M y c ) . The first 41 amino acids are acidic and glutamine rich and have sequence homology with the T A domains of Sp-1 and VP-16 proteins, however, box II has no sequence homology to other known T A domains (Kato et al., 1990), (Mitchell and Tjian, 1989). Box I is not required for activation, repression, or cooperating with ras for transforming primary fibroblasts, however, box II is required for repression and transformation but not for activation (L i et al., 1994). Thus transcriptional activation does not appear to be required for transformation, however, repression and oncogenic activity are inseparable and shed new light on the biochemical role of M y c . Other we l l -conserved domains in the carboxy terminus are: the basic (b) region between amino acids 355 and 367; the helix-loop-helix ( H L H ) at amino acids 368 to 410 and the leucine zipper (LZ) residing between positions 411 and 439 in humans. The structural features of these domains were discussed earlier. M y c prefers to bind the core sequence C A C G T G referred to as the M y c E-box. More recent studies have revealed that M y c wi l l bind to noncanonical E-boxes such as C A T G T G and C A T G C G (Blackwell et al., 1993). 9 1.3.3 M y c Fami ly Members L i k e most transcriptional activators M y c has a D N A binding partner called M a x , also a b H L H / L Z protein, however M a x does not contain a T A domain. Except for a nuclear localization signal (NLS) , Max has no other known functional domains. In contrast to M y c , M a x is a constitutively transcribed and translated protein having a half-life of approximately 24 hours (Blackwood et al., 1992). M y c / M a x heterodimers are kinetically favored over M a x homodimers. It is l ikely that M a x homodimers are predominant in resting cells, but after stimulation of M y c synthesis by a mitogen, heterodimerization occurs (Littlewood et al., 1992). The essential role for M a x in M y c transcriptional activation has been demonstrated in P C 12 cells, which lack a functional Max protein due to aberrant splicing of Max m R N A . Transactivation by M y c occurs only upon the ectopic expression of M a x (Hopewell and Ziff, 1995). However, M a x may be considered both an activator and an inhibitor of M y c function, depending on the relative expression levels of the two proteins. M a x can inhibit the transactivation by M y c and results from competition between the M y c / M a x heterodimer and the transcriptionally inactive M a x homodimer for binding to the E-box sequence element (Kretzner et al., 1992). Myc 1 0 CTG T A D I Boxl Boxll' bHLH/LZ t ATG NLS 439 a.a. bHLH/LZ Max 1 I 151 a.a. NLS SID bHLH/LZ Mad1 n 221 a.a. NLS Figure 1.1 Functional domains of Myc, Max and Madl. The abbreviations are: T A D (transcriptional activation domain); Box I and Box II (conserved amino terminal sequences in all Myc family members); b H L H / L Z (basic helix loop helix/leucine zipper); N L S (nuclear localization sequence); SID (Sin 3 interaction domain); CTG, A T G (alternative translation start sites). 11 Max also interacts with other bHLH/LZ proteins. The Mad family consisting of Mad l , M x i l (also known as Mad2), Mad3, Mad4, and Mnt (also known as Rox) all form heterodimers with Max and are important members of the Myc network (Ayer et al., 1993), (Hurlin et al., 1996). The Mad family homodimerize poorly and do not interact with any of the Myc family members, however they do bind strongly to Max. Consequently Mad proteins compete with Myc proteins for binding to Max, and Mad/Max heterodimers bind the same E-box site with equal affinity as Myc/Max dimers. Mad members do not contain T A domains instead they contain a SIN3 interaction domain (SID) and function as transcriptional repressors. Interactions of Myc or Mad with M a x are associated with the state of cellular proliferation or differentiation. While the half life of Max protein is approximately 24 hours, it's dimerization partners have very short half lives and their expression is tightly regulated throughout the cell cycle (Ayer and Eisenman, 1993). When quiescent cells are treated with a mitogen, Myc expression is induced, and the equilibrium shifts from Max/Max homodimers to Myc/Max heterodimers. Conversely addition of agents that stimulate differentiation repress Myc expression and induce Madl and M x i l expression (Larsson et al., 1994), (Zervos et al., 1993). Thus the relative abundance of Myc and Mad proteins provides a mechanism by which cell cycle entry or exit may be regulated. The mechanism by which the Mad family of proteins control transcriptional repression is thought to occur through their interactions with mSin3A and mSin3B. The interaction occurs through the amino terminal portion of Mad family members and four paired amphipathic oc-helixes (PAH) domains of mSin3. As previously discussed mSin3A/B are the mammalian homologues of the known yeast transcriptional repressor. Through its direct interaction with N -CoR, mSin3 recruits histone deacetylase, which stabilizes chromatin structure and thus inhibits transcription. Transactivation by Myc can be inhibited by the pl05-Rb related tumor suppressor protein pl07, which interacts with the amino-terminus of Myc in vivo (Beijersbergen et al., 12 1994), (Gu et al., 1994). The hydrophobic pocket of the Rb protein mediates interaction with M y c and is also required for binding to the adenovirus E 1 A molecule (Hu et al. , 1990). Transactivation by M y c is thought to occur through direct interaction with T B P (Hateboer et al., 1993), (Maheswaran et al., 1994). Thus M y c has the ability to interact physically with a key component of the basal transcription machinery. Co-immunoprecipitation of M y c with T B P from cell lysates can be blocked by the addition of a peptide sequence corresponding to the hydrophobic pocket of the Rb protein (Rustgi et al., 1991), (Hateboer et al., 1993). Thus the ability of Rb to inhibit transactivation by M y c may be due to direct competition for M y c - T B P binding. A list of genes reported to be activated by M y c is shown in Table 1.1. 13 Genes Possibly Act ivated by M y c Gene Reference alpha-prothymosin (Eilers et al., 1991); (Wu et al., 1997) cad (Miltenberger et al., 1995); (Fukasawa et al., 1997) p34 c d c 2 (Born etal., 1994) cdc25A (Galaktionov et al., 1996) cyclin A (Jansen-Durr et al., 1993); (Domashenko et al., 1997) cyclin E (Domashenko et al., 1997) D H F R (Mai, 1994); (Taylor et al., 1997) E C A 39 (Benvenisty et al., 1992); (Ben-Yosef et al., 1996) e I F " 2 a (Rosenwald, 1996; Rosenwald et al., 1993) eIF4E (Rosenwald, 1996; Rosenwald et al., 1993) ISGF3y/p48 (Weihua etal., 1997) L D H - A (Tavtigian et al., 1994); (Shim et al., 1997) M r D b (Grandori etal., 1996) O D C (Bello-Fernandez et al., 1993); (Walhout et al., 1997) PAI-1 (Prendergast et al., 1990) p53 (Reisman et al., 1993); (Prasad et al., 1997) rcc 1 (Tsuneoka et al., 1997) rcl (Lewis et al., 1997) T K (Pusch etal., 1997) Table 1.1 Genes positively regulated by Myc. Listed above are genes that have been implicated as being targets of M y c and the references pertaining to the evidence. 14 1.3.4 Myc as a Transcriptional Repressor. In addition to its transactivation abilities, M y c can also directly repress transcription via a mechanism that is dependent on the INR promoter region of susceptible genes. Examples of such promoters are shown in Table 1.2 but the two most characterized promoters are the adenovirus major late promoter ( A d M L P ) and the cyclin D l promoter. Expression of the b H L H / L Z protein U S F , which also binds to the M y c E-box, transactivates these genes but is inhibited in a dose-dependent manner following the expression of M y c ( L i et al., 1994), (Philipp et al., 1994). Transcriptional initiation by R N A Pol II occurs at the INR region in promoters that lack a T A T A box. This region contains the A T G start of transcription and is bound by TFII-I, a basal transcription factor that initiates transcription from INR elements (Smale and Baltimore, 1989). M y c directly interacts, through its b / H L H / L Z region, with TFII-I and provides a potential mechanism by which M y c may interfere with PIC assembly by its binding of TFII-I (Roy et al., 1993). A competing theory is based on the observation that repression seems to occur by M y c binding with M i z - 1 (Myc interacting zinc finger protein). M i z - 1 also binds the I N R and transactivates the A d M L P through interaction with p300. However in the presence of M y c , p300 is displaced and Miz-1 induced transactivation is proposed to be blocked (Peukert et al., 1997). Yin-Yang-1 ( Y Y 1 ) is a multifunctional zinc finger protein that can activate or repress gene transcription (Shrivastava and Calame, 1994) and interacts with the b H L H / L Z portion of M y c (Shrivastava et al., 1993). The interaction blocks both the activator and repressor functions of Y Y - 1 without interfering with its D N A binding ability (Shrivastava et al., 1993). These observations have led to the hypothesis that M y c might interfere by preventing interactions between Y Y - 1 the basal transcription machinery. 15 Genes Possibly Repressed by M y c Gene Reference albumin p l , a2 , a3-integrin C / E B P a c-myc collagen al ,cc2,a3 cyclin D l gadd 45, 153 gas 1 IgA. L F A - 1 M H C class I M T - 1 N C A M neu Tdt thrombospondin (L i et al., 1994) (Judware and Culp, 1997) (L i et al., 1994) (Nishikura and Murray, 1988), (Penn et al., 1990) (Yang et al., 1991; Yang et al., 1993) (Daksis et al., 1994), (Philipp et al., 1994) (Chen et al., 1996), (Marhin et al., 1997) (Lee etal., 1997) (Mai and Martensson, 1995) (Inghirami et al., 1990) (Bernards et al., 1986), (Manabe et al., 1996) (Kaddurah-Daouk et al., 1987) (Akeson and Bernards, 1990) (Suen and Hung, 1991) (Mai and Martensson, 1995) (Tikhonenko et al., 1996) Table 1.2 Genes negatively regulated by Myc. Listed above are genes that have been implicated as being targets of M y c and the references pertaining to the evidence. Recently fibroblasts homozygous null for c-myc have been generated and used to demonstrate that only cad and gadd45 regulation were altered (Bush et al., 1998). However it is possible that 16 many genetic alterations took place during the selection of these cells, which could have compensated for the lack of c-myc. The predictable alterations would be the genes that c-myc normally regulates. Thus altered cad and gadd45 expression levels could be an indirect result of the absence of c-myc. 1.3.5 Myc Proliferation One of the best-documented functions of M y c is its role in cellular proliferation. M y c belongs to the family of immediate early genes, which are rapidly induced in response to mitogenic stimulation of resting (GO) cells. Expression of M y c alone is reported to be sufficient to induce the transition from GO to G l , without a requirement for other immediate early response genes (Eilers et al., 1991). However the possibility of autocrine stimulation of other signaling paths had not been investigated. The injection of M y c anti-sense oligonucleotides does not block G l entry but rather prevents cell cycle progression past the G l phase (Coffey et al., 1988), (Heikkila et al., 1987), (Holt et al., 1988), (Loke et al., 1988) and ectopic expression of M y c relieves this inhibition (Coppola and Cole, 1986), (Eilers et al., 1991). Roussel and co-workers created a point mutation in the CSF-1 receptor, which upon stimulation with CSF-1 no longer lead to increased c-myc m R N A and did not result in cell proliferation; however ectopic expression of M y c restored proliferation (Roussel et al., 1991). The kinetics of c-myc induction is slower and more prolonged in comparison with other immediate early genes such c-fos or c-jun. Whi le its m R N A is absent in quiescent cells and induced maximally after 2 to 3 hours following mitogenic stimulation, its levels return to a basal level that is maintained continuously throughout the cell cycle (Hann et al., 1985), (Rabbitts et al., 1985), (Thompson et al., 1985). 17 1.4 Signal Transduction Pathways Regulating c-myc Induction Induction of c-myc by mitogen stimulation has been studied in many cell types, but mostly in fibroblasts, where c-myc is induced in response to platelet-derived growth factor (PDGF) , epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (Kelly et al., 1983), (Dean et al., 1986), (Cutry et al., 1989). Although several pathways have been implicated in the regulation of c-myc induction no clear mechanism that controls this process has been described. This thesis w i l l focus on this mechanism and w i l l provide evidence that PI 3-kinase is a key component of this process, however a summary of molecules previously described, as being involved, is necessary. 1.4.1 Tyrosine Kinases A s discussed earlier the ectopic expression of M y c restored the ability of a mutant CSF-1 receptor mutant to proliferate in response to CSF-1 . This tyrosine to phenylalanine substitution at amino acid position 809 failed to induce c-myc m R N A although c-fos and c-jun induction was normal (Roussel et al., 1991). A link between the nonreceptor tyrosine kinase src and c-myc was established subsequently when this tyrosine was found to be critical for the recruitment and activation of src in response to CSF-1 treatment (Courtneidge et al., 1993). 1.4.2 Ras/MAP Kinase Pathway Regulation of c-myc by the GTPase Ras was investigated by fusing of the estrogen receptor to the serine/threonine kinase Raf. Following stimulation of these fibroblasts with an analogue of estrogen, c-myc but not c-fos m R N A was induced (Kerkhoff et al., 1998). This study also showed that serum-induced c-myc and c-fos induction could be blocked with a dominant interfering mutant of Raf. This involved the inducible expression of the Ras binding domain of Raf. This evidence suggests that Raf is necessary and sufficient for c-myc, however 18 Raf is only required but not sufficient for c-fos induction. These results would appear to contradict the work from Roussel and Courtineidge as in their system R a s / M A P kinase activation was normal as evident from c-fos and c-jun induction. However, clearly R a s / M A P kinase activation was not sufficient for c-myc induction in this system. 1.4.3 v-abl M y c has also been implicated as being regulated by the nonreceptor tyrosine kinase abl. Activation of a temperature sensitive mutant of A b l results in the induction of c-myc m R N A (Cleveland et al., 1989), and the expression of a dominant negative M y c , lacking its T A domain, blocked transformation of fibroblasts by v-abl (Sawyers et al., 1992). The src homology-2 (SH-2) domain of v-abl is'required for both transformation and c-myc induction (Wong et al., 1995). Consistent with the observation that ectopic expression of M y c complements mutations in the SH-2 domain of B C R / A b l in transformation assays (Afar et al . , 1994). Further study demonstrated that an SH-2 domain containing mutant of B C R / A b l failed to induce c-myc and this correlated with the loss of PI 3-kinase activation (Skorski et al., 1997). However interaction with other signaling molecules may have also been compromised by this mutation. 1.4.4 Viral Induction of c-myc A conserved E 2 F binding site lies between the P l and P2 c-myc promoters and is required for c-myc transcription by the adenovirus E 1 A protein. The mechanism for c-myc induction involves the binding of viral E l A to the Rb tumor suppressor and thus displacing E2F, which is free to bind the myc promoter (Oswald et al., 1994). Several other viral oncogene products such as S V 4 0 large T-antigen and the human papilloma virus E7 protein can also disrupt the E2F-Rb complex (DeCaprio et al., 1988), (Whyte et al., 1988), (Dyson et al., 1989). 19 1.5 Regulation of c-myc during Development Wnts are secreted proteins expressed in adults and developing embryos (Dickinson and McMahon , 1992). In the developing embryo Wnt expression correlates with the formation of the animal/vegetal axis. It now appears that Wnts function through binding to the Frizzled family of serpentine receptors (Bhanot et al., 1996) and activation of the P D Z domain containing protein Dishevelled (Dsh) which inactivates G S K - 3 (Sokol, 1996). G S K - 3 is a negative regulator of a protein called p-catenin a protein that interacts with H M G - b o x transcription factors that are thought to regulate the genes involved in the formation of the animal/vegetal axis (Yost et al., 1996). Phosphorylation of p-catenin decreases its stability, a process recently shown to involve the tumor suppressor protein adenomatous polyposis coii ( A P C ) . In the absence of A P C , p-catenin accumulates and binds the H M G box transcription factor called T-cell factor-4 (Tcf-4) which is thought to induce transcription of the c-myc gene through Tcf-4 binding sites in the c-myc promoter (He et al., 1998). 1.6 Induction of Apoptosis by Myc It has been well documented that ectopic expression of M y c induces apoptosis under suboptimal growth conditions such as low serum concentrations in the case of fibroblasts (Evan et al., 1992). Increased levels of apoptosis are observed in lymphocytes of transgenic mice constitutively expressing M y c under the control of the E| i - immunoglobulin heavy chain enhancer (Dyall-Smith and Cory, 1988). The majority of studies have used an inducible M y c mutant which is a fusion of otherwise normal M y c to the hormone binding domain of the estrogen receptor (Eilers et al . , 1989). Molecules that are involved in M y c / E R induced 20 apoptosis, under low serum conditions, include B c l - 2 , Ras, p53 and Fas/FasL. Ectopic expression of the anti-apoptotic molecule bcl-2, suppresses M y c induced apoptosis in serum-starved fibroblasts although the exact mechanism is unknown (Bissonnette et al., 1992), (Fanidi et al . , 1992). Furthermore bcl-2 synergizes with M y c in the transformation of primary fibroblasts allowing cells aberrantly expressing M y c to proliferate unchecked (Vaux et al., 1988), (Strasser et al., 1991), (Nunez et al., 1990). Many growth factors such as P D G F , E G F , b F G F , and IGF-1 also block M y c induced apoptosis under low serum conditions (Harrington et al., 1994). However the molecules activated by these growth factors, that were required for inhibition of apoptosis, were not identified until rather recently. Point mutations of activated Ras (V12Ras) that only activate certain effectors have been used to address which of these downstream molecules can antagonize M y c induced apoptosis. A particular mutant of Ras (V12C40Ras) binds the catalytic subunit of PI 3-Kinase (p l 10) and increases PI 3-Kinase activity in vitro, however it no longer binds or activates Raf or the exchange factor R a l G D S (Rodriguez-Viciana et al., 1994), (Rodriguez-Viciana et al., 1996). This particular mutant was later shown to repress M y c induced apoptosis under low serum conditions implying the anti-apoptotic signal was delivered by PI3-kinase (Kauffmann-Zeh et al., 1997). The tumor suppressor p53 contains an E-box myc site in its promoter and is thus a potential target gene of M y c (Reisman et al., 1993). Indeed, expression of M y c induces p53 m R N A and protein expression and may have a role in M y c induced apoptosis as cells homozygous null for p53 fail to undergo apoptosis but instead enter the cell cycle (Hermeking and Eick , 1994). Moreover ectopic expression of p53 leads to increased expression of Fas [Muller, 1997 #580], and the promoter of Fas ligand (FasL) contains and E-box element suggesting that FasL might be a target gene for M y c (Doug Green Pers. Comm.). Strikingly, CD95 , also known as Fas or A P O - 1 , also has a role in M y c induced apoptosis. This observation was made fortuitously when a dominant negative mutant of Fadd blocked 21 apoptosis induced by Fas ligation or by M y c expression, suggesting that Fas action was required for cell ki l l ing by M y c . Consistent with this theory, activation of M y c / E R in Ipr (Fas mutant) or gld (Fas ligand mutant) fibroblasts failed to induce cell death. Additionally, Rat-1 cells that are normally refractory to cell kil l ing, by an agonistic antibody to Fas (called Jo2), became sensitive to ki l l ing by Jo2 after the activation of M y c / E R (Hueber et al., 1997). Thus M y c also has a role in Fas mediated apoptosis, suggesting that cell death requires two signals, one from M y c and the other from the Fas network of signaling molecules. However in these experiments the induction of apoptosis by estrogen stimulation requires reduced serum concentrations and this alone could have reduced PI 3-Kinase activity sufficient for Jo2 to induce ki l l ing. Thus an autocrine model emerges for apoptosis caused by the aberrant expression of M y c . This involves the increased expression of p53 and FasL by M y c and increased Fas expression regulated by p53. It is clear that no universal signaling mechanism, for regulation of the c-myc gene, has emerged from the previous studies. However during the course of this thesis an observation was made that provided a clue that PI 3-kinase could be involved in the induction of c-myc. 1.7 Phosphatidylinositol 3-Kinase The PI 3-Kinases are a diverse family of l ip id kinases originally discovered as being associated with the viral oncoproteins polyoma middle T (mT) antigen and v-src (Cantley et al., 1991). The family consists of three classes divided by their different structural domains, however all members contain what is called a PI Kinase (PIK) domain and a kinase domain (Figure 1.2). A l l members phosphorylate PI on the 3 position of the inositol ring, however the classes do prefer slightly different substrates. This thesis w i l l refer to the different classes of PI 3-Kinase using the nomenclature of Domin and Waterfield (Domin and Waterfield, 1997). AB RB PIK Kinase Class IA • RB PIK Kinase Class IB • PIK Kinase C2 Class II Class III F i g u r e 1.2 Classification of PI 3-Kinase family members. Functional domains are abbreviated as: A B (adapter binding); R B (Ras binding); P I K (PI Kinase); Kinase (PI 3-Kinase catalytic) 23 1.7.1 Class I PI 3-Kinases These phosphorylate phosphatidylinositol (PI) lipids producing the l ip id products phosphatidylinositol-3 phosphate (PI3P), phosphatidylinositol-3,4 bisphosphate (PI3,4P 2) and phosphatidylinositol-3,4,5 trisphosphate (PI3,4,5P 3) (Vanhaesebroeck et al. , 1997). These heterodimeric kinases all contain a catalytic subunit of 110-120 kDa and either a 50, 55 or 85 k D a regulatory subunit. So far three members have been cloned from humans and mice and share 42-58% amino acid identity and are referred to as pllOoc, p and 8 isoforms, the last being unique to cells of hemopoietic origin. Each of these molecules contains, at its amino terminus, a region required for binding the regulatory subunit. Moving towards the carboxy terminus these proteins also have a region that are required for binding the small GTPase p21 R a s , the P I K domain and a catalytic domain which define the class I A PI 3-Kinases. Homologues of these molecules have been cloned from multiple eukaryotes including D. discoideum, D . melanogaster, and C. elegans. The p85oc and p85p regulatory subunits, as implied, contain no catalytic activity, however they do contain several well characterized modular domains. The amino terminus contains an SH-3 domain followed by two poly-proline domains, a region of homology to the rho family of exchange factors, referred to as the double (dbl) domain, and two SH-2 domains separated by a region required for binding to the amino terminus of the p 110 catalytic subunit. The p85oc gene has at least two splice variants that give rise to p50oc and p55oc. These smaller molecules lack the amino terminal SH-3, poly-proline and the dbl like domains but are otherwise identical (Fruman et al., 1996), (Antonetti et al., 1996), (Inukai et al., 1997). The highly homologous p85p gene contains an additional small carboxy terminal sequence of unknown function and has no reported splice variants. A third gene called p55y encodes a protein with similar structure to the p55a gene product (Inukai et al., 1997). Regulatory subunits have also been cloned in D. melanogaster, and this molecule also lacks the amino terminal SH-3, 24 poly-proline and dbl like domains but is otherwise structurally similar to the p55oc gene product (Weinkove et al., 1997). A 1 1 0 k D a molecule has been cloned, called p l 10y, that is similar to p l 10a except that it lacks the very amino terminal domain required for binding to the p85 regulatory proteins (Stephens et al., 1994), (Stoyanov et al., 1995). It has therefore been classified as a class I B PI 3-Kinase. A possible regulatory molecule of 101 kDa has been cloned however it contains no homology to any other proteins but is considered a possible regulatory molecule as binding of p l O l to p l 10y increases the in vitro PI 3-Kinase activity of p l 10y by 50-fold (Stephens et al., 1997). The function of p l O l may be to increase the stability of p l 10y in a similar manner to that of p85 for the class I A PI 3-Kinases (Yu et al., 1998). The catalytic domains of the class I A PI 3-Kinases are recruited to the plasma membrane, in response to mitogenic stimulation. The SH-2 domains of the adapters are thought to mediate this translocation by binding to membrane proximal proteins that contain a tyrosine phosphorylated T y r - X - X - M e t motif. This class of PI 3-Kinase has also been shown to bind directly (Kodaki et al., 1994) and be activated by the p21 R a s family of small GTPases (Rodriguez-Viciana et al., 1994). Synergistic activation occurs with the binding to both p21 R a s and tyrosine phosphorylated peptides (Rodriguez-Viciana et al., 1996). In addition to l ip id kinase activity these enzymes also possess an intrinsic protein serine/threonine kinase activity (Carpenter et al., 1993), (Stoyanova et al., 1997). Substrates for phosphorylation appear to be the catalytic subunit itself and serine 608 on the p85a regulatory subunit. While phosphorylation of p l 108 decreases its l ipid kinase activity, no functional role for the phosphorylation of pllOcc, p or y subunits has been described. Phosphorylation of p85oc however appears to lower the l ipid kinase activity of the associated pllOoc (Carpenter et al., 1993), (Carpenter et al., 1993). The insulin receptor substrate-1 (IRS-1) is also described as an in vitro substrate (Lam et al., 1994). The SH-3 25 domain of p85 has been used to probe bovine brain lysate and has affinity for the GTPase dynamin which is involved in clathrin mediated endocytosis; however, what role PI 3-Kinase plays in this process is unclear (Booker et al., 1993). The p85 subunit is also a target for binding by the SH-3 domains of other molecules to its two poly-proline rich regions. The SH-3 domains of the oncoproteins src, fyn, lck, lyn and abl for example bind p85ot; however, no interactions with these kinases have been reported for p85p or, obviously, the p50/55 molecules (Pleiman et al., 1994). It should be noted that PI 3-Kinase is not constitutively associated with these kinases. In fact these tyrosine kinases can not bind PI 3-Kinase unless in an activated state, either through phosphorylation or oncogenic mutation (Liu et al., 1993). The fungal metabolite wortmannin (Wymann et al., 1996) or the synthetic molecule LY294002 (Vlahos et al., 1994) inhibit both the PI 3-Kinase and the protein kinase activities by blocking the ATP binding site of the catalytic subunit. However wortmannin differs from LY294002 in that it irreversibly inhibits the enzyme by covalently reacting with lysine 802 and it interferes with the lipid-binding site as well (Wymann et al., 1996). pllOy is thought to be directly recruited to the plasma membrane by Py subunits of heterotrimeric G-proteins. The recruitment probably takes place through an N-terminal GTPase binding region as this region has been shown to bind Ras in vitro (Rubio et al., 1997). 1.7.2 Class II P I 3-Kinases The class II PI 3-Kinases range in size from 170 to 210 kDa and contain a PIK and a catalytic domain similar to class IA PI 3-Kinases. While the class II molecules lack regulatory and GTPase-binding domains they do contain a region with homology to the C2 domain found in conventional PKC members at their carboxy terminus. The C2 domain in PKC has been shown to be the calcium-binding region, however critical aspartate residues important for calcium 26 binding are absent in the C2 domain of these PI 3-Kinases. To date no functional evidence exists that this class of PI 3-Kinase is activated by calcium. Class II PI 3-Kinases have been cloned in humans, mice, D. melanogaster, C. elegans and D. discoideum (Zhou et al., 1995), (MacDougall et al., 1995), (Molz et al., 1996), (Virbasius et al., 1996) , (Domin et al., 1997). O f note these molecules are highly resistant to the inhibitors wortmannin and LY294002 , with I C 5 0 values approximately 50 fold higher than class I PI 3-Kinases. However, their contribution to phospholipid production in response to mitogens is unknown (Fruman et al., 1998). 1.7.3 Class III PI 3-Kinases The yeast Vps34, and the mammalian homologue, are examples of class III PI 3-kinases. These molecules appear to contain only the P I K and catalytic domains. D. discoideum and D. melanogaster homologues have also be described (Zhou et al., 1995), (Vol in ia et al., 1995), (Linassier et al., 1997). They have no know adapters or mechanism of activation but the yeast molecule is thought to play a role in transporting newly synthesized proteins from the Golg i to the vacuole. These kinases are thought to be responsible for the formation of only PI3P since PI3,4P 2 and PI3,4,5P 3 lipids are absent in yeast. In addition the mammalian homologue is capable of forming only PI3P in vitro. While the yeast protein is highly resistant to wortmannin, the mammalian homologues have sensitivities similar to class I A PI 3-Kinases (Fruman et al., 1998). 1.7.4 P K B and PDK Recently a serine/threonine protein kinase, containing a plekstrin homology (PH) domain, has been described as being activated by the products of PI 3-Kinase. This kinase is know as Protein Kinase B ( P K B ) due to its homology to the protein kinases A ( P K A ) and C ( P K C ) (Marte and Downward, 1997). Two other groups have also described this molecule and have 27 referred to it as Related to A and C Protein Kinases ( R A C - P K ) , and c-akt, as it is the cellular homologue of the v-akt gene product from a transforming avian virus (Jones et al., 1991), (Bellacosa et al., 1991). In addition to the first identified member of the family (PKBoc), two other genes have been cloned, P K B p and P K B y (Jones et al., 1991), (Konishi et al., 1995). P K B p has two alternately spiced forms, P K B p 2 is very similar in size and structure to PKBcc, however, P K B P ! contains an additional 40 amino acids, of unknown function, at its carboxy terminus. P K B is phosphorylated and activated by a wide variety of mitogens such as P D G F , E G F and serum. The P H domain of P K B has been shown to interact with multiple proteins such as P K C isoforms and Py subunits of heterotrimeric G proteins (Konishi et al., 1995). More recent experiments have also shown interactions with the 3-phosphoinositol l ip id products of PI 3-kinase, specifically PI3,4P 2 (Klippel et al., 1997),(Franke et al., 1997) and possibly PI3,4,5P 3 (Stokoe et al., 1997) suggesting that P K B may be regulated by PI 3-Kinase activity. Further evidence for this is provided by the observation that wortmannin or L Y 2 9 4 0 0 2 as well as dominant interfering mutants of PI 3-Kinase block activation of P K B (Burgering and Coffer, 1995). In vitro studies have shown the P H domain binds with high affinity and with a stoichiometry of one l ip id product for one P H domain (Freeh et al., 1997). Further in vitro studies have shown P K B to be preferentially activated by PI3,4P 2 but not PI3,4,5P 3 (Klippel et al., 1997), in fact some studies have suggested PI3,4,5P 3 is inhibitory (Franke et al., 1997). Recently, however, the examination of cells derived from mice lacking the PI5P phosphatase, called SHIP (SH-2 containing inositol-5 phosphatase), have been used to address this issue in vivo. S H I P dephosphorylates PI3,4,5P 3 at the 5-inositol position to generate PI3,4P 2 . Predictably cells lacking this phosphatase generate high levels PI3,4,5P 3 upon mitogenic stimulation (Huber et al., 1998). However they also display a greater fold activation of P K B than cells derived from normal littermates ( M . Scheid pers. Comm.). 28 There is some disagreement as to whether the P H domain is required for P K B activation. A point mutation (R25C) or a small deletion ( A l l - 3 5 ) in the P H domain abolishes P K B activation in response to P D G F (Franke et al., 1995). However, Roth et. al. have shown that the same mutant can be activated by insulin, although to a reduced level compared to the wi ld type protein. These two groups are in agreement however with respect to the A l 1-35 deletion mutant as not being inducible. To add to the confusion, Roth et. al. have demonstrated that the entire P H domain can be removed without affecting P K B activation (Kohn et al. , 1996). This latter observation might be most easily explained by the possibility that over-expression of P K B promotes P K B to interact with P D K s (discussed below). Wi th the exception of Roth et al., the vast majority of investigators have shown that mutations in the P H domain results in a kinase that is no longer active. In fact studies have shown that these mutants of P K B no longer serve as substrates for phosphorylation (Stokoe et al., 1997). There are two sites of phosphorylation on P K B , threonine 308 and serine 473 (Alessi et al., 1996). Mutation of both sites to alanine residues blocks activation of the kinase. Conversely mutating either residues to acidic amino acids is sufficient to produce a constitutively active enzyme, although the highest activity is obtained when both sites are mutated to acidic amino acids (Alessi et al., 1996), (Stokoe et al., 1997). The function of serine 473 phosphorylation is further complicated by the fact that the carboxy terminus of P K B y terminates at amino acid 454, and thus P K B y lacks this phosphorylation site. Perhaps this produces a kinase dependent on PI 3-Kinase activity but independent of phosphorylation at Ser473. Whi le phosphorylation is required for P K B activation, it is the P H domain that facilitates the translocation of P K B to the plasma membrane. Membrane localization can be mimicked by the addition of a myristoylation sequence to P K B , which results in P K B activation due to its constitutive phosphorylation (Andjelkovic et al., 29 1997), (Kohn et al., 1996). The kinase responsible for this phosphorylation is most likely to be the constitutively active kinases P D K 1 and possibly P D K 2 . Recently two groups have identified or cloned a 3-phosphoinositide dependent kinase, referred to as P D K 1 , that is capable of phosphorylating Thr308 and activating P K B in vitro (Alessi et al., 1997), (Stephens et al., 1998). The D. melanogaster gene has also been cloned and is referred to as D S T P K 6 1 (Alessi et al., 1997). This phosphorylation does not result in the phosphorylation of Ser473, suggesting that this latter site is not a target for P D K 1 . Instead this site is phosphorylated in vivo by an unidentified kinase, the activity of which is attributed to an as yet uncloned kinase termed P D K 2 . PI 3-Kinase regulates both P D K 1 and P D K 2 as wortmannin or LY294002 block phosphorylation of P K B in vivo, but not in vitro, suggesting that at least P D K 1 is not a direct target of these inhibitors. Since P D K 1 contains a P H domain in its carboxy terminal region it was proposed that PI 3-Kinase might activate this kinase through the generation of 3-phospholipids that bind this site on P D K 1 . Predictably the activity of recombinant P D K 1 ( r P D K l ) towards recombinant P K B ( rPKB) is stimulated by the addition of PI3,4,5P 3 or PI3,4P 2 in vitro. However r P D K l activates a mutant of r P K B that lacks a P H domain in a phospholipid-independent manner. Additionally P D K 1 appears to be constitutively active as immunoprecipitated P D K 1 from either starved or stimulated cells phosphorylates and activates this mutant of P K B to equal levels. Moreover P D K 1 lacking its P H domain fails to phosphorylate or activate r P K B unless PI3,4,5P 3 is present (Alessi et al . , 1997). These observations suggest that the P H domain of P K B serves to mask the regulatory sites of phosphorylation from P D K 1 , and that PI3,4,5P 3 binding by P D K 1 is not required for its activity. Thus the inducible activation of r P K B by PI3,4,5P 3 in the presence of r P D K l is most likely due to conformational constraints of the P H domain of P K B . Taken together the current model for in vivo P K B activation, is that the P H domain of P K B not only functions as a means to recruit P K B 30 to the plasma membrane, but it also undergoes a conformational change that permits access to P K B b y P D K l . This conflicts with the original observations that demonstrated immunoprecipitated P K B was activated in vitro by the addition of PI3P lipids alone (Klippel et al., 1997), (Franke et al., 1997). Since this does not occur with recombinant P K B (James et a l . , 1996) and phosphorylation is required for activation, it is possible that these studies were complicated by the presence of contaminating P D K 1 in the P K B immunoprecipitates. Alternatively endogenous P D K s may already have phosphorylated the immunoprecipitated P K B . Clearly P K B activation is a two step process where the first step is the binding of PI3,4,5P 3 (but not PI3,4P 2) to the P H domain of P K B and induction of a conformational change which allows as a second step, the phosphorylation of P K B by P D K s . A n active P K B is produced only after both of these events have occurred. Mitogen activated protein kinase-activated protein ( M A P K A P ) kinase-2 and integrin linked kinase (Ilk) phosphorylate P K B at Ser473 in vitro and are thus two potential P D K 2 kinases (Alessi et al., 1996), (Antonetti et al., 1996). What role the M A P kinase family member p38 or molecules thought to be involved in regulating matrix adhesion could have in regulating P K B is unknown. Perhaps the M A P K A P kinase-2 observation explains why P K B can be activated by cellular stresses such as heat shock and hyperosmolar!ty in a PI 3-Kinase independent manner (Konishi et al., 1996). The preferred substrate sequence for P K B to phosphorylate a synthetic peptide is RxRyy(S/T)h. Where x is any amino acid, y is any small residue other than glycine and h is a bulky hydrophobic amino acid (Alessi et al., 1996). The first substrate identified was glycogen synthase kinase-3 (GSK3) . Insulin or IGF-1 stimulation activates P K B , which phosphorylates Ser9 of G S K 3 resulting in its inactivation and consequent activation of glycogen synthesis. Expression of a constitutively active form of P K B results in the translocation of the glucose transporter, called G L U T 4 , from the cytosol to the plasma membrane resulting in glucose uptake 31 in 3T3-L1 preadipocytes (Kohn et al., 1996). A n additional enzyme involved in the regulation of metabolism that is phosphorylated and activated by P K B is 6-phosphofructo-2-kinase (PFK-2) (Deprez etal. , 1997). There is evidence implicating P K B in regulating cel l survival. Expression of a constitutively active form of P K B protects cells from apoptosis induced by the withdrawal of serum while M y c is artificially expressed (Kauffmann-Zeh et al., 1997). The mechanism by which P K B protects cells from apoptosis is contentious. P K B has been reported to phosphorylate and inactivate the proapoptotic bcl-2 family member Bad at Ser l36 creating a binding site for 14-3-3 proteins (Datta et al., 1997). When these chaperones are bound, Bad is unable to heterodimerize with and inhibit Bcl -2 or B c l - X L (Zha et al., 1996). Over-expression of Bad induces cell death, which can be blocked by the expression of constitutively active P K B . However Bad appears to be expressed in only a small range of tissue types and cel l lines suggesting that there are other substrates that P K B regulates that are responsible for blocking apoptosis. Moreover there is convincing evidence that P K B is not the kinase responsible for phosphorylation of Bad in vivo (Scheid and Duronio, 1998). 1.7.5 S6-Kinase Another molecule that appears to be regulated by PI 3-Kinase is the 40S ribosomal protein S6 kinase (p70/p85 s 6 K). The regulation of S6 kinase is complex although wortmannin or LY294002 also block its activation giving rise to the suggestion that p 7 0 s 6 K is also regulated by PI 3-Kinase activity. The p70/p85 isoforms are produced from alternate translation start sites from the same transcript. The two molecules are regulated in an identical fashion and seem to differ only by an additional 23 amino acid extension at the amino terminus of p85, which targets this isoform constitutively to the nucleus (Reinhard et al., 1994). Although phorbol esters can activate P 7 0 s 6 K w vivo and E R K M A P kinase can phosphorylate P 7 0 S 6 K m vitro, dominant 32 interfering mutants of Ras fail to block P70 S 6 Kactivation and constitutively active mutants of Ras fail to activate P70 S 6 K as well (Ming et al., 1994). Thus E R K M A P kinase activation is neither required nor sufficient for P70 s 6 Kactivation. Another pharmacological approach has been used to address the regulation of this kinase. The immunosuppressant rapamycin is another potent inhibitor of p70 s 6 K , blocking its activation by all known stimuli (Chung et al., 1992) (Price et al., 1992) (Kuo et al. , 1992). The target of rapamycin was first identified in yeast as the serine/threonine kinase T O R I / 2 (Heitman et al., 1991), and the mammalian homologue is referred to as m T O R , F R A P or R A F T 1 (Brown et al., 1994). Rapamycin first binds to its receptor F K B P 1 2 and this complex then binds and inhibits m T O R kinase activity. Expression of an m T O R mutant that no longer binds FKBP12/rapamycin protects p 7 0 s 6 K from inhibition by rapamycin suggesting m T O R regulates p70 S 6 K activation in vivo (Brown et al., 1995). m T O R , like PI3-Kinase, is a member of the PIK-related family of kinases although no l ip id kinase activity has been reported for mTOR. It is however susceptible to inhibition by wortmannin or L Y 2 9 4 0 0 2 (Brunn et al. , 1996). However there is evidence that m T O R itself may lie downstream of PI 3-Kinase. The expression of a gag/PKB fusion leads to constitutive p 7 0 s 6 K activity that rapamycin can still inhibit (Burgering and Coffer, 1995). Moreover, there is no evidence that P K B directly phosphorylates p70 s 6 K . This activation does not appear to be autocrine as expression of the gag/PKB fusion does not constitutively activate E R K M A P kinase. L ike P K B , p70 S 6 K is also regulated by two critical phosphorylation events which in p 7 0 s 6 K occur at Thr229 and Ser389 (Pullen and Thomas, 1997). Interestingly the sequence of amino acids surrounding Thr229 is similar to Thr308 in P K B . Predictably P D K 1 does phosphorylate Thr229 on p70 S 6 K but only after Ser389 is phosphorylated by an as yet uncharacterized kinase (Pullen et al., 1998). Mutation of Ser389 to an acidic amino acid results in constitutive phosphorylation of Thr229 and an active kinase, however mutation of Thr229 to an acidic amino acid does not result in phosphorylation of Ser389 nor does it produce an active kinase. Thus Ser389 phosphorylation 33 regulates Thr229 in a coordinated and hierarchical fashion. Alteration of either of these sites to alanine residues ablates kinase activity (Pearson et al., 1995; Pullen and Thomas, 1997). How P70 S 6 K and m T O R are regulated by mitogens is not clear, however for the reasons mentioned above, PI 3-Kinase appears to play an important role. A s already mentioned p70 /85 S 6 K phosphorylates the 40S ribosomal protein S6 at multiple sites in vivo. Inhibition of mitogen-stimulated p 7 0 s 6 K by rapamycin blocks these phosphorylation events and cell cycle progression at the G l phase of the cell cycle (Chung et al., 1992) (Price et al., 1992) (Ferrari et al., 1993). Ribosome phosphorylation is thought to be important for the efficient translation of a certain class of m R N A message that contains a polypyrimidine tract at the start of transcription (Pullen and Thomas, 1997). These messages code for many of the enzymes involved in protein synthesis which are required for cell cycle progression (Nasmyth, 1996). 1.8 Ras and MAP kinase The Ras super-family are small membrane associated 21 k D GTPases. A process of proteolysis, carboxymethylation and finally farnesylation of the carboxy terminal C A A X amino acid sequence mediate membrane association (Der and Cox, 1991) (de Gunzburg, 1991). The Ras super-family is divided into families based on amino acid homology. Harvey (Ha), Kirsten (K) and N-Ras make up the classical Ras family. These molecules are active when bound with G T P and inactive when in a G D P bound state. There are several types of molecules that regulate the state of Ras, such as guanine nucleotide exchange factors (GNEFs) which promote the exchange of G D P for G T P , GTPase activating proteins which stimulate the hydrolysis of G T P to G D P and guanine nucleotide dissociation inhibitors which lock Ras in either a G D P or G T P bound state (McCormick, 1995). Ras proteins have been implicated in regulating a wide variety of biochemical and biological events (de Vries et al., 1996). These events have been attributed to the ability of Ras to activate many signaling pathways such as, PI 3-Kinase, other Ras super-34 family members, and a proto-oncogene serine/threonine kinase c-Raf (Rodriguez-Viciana et al., 1996). PI 3-Kinase activation, as mentioned above, is thought to occur through direct interaction, which results in the membrane localization of PI 3-Kinase and consequent production of 3-phosphoinositol lipids (Rodriguez-Viciana et al., 1996). Direct association also occurs with Raf, which also places Raf at the plasma membrane. Oncogenic mutants of Raf are constitutively active due to its fusion with the retroviral gag protein, which localizes Raf to the plasma membrane which is thought to circumvent regulation by Ras (Heidecker et al., 1990). Active Raf phosphorylates and activates the dual specificity serine/threonine and tyrosine kinase M A P kinase kinase 1 ( M E K 1 ) which then phosphorylates Erk M A P kinase, at a Thr-Glu-Tyr motif, resulting in Erk activation (Marais and Marshall, 1996). Rho family of GTPases, which include Cdc42, R a c l and Rho, are also members of the Ras super-family. These molecules regulate J N K M A P kinase through a cascade of kinases involving M A P kinase kinase kinases ( M E K K s ) and dual specificity M A P kinase kinases ( M K K s ) in a similar cascade as for R a f / M E K / E r k (Chan-Hui and Weaver, 1998). Since J N K and p38 activation appear to be inseparable events, the activation of p38, possibly by M K K 4 , might also be regulated by the Rho family of GTPases. 1.9 Cytokine Signal Transduction IL-3 , IL-4 and S L F , are members of a large family of structurally related polypeptide hematopoietic growth factors that regulate the growth and survival of many different cell types of the immune system. 1.9.1 c-kit Signaling The receptor for S L F , also known as c-kit, is a 150 k D a homodimeric protein tyrosine kinase which contains five immunoglobulin like regions in its extracellular region and a split or 35 interrupted kinase domain on its intracellular region (Blechman et al., 1993). Its ligand, S L F , exists as a dimer and its binding to the receptor is thought to initiate c-&z'r-signaling events by inducing the dimerization of the receptor. The two receptor chains then thought to autophosphorylate, due to their close proximity, on multiple tyrosine residues, which create binding sites for molecules containing either SH-2 or P T B (phosphotyrosine-binding) domains. Such molecules that bind directly include PLC-y, Grb2/SOS, PI 3-Kinase, and S H C , (Matsuguchi et al., 1994), (Welham and Schrader, 1992), (Rottapel et a l , 1991). Stimulation with S L F leads to the activation of: p21 R a s (Duronio et al. , 1992),Raf, M E K 1 / 2 , Erk (Okuda et al., 1992), J N K (Foltz and Schrader, 1997), and p38 (Foltz et al., 1997) M A P kinases, PLC-y , as well as increased PI 3-Kinase activity (Rottapel et al., 1991). J A K 2 activation and vav tyrosine phosphorylation has been reported in the M07e and TF-1 cell lines (Ala i et al . , 1992), however S L F stimulation probably does not activate any of the J A K molecules in mast cells or any of the S T A T transcription factors. 1.9.2 IL-3 Signaling The receptor for IL-3 is a heterodimer consisting of a p-chain (IL-3Rp or p c ) that is shared with the IL-5 and granulocyte macrophage-colony stimulating factor ( G M - C S F ) receptors and a unique 70 kDa a-chain (also known as SUT-1 or IL-3Ra) specific for IL-3 (Miyajima et al., 1992). G M - C S F and IL-5 also have their own specific a-chains. In mice, but not in humans, there are two different types of high affinity IL-3 receptors based on the use of two different p-chains known as A i c - 2 A and A i c - 2 B . A i c - 2 A alone has a low affinity for IL-3 . G M - C S F and IL-5 receptors however utilize only A i c - 2 B and their respective a-chains (Duronio et al., 1992). Based on their high degree of homology both in the coding and non-coding regions, and their close genomic proximity, these two p-chain genes are thought to have evolved from a gene 36 duplication event that occurred after humans and rodents diverged during evolution (Gorman et al., 1992). Both chains contain the hallmarks of the type I cytokine receptor family, specifically four conserved cysteine residues and a tryptophan-serine-X-tryptophan-serine ( W S X W S ) motif in the extra cellular region, except that the p-chain has two repeats of these cysteine/tryptophan motifs. Whi l e these receptors exhibit no intrinsic tyrosine kinase activity they have two membrane proximal proline-rich regions known as box-1 and box-2 that (bind and) are required for the activation of the tyrosine kinases L y n , Fes and J A K 2 (Rao and Mufson, 1995). These kinases are thought to mediate tyrosine phosphorylation of the IL-3 receptor (Ihle et al., 1995), creating binding sites for signaling molecules with SH-2 and P T B domains, and phosphorylating and activating the transcription factor S T A T - 5 (Hara and Miyajima, 1996) (Chin et al., 1996). The heterodimerization of the receptor is thought to bring the J A K kinases into proximity where they cross phosphorylate and activate each other. This model is similar to the mechanism of activation for receptor tyrosine kinases such as c-kit or the P D G F receptor. However, it should be noted that both positive and negative regulatory sites of tyrosine phosphorylation have been identified on J A K molecules (Zhou et al., 1997) indicating that tyrosine phosphorylation alone is not a clear indicator of activation. There is some disagreement, however, as to whether vav is involved in IL-3 signaling (Matsuguchi et al., 1995), (Ala i et al., 1992). L i k e S L F , IL-3 stimulation also results in the activation of the Ras/Raf/Erk cascade and the J N K and p38 M A P kinases as well as increased PI 3-Kinase activity (Gold et al., 1994), (Craddock and Welham, 1997). IL-3 signaling differs from events induced by S L F in that there is no activation of P L C y or c P K C isoforms in response to IL-3 (Hallek et al., 1992). The proto-oncogene pim-l, a serine/threonine kinase, is activated by IL-3 , but not S L F (Yip-Schneider et al., 1995), and cells derived from mice deficient in pim-\ display reduce proliferation when cultured in IL -3 , 37 indicating pim-l is involved but not essential (Domen et al., 1993). Clearly IL-3 and S L F activate an overlapping set of molecules. 1.9.3 IL-4 Signaling There are two types of high affinity IL-4 receptors (IL-4R). One is composed of a 140 k D a I L - 4 R a chain that has affinity for IL-4 and the 65 kDa y c chain that has none. The latter was originally cloned as a component of the IL-2 receptor and was later determined to also participate in the receptors for IL-4, 7, 9 and 15 (Kondo et al., 1993). A second type of high affinity IL-4 receptor is composed of the same I L 4 R a chain but contains the IL -13Ra chain instead of the y c . When expressed alone the LL-13Ra chain binds EL-13 but not LL-4, but IL-13 is bound with higher affinity in the presence of the IL-4Rp chain (Hilton et al., 1996), (Caput et al., 1996). The IL -4a chain, and IL-13oc, also have the conserved four cysteine residues and a W S X W S motif characteristic of type I cytokine receptors as well as a cytoplasmic domain that lacks catalytic activity. The intracellular portion of the I L - 4 R a chain, and the y c , contain b o x l and box2 domains, which are required for J A K activation (Tanner et al., 1995). J A K 1 is constitutively associated with the DL-4Ra and J A K 3 with the y c (Yin et al., 1994) and removal of the box 1/2 domains results in the loss of J A K activation. In cells lacking jc, but expressing I L -13Ra, IL-4 fails to induce activation of J A K 3 but instead activates T Y K - 2 suggesting that the IL-13Rct chain recruits T Y K 2 instead of J A K 3 recruited by the y c (Orchansky et al., 1997). The cytoplasmic domain of the IL-13Ra is essential for its activity and expression of a mutant I L -13Roc lacking the cytoplasmic domain inhibits IL-4 signaling by competing with y c for EL-4/IL-4Roc complexes. This dominant inhibiting effect is enhanced by the presence of IL-13, indicating that complexes of IL-13 and IL-13Ra compete with IL-4 for I L - 4 R a (Orchansky et al., 1997). 38 IL-4 stimulation also results in the tyrosine phosphorylation and recruitment of insulin receptor sustrate-1 and 2 (IRS2) (Welham et al., 1997), (Johnston et al., 1995), which bind to tyrosine 497 in the I L - 4 R a chain (Wang et al., 1998). This residue is also important for the activation of S T A T - 6 (Harada et al., 1998) although recent evidence has suggested there is a redundant mechanism for S T A T - 6 activation through J A K 1 in the absence of this tyrosine (Moriggl et al., 1998). The SH-2 domains of the p85 subunit of PI 3-Kinase bind phosphorylated IRS2 in response to IL-4 stimulation (Welham et al., 1997), (Wang et al., 1995) and provide one potential mechanism for the increased PI 3-Kinase activity observed in response to IL-4 . Another mechanism for increased PI3-Kinase activity is through the tyrosine phosphorylation and recruitment of the tyrosine kinases c-fes (Izuhara et al., 1996). c-fes contains a Y X X M sequence and is bound by the regulatory subunit of PI 3-Kinase upon IL-4 stimulation (Izuhara et al., 1994). The IRS proteins are large 170-190 kDa proteins with P H and P T B domains and are heavily tyrosine phosphorylated in response to IL-4 stimulation. IRS molecules contain tyrosine residues in consensus sites for the binding of the SH-2 domains of other molecules such as Grb2 and SHP2 (Wang et al., 1995), thought to be involved in the activation of Ras. However IL-4 does not induce activation of Ras (Duronio et al., 1992) or Erk M A P Kinase (Welham et al., 1994). Additionally IL-4 stimulation does not result in tyrosine phosphorylation of SHP2 or increases in its phosphatase activity (Welham et al., 1994). The role of IRS molecules in IL-4 signaling is further complicated by the observation that primary bone marrow derived mast cells do not express detectable levels of IRS 1/2 proteins (Welham et al., 1997). The expression and activity of Lsk increase in response to IL-4 stimulation (Musso et al., 1994). L s k has homology with the tyrosine kinase known as C-terminal Src kinase (csk), suggesting that IL-4 might negatively regulate Src family kinases, however this possibility has not been explored. 39 1.10 Induction of Nuclear Proto-oncogenes by IL-3, IL-4 or SLF The study of the nuclear proto-oncogenes c-myc, c-fos and c-jun has been the focus of this thesis with particular attention to the signal transduction events regulating c-myc induction. When I started my thesis work there was no information on the regulation of these genes by IL-4, although there was evidence that expression of c-myc and c-fos were induced in response to IL-3 (Conscience et al., 1986) or S L F (Horie and Broxmeyer, 1993). Activator protein-1 (AP-1) is composed of homodimers of the Jun family (Jun, JunB, and JunD) or heterodimers consisting of Jun and Fos (Fos, FosB, F r a l and Fra2) family members. There are thus 15 different possible combinations for producing a dimer which has AP-1 activity, and is capable of binding the consensus D N A sequence T G A C / G T C A and stimulating the transcription of genes that contain this sequence in their promoter region. The Ras /Raf /MEK/Erk M A P kinase signaling pathway is sufficient for the induction of the c-fos and c-jun genes. Activated Erk phosphorylates the Ets family transcription factor Elk-1 which then binds the transcription factor called serum response factor (SRF) (Janknecht and Hunter, 1997). S R F is pre-assembled at the serum response element (SRE) in the c-fos promoter and the binding of Elk-1 is sufficient for transcriptional activation of the c-fos gene (Rivera et al., 1993). The c-jun gene is thought to be regulated by AP-1 composed of Jun/Jun homodimers. Jun is itself regulated by both phosphorylation and dephosphorylation events mediated by M A P kinase members. The Jun protein is detectable in resting cells and upon stimulation with mitogens, or phorbol esters, becomes dephosphorylated primarily at the carboxy terminus (Boyle et a l . , 1991). This region is the D N A binding portion of the molecule and when dephosphorylated binds to AP-1 sites and can stimulate transcription through its amino terminal transcriptional activation domain. The promoter of the c-jun gene contains two AP-1 sites and in response to phorbol ester treatment, Jun is dephosphorylated at the carboxy terminus but is not 40 phosphorylated at the amino terminus, which allows Jun to bind D N A and activate c-jun gene transcription. Thus the Raf/Erk M A P kinase signaling path is sufficient for the transcriptional activation of the c-jun and c-fos promoters. A s expected, IL-3 or S L F stimulation leads to increased c-fos and c-jun m R N A levels. However, IL-4 treatment does not lead to the transcriptional activation of these genes probably due to the inability of IL-4 to activate this kinase cascade. A s discussed, many molecules have been implicated in the regulation of c-myc induction. Stimulation with IL-3 or S L F also leads to the induction of c-myc but through an unknown mechanism. 41 CHAPTER 2. Materials and Methods 2.1 Cloning of Steel locus factor in pGEX-2T S L F c D N A from p M G F 10.1 (Melanie Welham) was digested with BgUI and Bsu36I, heat inactivated and filled in with Klenow (2.5 m M dNTPs/20 | i M DTT/for 1 hour at R/T) . This fragment was cloned into the Smal site of p G E X - 2 T . 2.2 Purification of GST-SLF p G E X - 2 T / S L F was transformed into E. coli strain DH5cc or UT5600 . 1 m L of transformed cells of an overnight culture were inoculated into I L of 2 X Y T media containing 50 ug/ml ampici l l in and grown to an O . D . 6 0 0 of 0.5. Expression was induced with 0.1 m M isopropyl-|3-D-thiogalactopyranoside (IPTG) at 28° C for 4 hours. The cells were collected by centrifugation and resuspend in 10 ml of: phosphate buffered saline (PBS), 0.1% Tween-20, 1 u M phenylmethylsulfonyl fluoride (PMSF) , 2 ug/mL leupeptin, 0.7 ug/mL pepstatin, 10 ug/mL aprotinin, 10 ug/mL soybean trypsin inhibitor (STI), I m M ethylenediamine tetra-acetic acid ( E D T A ) , I m M E G T A , 1 mg/mL lysozyme, 15 m M 2-mercaptoethanol and 0.1 mg/mL DNase L , and incubated on ice for 20 minutes. Cells were lysed by sonicating 3 times for 30 seconds each and the extract was centrifuged at 10,000 R P M for 30 minutes at 4° C . 1.5mL (50% slurry) GST-Agarose beads (Pharmacia) were prepared by washing 3 times with P B S . The lysate was filtered through a 0.8 u, filter and added to the pre-washed GST-Agarose beads. The mixture was incubated with rotation at 4°C for 30 minutes. Beads were transferred to a B i o R a d disposable column and flow through was discarded. The beads were washed with 100 m L PBS/0.5 M 42 N a C l / 1 % NP-40/15 m M 2-Mercaptoethanol at 4°C by gravity column filtration and then with lOOmL PBS/15 m M 2-Mercaptoethanol. The column was either eluted with 0.5 m L of 150 m M Tris p H 7.5/10 m M glutathione/10 m M 2-Mercaptoethanol or cleaved off with 0.1 mg human thrombin (Boehringer-Mannheim) in 50 m M Tris p H 7.5/150 m M NaCl/2.5 m M CaCL, for 1 hour at room temperature. G S T - S L F purified as two proteins with molecular weights of 45 and 30 kDa. The larger was absent and the lower more prevalent post digest along with an expected 18 k D a band corresponding to S L F . This was concentrated using Centricon concentration filters. 2.3 C e l l S t i m u l a t i o n C o n d i t i o n s A l l hemopoietic cells except for Jurkat T-cell lymphoma cells were growth arrested by culturing in R P M I 1640, 10% F C S , 0.3% W E H I - 3 B conditioned media for 16 hours at 37°C in a 5% C 0 2 incubator. Cells were then washed 3 times with serum free R P M I 1640 and incubated in serum free R P M I 1640 at 37°C. For kinase assays cells were used at a density of 5 x l 0 6 cells /mL in Eppindorf tubes in a 37°C water bath, for RNase protection or Northern analysis cells were used at a density of 2 x l 0 6 cells/mL in 5 m L tissue culture dishes in 37°C, 5% C 0 2 tissue culture incubator. Cells were stimulated with 5 u g / m L synthetic murine IL-3 , 10 |ig/mL synthetic murine IL-4 or 1 (Xg/mL synthetic murine G M - C S F . A l l synthetic cytokines were provided by Ian Clark-Lewis (Biomedical Research Centre, U B C , Canada). Recombinant murine S L F was assayed by 3[H]-Thymidine incorporation to give half-maximal response at a 1:20,000 dilution. A l l stimulations were performed with a 1:1000 dilution of r S L F since maximal tyrosine phosphorylation was observed at this dilution. X / 0 6 3 murine IL-2 conditioned media was used as the source of interleukin-2 and was used at 20%. T P A (Sigma, U S A ) was dissolved in D M S O and used at 100 ng/mL final. 43 A l l adherent cell types were growth arrested by washing 3 times in serum free R P M I 1640 and culturing in R P M I 1640, 1% F C S for NIH-3T3 cells or Optimem media (Gibco B R L ) for H e L a cells for 16 hours. Cells were then washed 3 times with serum free R P M I 1640 and incubated for an additional 1 hour in serum free R P M I 1640 at 37°C in a 5% C 0 2 tissue culture incubator prior to stimulation. NIH-3T3 cells were stimulated with 50 ng/mL recombinant P D G F and H e L a cells were stimulated with 100 ng/mL recombinant E G F ( R & D Research). Jurkat T-cell lymphoma cells were growth arrested by washing 3 times in serum free R P M I 1640 and culturing in R P M I 1640, 1% F C S for 16 hours. Cells were then washed 3 times with serum free R P M I 1640 and incubated for an additional 1 hour in serum free R P M I 1640 at 37°C in a 5% C 0 2 tissue culture incubator prior to stimulation. Cells were stimulated with synthetic SDF1 (provided by Ian Clark-Lewis) at a final concentration of 250 ng/mL. 2.4 Kinase Inhibitors L Y 2 9 4 0 0 2 and rapamycin (Calbiochem, U S A ) were dissolved in D M S O at stock concentrations of 50 m M and 10 m M and used at 25 u M and 100 n M final respectively. Wortmannin (Sigma, U S A ) was also dissolved in D M S O at a stock concentration of 100 u M and used at 100 n M final. 2.5 SDS-PAGE and Immunoblotting Whole cell lysates from 2-4 x 105 cells or the supernatant of a kinase assay were resolved by S D S - P A G E . Electrophoresis buffer consisted of 25 m M Tris, 192 m M Glycine, 0.1% SDS. Electrophoresis was carried out at 80 Volts (constant voltage) until the bromophenol-blue dye entered the separating gel. The voltage was then increased to 150 Volts until this dye was at the bottom of the gel. Gels were transferred to nitrocellulose membranes (0.45 | i pore size from 44 Schleicher and Schull , Germany) at 0.8 Amps/cm 2 (constant current) for 75 minutes using a semi-dry transfer apparatus (Pharmacia, Sweden) in transfer buffer containing: 39 m M Glycine, 48 m M Tris, 0.0375% (w/v) SDS, 20% (v/v) methanol. After transfer the membranes were stained with Ponceau S (Sigma, U S A ) to identify the molecular weight standards (BioRad, U S A ) . 2.6 P K B Kinase Assays Cells starved and stimulated as described in stimulation conditions section. Fol lowing stimulation 5 X 10 6 M C / 9 or primary mast cells/sample were lysed in: 50 m M Tris p H 7.5, 0.25% Nonidet P-40, 1 m M E D T A , 50 m M NaF, 20 m M p-glycerophosphate, 100 u M m M N a V 0 4 , 100 \iM N a M o 0 4 , 40 n-g/mL phenylmethyl sulfonylfluoride (PMSF) , 2 ng/ml soybean trypsin inhibitor (STI), 1.4 mg/mL pepstatin, 2 ug/mL leupeptin, 1 ug/mL microcystin-LR. After centrifugation for 2 minutes at 14000 R P M at 4°C, the transferred supernatant was pre-cleared with 20 uL of 50% slurry of protein G beads at 4°C for 20 minutes. The mixture was centrifuged briefly to pellet the beads, and the supernatant was immunoprecipitated with 4 ug of sheep a-P K B antibody from Upstate Biotechnology Inc. (cat # 06558) at 4 °C for 45 minutes. 20 uL of a 50% slurry of protein G beads were added and rotated for 30 minutes at 4°C. The beads were then washed 3 times with the above lysis buffer except 0.35 M N a C l was added. The beads were washed once with kinase assay buffer (20 m M H E P E S p H 7.4, 20 m M p-glycerolphosphate, 1 m M dithiothreitol (DTT) , 1 m M M g C l 2 , 1 ug/mL microcyst in-LR, 1 m M N a V 0 4 , 1 m M N a M o 0 4 ) , and then resuspend in 25 | i L of kinase assay buffer (50 ug/mL histone H 2 B (12 kDa), 200 u M A T P and 10 uCi 3 2 P - A T P ) and incubated at 30°C for 15 minutes. The assay was resolved on a 15% poly acrylamide gel, transferred to nitrocellulose by semi-dry blotting and visualized by audioradiography. 45 2.7 P K B Western Blotting Blots were blocked with 5% skim milk powder in Tris Buffered Saline (TBS) (20 m M Tris p H 7.5, 150 m M NaCl ) for 2 hours at room temperature. Then incubated with primary sheep ant i -PKB antibody from Upstate biotechnology (cat # 06558) which was diluted to 1.0 ug/mL in 5% low fat mi lk /TBSN/0.05% N a N 3 overnight at 4°C. Following 3 washes with T B S N (TBS, 0.05% NP-40) and 2 washes with T B S , secondary horseradish peroxidase (HRP) -coupled goat anti-sheep antibody (Dako, Denmark) diluted 1:20,000 in T B S N was added for 1 hour at room temperature. Excess secondary was removed with three washes of T B S N and 3 washes of T B S for 10 minutes each at room temperature. H R P was developed using enhanced chemiluminescence (ECL) from Amersham, U S A . 2.8 J N K Kinase Assays Cells were starved and stimulated as described in the stimulation conditions section. Fol lowing stimulation, 5 X 10 6 M C / 9 or primary mast cells/sample were lysed in: 50 m M Tris p H 7.7, 1% Triton X-100, 100 m M N a C l , 5 u M p-Methylaspartate, 200 u M N a V 0 4 , 10 m M NaF, 20 m M p-glycerophosphate, 5 m M E D T A , 40 ug/mL P M S F , 2 ug/mL STI, 1.4 ug/mL pepstatin, and 2 u.g/mL leupeptin. After centrifugation for 2 minutes at 14000 R P M at 4°C, the supernatant was incubated with 20 uL of 50% slurry of protein G beads at 4 °C for 20 minutes. The beads were centrifuged briefly to pellet beads, and the supernatant was immunoprecipitated with 2 ug/sample of agarose-conjugated goat an t i - JNKl from Santa Cruz biotechnology (SC-474) for 45 minutes at 4°C. Beads were washed 3 times with the above lysis buffer and once with kinase assay buffer (25 m M H E P E S p H 7.4, 2.5 u M N a V 0 4 , 2 m M D T T , 25 m M p-glycerolphosphate, 46 25 m M M g C l 2 ) , and then resuspend in 20 uL of kinase assay buffer containing 1 ug GST/c-jun (42 kD) , and 10 uCi 3 2 P - A T P for 20 minutes at room temperature. The assay was then resolved on a 12 % polyacrylamide gel, and transferred to nitrocellulose by semi-dry blotting and visualized by audioradiography. 2.9 Western Blot for J N K 1 Loading Blot was blocked with 5% B S A fraction V (Boeringer Mannheim, Germany) in Tris buffered saline (TBS) for 2 hours at room temperature. The blot was then incubated at room temperature with primary rabbit a n t i - J N K l antibody from Santa Cruz (SC-474) which was diluted 1:2000 in T B S N for 2 hours. Following 3 washes with T B S N and 2 washes with T B S , secondary horseradish peroxidase (HRP) -coupled goat anti-rabbit (Dako, Denmark) diluted 1:20,000 in T B S N was added for 1 hour at room temperature. Excess secondary was removed with three washes in T B S N and 3 washes with T B S for 10 minutes each at room temperature. H R P was developed using enhanced chemiluminescence (ECL) from Amersham, U S A . 2.10 p70 S6 Kinase G e l Shift Cel l lysate was prepared as described in the cell stimulation section above. S D S - P A G E procedure was the same as above except for the following changes. Acrylamide to bis-acrylamide ration was 118.5:1 instead of 37.5:1 and samples were resolved with an 8% mini-gel: 3.3 m L water, 2.13 m L 30% acrylamide, 0.54 m L bis-acrylamide, 2 m L 1.5 M Tris p H 8.8, 80 uL 10% SDS, 56 uL 10% ammonium persulfate (APS), and 5.6 uL T E M E D . The gel was then transferred to nitrocellulose and blocked with 5% (w/v) skim milk/0.05% sodium azide for 1 hour at room temperature and probed with an anti-p70 s 6 K antibody purchased from Santa Cruz biotechnology (Santa Cruz, California). Fol lowing 3 washes with T B S N and 2 washes with 47 T B S , secondary horseradish peroxidase (HRP) -coupled goat anti-rabbit (Dako, Denmark) diluted 1:20,000 in T B S N was added for 1 hour at room temperature. Excess secondary was removed with three washes of T B S N and 3 washes with T B S for 10 minutes each at room temperature. H R P was developed using enhanced chemiluminescence ( E C L ) from Amersham, U S A . 2.11 T r a n s i e n t T r a n s f e c t i o n o f B a f / 3 cells Murine Ba/F3 pre-B-cells were grown in R P M I 1640, 10% F C S , 2% W E H I - 3 B conditioned media and growth arrested by culturing in R P M I 1640, 10% F C S , 0.2% W E H I - 3 B conditioned media for 16 hours. Cells were washed 3 times with serum free R P M I 1640 and 1 X 107 cells/sample were electroporated (250V, 960 uF) with 10 ug of pH3P21uc (which was created by substituting the gene for C A T (Watanabe et al., 1995) with the gene encoding luciferase), 20 ug of empty pSG5 vector or pSG5Ap85 in a 250 ul volume of serum free RPMI-1640 containing 10 ug/ml DEAE-dext ran (Amersham Pharmacia; Uppsala, Sweden). The cells were then cultured with or without 2% W E H I - 3 B or 3% x/o63 mIL-4 conditioned media for 10 hours lysed and analyzed for Luciferase (Promega; Madison, Wisconsin) and p-galactosidase (Clonetech; Palo A l t o , Cal i fornia) activities with a luminometer according to manufacturers' recommendations. 2.12 I s o l a t i o n a n d C u l t u r e o f P r i m a r y M a s t C e l l s S T A T - 6 mice were from Michael Grusby (Harvard Medica l School) and wild-type Ba lb /C mice from Jackson Labs. Femurs from age and sex matched mice were harvested and flushed with R P M I 1640. Cells were cultured in R P M I 1640, 10% F C S , 2% W E H I - 3 B and 3% 48 x/o63 mIL-4 conditioned media for a minimum of 3 weeks. Cells were growth arrested and prepared for stimulation as described above for hemopoietic cells. 2.13 N o r t h e r n B l o t P r o c e d u r e Cells were growth arrested, prepared for stimulation and treated with cytokines or growth factors as described above. Cells were harvested by washing 2 times in sterile P B S , and resuspended in 2 m L of 10 m M Tris p H 7.4, 100 m M N a C l , 10 m M E D T A and transferred to a fresh 50 m L Falcon tube. 2 mg of Proteinase K and 8 m L of 10 m M Tris p H 7.4, 100 m M N a C l , 1 m M E D T A , 0.5% SDS were added and the sample was mixed by drawing through an 18 gauge needle on a 20 cc syringe followed with a 22 gauge needle until sample was no longer viscous. Samples were incubated at 37°C for 1 hour and oligo dT was prepared by washing 200 |xL of beads/sample with 0.1 M N a O H , followed by 3 washes with 0.5 M N a C l , 10 m M Tris p H 7.4, 1 m M E D T A , 0.1% SDS. A n additional 0.4 M N a C l was added to the cell lysate and then the lysate was added to the prepared oligo dT beads. Samples were rotated at room temperature overnight, spun at 1500 R P M for 5 minutes at room temperature, to pellet the beads, and the supernatant was discarded. Beads were resuspended in 0.5 M N a C l , 10 m M Tris p H 7.4, 1 m M E D T A , 0.1% SDS and transferred into disposable columns (BioRad), washed with one column volume of 100 m M N a C l , 10 m M Tris p H 7.4, 1 m M E D T A , 0.1% SDS and eluted with 2 m L of 10 m M Tris p H 7.4. Four m L of 100% ethanol, 0.2 m L of 3 M N a O A c p H 5.2 were added and samples were incubated at - 2 0 ° C overnight. The m R N A was pelleted by centrifugation at 10,000 R P M in a Sorval SS34 rotor for 30 minutes at 4°C. The supernatant was carefully removed and the pellet allowed to air dry and then resuspended in 50-100 uL mi l l i -Q filtered, double distilled water. The m R N A concentration was determined by diluting an aliquot 1:250 in water and measuring the optical density (OD) at 260 and 280 nm. A n O D of 0.1 was 1 ug/uL. 1-49 2 ug mRNA7sample were mixed with 3 volumes of 20 m M M O P S p H 7.0, 1 m M E D T A , 5 m M N a O A c , 50% formamide and 6% formaldehyde (this solution was sterilized before adding to m R N A by filtrating through a 0.2 u filter), heated to 60°C for 5 minutes and placed on ice. Six volumes of 50% glycerol, 20 m M M O P S p H 7.0, 1 m M E D T A , 5 m M N a O A c and a few uL of concentrated bromophenol blue (BPB) were then added. Samples were loaded on a 15x15 cm gel (150 mL) containing 20 m M M O P S p H 7.0, 1 m M E D T A , 5 m M N a O A c , 1% agarose and 18% formaldehyde and run at 100 Volts (constant voltage) in 20 m M M O P S p H 7.0, 1 m M E D T A , 5 m M N a O A c untill B P B was 3/4 of the way down. The m R N A was transferred to hybond-nitrocellulose (Amersham) using the traditional Northern wick transfer method with 20xSSC ( 3 M N a C l and 0.3 M Na3citrate p H 7.0) for 48 hours at room temperature. The blot was rinsed with d H 2 0 and cross-linked with 1200 J in a UV-stratalinker ( L K B Instruments). The blot was prehybridized with 50% formamide, 5xSSC, 50 m M N a 3 P 0 4 p H 6.4, 0.1 g F ico l l 400, 0.1 g polyvinylpyrrolidone, 0.1% B S A fraction V (these previous 3 reagents are also known as l x Denhardts), 50 ug/mL herring sperm D N A and 0.1% SDS at 42°C for a minimum of 4 hours to overnight. The c-myc probe was obtained by digesting pMc-myc54 with Xhol to obtain a 1.4 kb probe encompassing the 3' half of exon 2 to the 3' end of exon 3 of the c-myc gene. The pSG5/c-jun and pUC19c-fos plasmids were a gift from Anna Zubiaga (Harvard Medical School). The 1.8 kb c-jun probe was obtained by digesting with EcoRl and 1.5 kb c-fos probe was obtained from a BglLVPvuII digest. The 750 bp G A P D H probe was obtained by a HindDI digest of pBS/Gapdh. Twenty-five ng of D N A probe was prepared by appropriate restriction enzyme digestion and gel purification. D N A probe was radioactively labeled by heating at 100°C for 10 minutes then adding 0.4 M H E P E S p H 6.6, 20 m M M g C l 2 , 2 m M D T T , 200 u M d(GAT)TP , 0.6 mg/mL 50 random D N A hexamers (Pharmacia #2166), 0.8 mg/mL B S A , 50 m C i a - 3 2 P-dCTP , and 2 U D N A polymerase I (Klenow fragment from N E B ) at 37°C for 1 hour. Probe was purified using Sephadex G-50 beads, packed into a 1 cc syringe.; Probe synthesis mixture was added to the top of this column and was centrifuged at 4000 R P M for 2 minutes in a clinical centrifuge. F u l l -length probe flowed though the column while unincorporated 3 2 P - d C T P was retained by the beads. Labeled probe was boiled again to denature and added to hybridization buffer, which was the same as prehybridization buffer described above except with 0.5% dextran sulfate added. This hybridization solution was added to the prehybridized blot in a rotating hybridization oven and incubated at 42°C overnight. The blot was washed 2 times in 2xSSC/0.1% SDS at 55°C for 15 minutes. If excessive background was detected by Geiger counter then additional washes of 0.5xSSC/0.1% SDS at 55°C for 15 minutes or 0.2xSSC/0.1% SDS at 55°C for 15 minutes were performed until no background was detected. The blot was then visualized by autoradiography overnight at - 7 0 ° C with intensifying screens. To reprobe the blot, the old probe was removed adding boiling 0.1% SDS and allowing it to cool to room temperature before discarding the SDS solution. The stripped blot was examined by autoradiography overnight at - 7 0 ° C with intensifying screens to confirm removal of the old probe. The blot was then prehybridized as described above and probed with the next probe using the same procedure as described above. 2.14 RNase protection assays Plasmids encoding c-myc, c-fos and c-jun in the anti-sense orientation were purchased from Ambion (Austin, Texas). The plasmid encoding G A P D H in the anti-sense orientation was purchased from Pharmingen (San Diego, U S A ) . Anti-sense R N A was synthesized from these plasmids using 5 |aL mi l l i -Q d d H 2 0 , 4 uL 5x Sp6 or T3/7 buffer (Gibco-BRL, Grand Island, New York) , 2 uL D T T (Gibco -BRL, Grand Island, New York) , 1 uL RNasin ( G i b c o - B R L , Grand 51 Island, New York) , 1 uL of 10 m M stock of r ( G U A ) T P (Pharmacia, Uppsala, Sweden), 1 uL of 5 u M stock of r C T P (Pharmacia, Uppsala, Sweden), 1 uL template plasmid, 50 m C i 3 2 P - r C T P , 1 uL Sp6 R N A polymerase (for Ambion templates) or 1 uL T7 R N A polymerase (for Pharmingen templates) and 40°C for 1 hour. Two uL RNase free DNase from Worthington Enzymes (New Jersey, U S A ) was added and incubated at 37°C for an additional 30 minutes. A n equal volume of loading dye (Ambion) was added and probe was purified by Urea 5% P A G E . The location of the full length probes were visualized by autoradiography and the gel area was excised and cut into 1 mm cubes. Four hundred uL of probe elution buffer (Ambion) was added and sample was rotated at 37°C overnight in hybridization oven, elution buffer containing the probe was then removed and 1 uL counted with a scintillation counter to assess incorporation. Total cellular R N A was purified using Trizol (Gibco-BRL) according to manufacturer's recommendations. For all hybridizations 5 x l 0 6 cpm of each of the c-myc, c-fos and c-jun probes and 2 x l 0 5 cpm of G A P D F f probe were used with 2-4 u.g of total cellular RNA/sample. Total R N A was mixed with these probes along with 1/10* volume of 3 M N a O A c (Ambion) and 2.5 volumes of 100% ethanol, incubated at - 2 0 ° C for 30 minutes and then spun at 14,000 R P M in Eppindorf bench top centrifuge for 10 minutes. The supernatant was removed and the pellet was air dried for 20-30 minutes. Twenty uL of hybridization buffer (Ambion) was then added and the samples were boi led for 10 minutes, spun briefly and incubated at 45°C overnight. Digestion, inactivation and resuspension were performed according to manufacturer's recommendations. Samples were then resolved using a 7% urea P A G E , and the gel was dried and visualized by autoradiography at - 7 0 ° C overnight with intensifying screens. 52 C H A P T E R 3. Regulation of c-myc induction by cytokines 3.1 Introduction IL-4 was originally characterized as B-cel l growth factor ( B C G F ) due to its ability to act as a co-stimulant for B-cells treated with anti-IgM antibody (Jansen et al., 1990). Further work demonstrated that proliferation of other cell types was enhanced by IL-4 such as T-cells and mast cells (Schmitt et al., 1987). However IL-4 failed to support proliferation of these cells types in long-term (several weeks) growth assays (Kelso and Troutt, 1992). Although there are several immortalized cell lines that can grow in response to IL-4, there is no normal cell type that displays this ability. During the process of becoming immortal many genetic alterations can occur that probably gave these cells the ability to grow in this environment. However, for normal cells, the biological role of IL-4 appears to as a survival factor, which allows cells to persist for several days but does not promote proliferation (Levings et al., 1999). The lack of growth in response to IL-4 is most likely due to its inability to activate the R a s / M A P kinase signaling path. Addit ional ly the inability of IL-4 to activate these molecules suggests that this signaling path is not required for IL-4 mediated survival. I therefore searched for biochemical events that could account for the biological activity of IL-4. 3.2 Results 53 3.2.1 Myc expression is associated with proliferation and survival Several investigators have demonstrated that enforced expression of M y c induces apoptosis (Evan et al., 1992) suggesting a novel function for M y c . However, using Northern blotting or RNase protection assays I had observed constitutive levels of c-myc m R N A in many immortal murine cell lines and human tumor derived cell lines, suggesting that c-myc expression correlated with the immortalization and transformation process and was thus providing a growth or survival advantage (Figure 3.1). It is not l ikely that the levels of c-myc m R N A in unstimulated samples was from incomplete growth arrest, as c-fos (Figure 3.1 A ) or c-jun (Figure 3.1 B and C) m R N A s were undetectable. Moreover the lack of c-myc induction is probably not due to an inability to respond to the indicated stimuli, as c-fos or c-jun genes were readily induced by either T P A (Figure 3.1 A ) or IL-2 (Figure 3.1 B and C) . Further evidence which links M y c expression to enhanced proliferation came during the generation of a murine mast cell line that could grow continuously in the absence of serum. The parental IL-3 dependent murine mast cell line, called R 6 X , was cultured in successively reduced concentrations of serum until a clone was obtained as being able to grow in serum free media, called serum free R 6 X or S F R 6 X . However this new cell line was still dependent on IL-3 for proliferation (data not shown). Northern analysis showed these cells to have constitutive c-myc expression, in contrast to the parental cell line (Figure 3.2). Again these S F R 6 X cells were factor-deprived, as c-jun message was undetectable in unstimulated cells but was induced in response to IL-3 , suggesting these cells were readily responsive (Figure 3.2). Together the observations of constitutive c-myc c-myc c-fos 54 A B T P A - IL-2 m IL-2 c-jun G A P D H c-myc G A P D H D E G F SDF1 f mm Figure 3.1 Constitutive expression of c-myc. A) CT4S, B) CTLL-2, C) HDK1, D) HeLa, E ) Jurkat cell lines were growth arrested by culturing in 1/10* the normal concentration of growth factor or serum for 16 hours. Cells were then washed three times with serum free RPMI 1640, incubated at 37°C in serum-free RPMI 1640 for 30 minutes, and then treated with saturating doses of Tetradecanoyl phorbol-13-acetate (TPA), Interleukin-2 (IL-2), Epidermal Growth Factor (EGF) or Stromal Derived Factor-1 (SDF1) for 1 hour or as a control an equivalent volume of PBS (-). Times chosen for analysis were determined in pilot experiments with these stimuli to be those at which mRNA for c-myc, c-fos and c-jun could be detected. Cells were then lysed and analyzed by Northern blot (A, B and C) for c-myc and either c-fos or c-jun mRNA expression or by RNase protection assay (D and E) for c-myc mRNA expression. Cells were also analyzed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels as a control for consistent loading. A l l data shown are representative of at least three experiments. 55 mRNA expression in many transformed or immortalized cell lines as well as in a cell line capable of proliferating in the absence of serum, strongly suggest that Myc expression provides either a proliferative advantage or the ability to grow in the absence of an appropriate external stimuli. A B - SLF IL-4 - TPA IL-3 c-myc c-jun Figure 3.2 Expression of c-myc in parental and serum free R6X mast cells. A) Parental R6X or B) serum free R6X cell lines were growth arrested and prepared as in figure 3.1. Cells were then treated with Interleukin-4 (IL-4) for 2 hours, Steel locus factor (SLF), Interleukin-3 (IL-3) for 1 hour, tetradecanoyl-phorbol myristic-acetate (TPA) for 30 minutes, or as an unstimulated control and equivalent amount of phosphate buffered saline (-). Cells were then lysed and analyzed by RNase protection assay (A) or by Northern blot (B) for c-myc, c-jun and G A P D H mRNA expression. A l l data shown are representative of at least three experiments. These results suggested that Myc expression could promote proliferation or survival, and correlated with the process of tumor formation. However, except for the tumor derived human cell lines, the other cell types were still factor dependent suggesting that Myc expression alone 56 did not result in a fully transformed, factor independent, phenotype. This was similar to the biological activity of IL-4, promoting survival but not proliferation, never the less in the presence of true growth factors, IL-4 could enhance proliferation. Thus it seemed possible that IL-4 may exert some of its biological effects through the induction of Myc expression. To examine this possibility, primary bone marrow derived mast cells were examined by RNase protection assay for c-myc mRNA expression following stimulation with IL-4. As shown in figure 3.3, IL-4 stimulation increased the level of c-myc message over unstimulated controls. However, unlike the case with IL-3, stimulation with IL-4 failed to induce c-fos or c-jun message, most likely due to its inability to activate Ras or MAP-Kinases family members (data not shown). - IL-3 IL-4 c-myc mm w c-fos W c-jun Figure 3.3 Induction of c-myc, c-fos and c-jun by IL-3 or IL-4. Murine primary bone marrow derived mast cells were growth arrested and prepared as in figure 3.1. Cells were stimulated with Interleukin-4 (IL-4) for 2 hours, Interleukin-3 (IL-3) for 1 hour or as an unstimulated control an equivalent amount of phosphate buffered saline (-), lysed and analyzed for c-myc, c-fos, c-jun and G A P D H mRNA levels by RNase protection assay. A l l data shown are representative of at least three experiments. A l l data shown are representative of at least three experiments. GAPDH mm wm mm 57 3.2.2 R o l e o f P I 3 - K i n a s e i n c-myc i n d u c t i o n i n r e s p o n s e to I L - 4 I next examined the molecular mechanism for c-myc induction in response to IL-4. A clue to the mechanism of c-myc regulation was provided by previous observations on IL-4 signaling. Although IL-4 failed to activate Ras (Duronio et al., 1992) or members of the M A P kinase family (Welham et al., 1994), (Foltz et al., 1997), (Foltz and Schrader, 1997), it did induce increases in both PI 3-kinase activity (Gold et al., 1994) and activate J A K 1 , J A K 3 (Wang et al., 1995) and S T A T 6 (Wang et al., 1998). I therefore examined the requirement of these two signaling paths using cells derived from S T A T - 6 deficient mice and two well-characterized inhibitors of PI 3-Kinase. Two mechanistically and structurally distinct inhibitors of PI 3-kinase, LY294002 or wortmannin were used to investigate the role of PI 3-kinase in the regulation of the c-myc gene. Pre-incubation of the murine M C / 9 mast cell line with LY294002 resulted in a dose-dependent abrogation of the increased levels of c-myc m R N A stimulated by IL-4 (Figure 3.4), suggesting that in response to IL-4, PI 3-Kinase was required for c-myc induction. 3.2.3 R o l e o f P I 3 - K i n a s e i n c-myc i n d u c t i o n i n r e s p o n s e to I L - 3 o r S L F I next assessed the requirement of PI 3-Kinase activity for the induction of c-myc in response to IL-3 or S L F . Pre-incubation of murine M C / 9 mast cells with wortmannin or LY294002 abrogated increased levels of c-myc m R N A in response to these stimuli in a dose-dependent manner (Figure 3.5 and data not shown). Optimal inhibition of c-myc induction in response to either IL -3 , IL-4 or S L F was obtained with 100 n M wortmannin or 25 pJvl LY294002. These observations suggest that PI 3-Kinase activity is universally required for these very different stimuli to induce c-myc. 58 3.2.4 Role of m T O R in c-myc induction by growth factors LY294002 or wortmannin also inhibit the serine/threonine kinase, mammalian Target Of Rapamycin, (mTOR), which has significant homology in its phosphotransfer domain with PI 3-kinase (Brunn et al., 1996). mTOR phosphorylates and activates the serine/threonine kinase p70 s 6 K. To exclude the possible involvement of these kinases in the regulation of the c-myc gene, we used a specific inhibitor of mTOR, rapamycin. Rapamycin, in conjunction with its cellular receptor FKBP12, binds to and inhibits the activity of mTOR (Chiu et al., 1994) thus preventing the activation of p70 S 6 K, but it does not effect the activity of PI 3-kinase or P K B (Figure 3.8). Pretreatment of murine MC/9 mast cells with rapamycin had no effect on the increased levels of I L - 4 : - + + + L Y 2 9 4 0 0 2 (LIM): - - 5 2 5 c-myc H * mm G A P D H • § Figure 3.4 PI 3-Kinase activity is required for the induction of c-myc by IL-4. Murine MC/9 mast cells were growth arrested and prepared as in figure 3.1 but prior to stimulation with IL-4 cells were pretreated for 10 minutes with 0.05% dimethyl sulfoxide (-) or the indicated concentrations of LY294002 (LY294002). Cells were then treated with a saturating dose of IL-4 for 2 hours (IL-4), or as an unstimulated control an equivalent amount of phosphate buffered saline (-). Cells were lysed and c-myc (upper panel) and G A P D H (lower panel) mRNA levels were analyzed by RNase protection assay. A l l data shown are representative of at least three experiments. A l l data shown are representative of at least three experiments. 59 c-myc mRNA induced by IL-3, IL-4 or SLF (Figure 3.6). Similar results were obtained using primary bone marrow-derived murine mast cells stimulated with IL-3, IL-4 or SLF or primary T-lymphocytes stimulated with IL-4 (data not shown). IL-3: - + + + + + + + Inhibitor: - - L Y ( L I M ) Wm(nM) Concentration: - - 1 5 25 10 50 100 c-myc GAPDH Figure 3.5 LY294002 or wortmannin block c-myc induction in response to IL-3 in a dose-dependent manner. Murine MC/9 mast cells were growth arrested and prepared as in Figure 3.1 but prior to stimulation with cytokine cells were pretreated for 10 minutes with 0.05% DMSO (-) or the indicated concentrations of LY294002 (LY) or Wortmannin (Wm). Cells were then treated with a saturating dose of IL-3 for 1 hour (IL-3) or, as an unstimulated control an equivalent amount of phosphate buffered saline (-). Cells were lysed and c-myc (upper panel) and G A P D H (lower panel) mRNA levels were analyzed by RNase protection assay. A l l data shown are representative of at least three experiments. A l l data shown are representative of at least three experiments. 60 A B Stimulat ion: - IL-3 SLF Inhibitor: - - L W R - L W R c-myc c-fos c-jun G A P D H Stimulation: - IL-4 Inhibitor: - L W R - L W R c-myc H mW G A P D H • • • • • • • • Figure 3.6 PI3-Kinase but not mTOR is required for c-myc induction in response cytokines. MC/9 mast cells were growth arrested and prepared as in Figure 3.1. However prior to stimulation with the indicated cytokines the cells were incubated with 0.05% DMSO (-), 25 u M LY294002 (L), 100 nM wortmannin (W) or 100 nM rapamycin (R) for 10 minutes. Panel A) cells were then stimulated with IL-3 (IL-3), SLF (SLF) for 1 hour, or panel B ) IL-4 (IL-4) for 2 hours. For the longer stimulation with IL-4, additional wortmannin (to 50 nM) was added after 1 hour. Levels of c-myc, c-fos, c-jun and G A P D H mRNA levels were analyzed by RNase protection assay. A l l data shown are representative of at least three experiments. 6 1 3.2.5 Role of PI 3-Kinase in c-myc induction in non-hemopoietic cells The requirement of PI3-Kinase for c-myc induction was not restricted to hemopoietic cells, as NIH-3T3 fibroblasts, that were pre-incubated with LY294002 or wortmannin, no longer upregulated c-myc in response to PDGF. As with hemopoietic cells, pre-incubation of NIH 3T3 fibroblasts with rapamycin had no effect on c-myc induction (Figure.3.7). Stimulation: - PDGF Inhibitor: - L W R - L W R c-myc m w # C-TOS c-jun GAPDH Figure 3.7 PI3-kinase but not mTOR is required for c-myc induction in NIH-3T3 fibroblasts. Murine NIH-3T3 fibroblasts were growth arrested by culturing in 1 % serum overnight, washed three times with serum free RPMI 1640, and incubated at 37°C in serum free RPMI 1640 for 1 hour. Prior to stimulation with PDGF the cells were pretreated for 10 minutes with 0.05% DMSO (-), 25 u\M LY294002 (L), 100 nM Wortmannin (W) or 100 nM Rapamycin (R). Cells were stimulated for 2 hours with recombinant PDGF or, as an unstimulated control, phosphate buffered saline (-). After 1 hour of stimulation, additional wortmannin (to 50 nM) was added. Levels of c-myc, c-fos, c-jun (upper panel) and G A P D H (lower panel) mRNA levels were analyzed by RNase protection assay. 62 3.2.6 Specificity of P I 3-Kinase inhibitors To control for non-specific toxicity of these PI 3-kinase inhibitors, we also investigated their effect on the induction of c-fos and c-jun. LY294002 and wortmannin had no effect on the levels of c-fos or c-jun m R N A induced by P D G F in NIH-3T3 fibroblasts (Figure 3.7). Likewise the levels of these transcripts induced by IL-3 or S L F in M C / 9 mast cells (Figure 3.6), or murine bone marrow-derived mast cells (data not shown) were unaffected by LY294002. Wortmannin, however, partially inhibited the induction of c-fos and c-jun in M C / 9 mast cells treated with IL-3 or S L F (Figure 3.6). However, since LY294002 had no effect on c-fos or c-jun m R N A levels, I concluded that transcription of the c-fos or c-jun genes did not require PI 3-Kinase activity. This is discussed further in chapter 4. The observation that wortmannin partially inhibited c-fos and c-jun induction was further explored as demonstrated in section 3.2.13. 63 3.2.7 Inhibition of PKB To confirm that the inhibitors were blocking PI 3-kinase activity, we assessed the activity of a downstream enzyme called PKB (Burgering and Coffer, 1995). Stimulation of MC/9 mast cells with IL-3, IL-4 or SLF resulted in increases in PKB kinase activity that were abolished by pretreating cells with LY294002 or wortmannin but not with rapamycin (Figure 3.8). Similar results were obtained using murine bone marrow-derived mast cells (data not shown). Thus inhibition of increases in the levels of c-myc mRNA induced by growth factors correlated with inhibition of PKB activation. Stimulation: - IL-3 IL-4 - SLF Inhibitor: - - L W R - L W R - - L W R H2B m « - m m Figure 3.8 Effect of LY294002, wortmannin or rapamycin on the growth factor induced activation of PKB. Murine MC/9 mast cells were growth arrested and prepared as in Figure 3.1 Prior to stimulation with the indicated cytokines the cells were treated with 0.05% DMSO (-), 25 njvl LY294002 (L), 100 nM wortmannin (W) or 100 nM rapamycin (R) for 10 minutes. Cells were then stimulated with the indicated cytokines for 10 minutes lysed and P K B was immunoprecipitated with an anti-PKB antibody. The relative kinase activity was assessed by an immune complex kinase assay using Histone H2B as the substrate. The reaction products were resolved by SDS-PAGE and transferred to nitrocellulose. Phosphorylation of H2B was assessed by autoradiography (top panel). The membrane was immunoblotted (IB) with an anti-PKB antibody (oc-PKB) to confirm equivalent amounts of kinase were loaded in each lane (bottom panel). A l l data shown are representative of at least three experiments. 64 3.2.8 Inhibition of S6-Kinase As a control for the effectiveness of rapamycin, I assessed the activation of a kinase downstream of mTOR, called p70 s 6 K . As expected rapamycin, as well as LY294002 and wortmannin, were effective in blocking the decrease in the electrophoretic mobility of p70 S 6 K (Fig. 3.9). Decreased mobility correlates with its activation (Cheatham et al., 1994). Since rapamycin blocked p70 S 6 K activation but failed to inhibit c-myc induction, I concluded that mTOR activity, and activation of p70 S 6 K, were not required for c-myc induction by any of these stimuli in the multiple cell types tested. Stimulation: - IL-3 IL-4 Inhibitor: - - L W R - L W R pp 7 0S6K p 7 0 S6K « i i c i i i f l i t i IB: a-p70 S 6 K Figure 3.9 Effect of LY294002, wortmannin or rapamycin on the growth factor induced activation of p7Cf6K. Murine MC/9 mast cells were growth arrested and prepared as in Figure 3.1 Cells were pre-incubated with 0.05% DMSO (-), 25 u M LY294002 (L), 100 nM wortmannin (W) or 100 nM rapamycin (R) for 10 minutes prior to stimulation with saturating doses of the indicated cytokines. The cells were stimulated with IL-3 (IL3) or IL-4 (IL-4) for 10 minutes, lysed and the activation of p70 S 6 K was detected after low bis-acrylamide SDS-PAGE by immunoblotting (IB) with an anti-p70S6K antibody (cc-p70s6K). Hyperphosphorylated p70 S 6 K (pp70S6K) was identified by its decreased electrophoretic mobility relative to that of hypophosphorylated p70 S 6 K (p70 S 6 K). A l l data shown are representative of at least three experiments. 65 3.2.9 Role of STAT-6 in c-myc induction by IL-4 Stimulation of cells with IL-4 also results in the activation of the transcription factor S T A T - 6 (Izuhara et al., 1996), which has been shown to be crucial for many of the biological and biochemical functions of IL-4 (Takeda et al., 1997). To examine whether S T A T - 6 was required in addition to PI 3-kinase activity for the induction of c-myc in response to IL-4 , we utilized primary bone marrow-derived mast cells from mice that were homozygous null for S T A T - 6 (a gift from M . Grusby). These experiments excluded a role for S T A T - 6 , as JJL-4 induced increased levels of c-myc m R N A in cells from S T A T - 6 null mice, (figure 3.10 A ) that were comparable with those observed with cells from wild type littermates (Figure 3.10 B) . Moreover pretreating cells with LY294002 or wortmannin but not rapamycin (Figure 3.10) abrogated those increases. 66 A Stimulation: - IL-4 SLF Inhibitor: - L W R - L W R - L W R c-myc m m mm> - #: G A P D H i t m iii i i m m m m M l # H B Stimulation: - SLF IL-4 Inhibitor: - L W R - L W R L W R c-myc G A P D H I I S I H f M H l Figure 3.10 STAT-6 is not required for increased levels of c-myc mRNA stimulated by IL-4 or SLF. Bone marrow-derived mast cells from STAT-6 null mice (A) or wild-type littermates (B) were growth arrested and prepared as in Figure 3.1 and pre treated with inhibitors as in Figure 3.4. Cells were stimulated with saturating doses of IL-4 for 2 hours (IL-4) or SLF for 1 hour (SLF) or as an unstimulated control an equivalent amount of phosphate buffered saline (-). Levels of c-myc and G A P D H mRNA were analyzed by RNase protection assay. A l l data shown are representative of at least three experiments. 67 3.2.10 D o m i n a n t i n h i b i t o r y m u t a n t o f P I 3 - K i n a s e b l o c k s i n d u c t i o n o f a c-myc r e p o r t e r Finally, we used a genetic approach to confirm our observations with enzyme inhibitors. The murine factor-dependent hemopoietic cell line BaF/3 has previously been shown to activate a c-myc reporter in response to IL-3 (Watanabe et al., 1995). This reporter contains a 2.3 K b fragment of the c-myc promoter. Transfection into Baf/3 cells and stimulation of cells with IL-3 or IL-4 resulted in 4.5 or 2.5 fold increases in reporter activity respectively (Figure 3.11 filled bars). However, co-transfection of a dominant inhibitory mutant of the p85 regulatory subunit of PI 3-kinase, called Ap85 (Rodriguez-Viciana et al., 1997) abolished the increased reporter activity stimulated by these factors (Figure 3.11 open bars). C IL-3 IL-4 F i g u r e 3.11 Dominant inhibitory mutant of the p85 subunit of PI 3-kinase (Ap85) blocks the activation of a c-myc luciferase reporter. Murine BaF/3 cells were electroporated with the c-myc reporter plasmid pH3P21uc, pEFBos/pGal and where indicated either empty vector ( | ) or pSG5 encoding Ap85 ( Q ). Cells were stimulated with 2% media conditioned with IL-3 (IL-3) (Welham and Schrader, 1992), 3% media conditioned with IL-4 (IL-4) (Welham and Schrader, 1992), or phosphate buffered saline as an unstimulated control (C) for 9 hours. Cells were lysed and luciferase and p-galactosidase activities were measured with a luminometer. Luciferase activities were normalized to p-galactosidase activity. A l l data shown are representative of at least three experiments. 68 3.2.11 Role of PI 3-Kinase in the constitutive expression of c-myc Next I assessed a possible role for PI 3-Kinase in the constitutive expression of c-myc in HeLa cells. Pre-incubation of HeLa human epithelial cervical carcinoma cells with LY294002, wortmannin or rapamycin had no effect on c-myc mRNA levels. Moreover EGF stimulation failed to increase the levels of c-myc mRNA suggesting that a mutation in these cells is likely to be distal to PI3-Kinase and renders the c-myc gene unresponsive to EGF stimulation. Stimulation: - EGF Inhibitor: - L W R - L W R c-myc GAPDH mmmmm m • mm mm mm mm mW^mW' Figure 3.12 Constitutive expression of c-myc in HeLa cells is not PI 3-kinase dependent. HeLa cells were growth arrested by culturing cells in Optimem media for 16 hours, then washed 3 times with serum free RPMI 1640 to remove serum and incubated in serum free media for 4 hours prior to pre-treatment with inhibitors as in figure 3.6. Cells were then treated with recombinant murine EGF (EGF) for 1 hour or an equivalent volume of phosphate buffered saline as an unstimulated control (-), lysed and analyzed for c-myc and G A P D H mRNA levels by RNase protection assay. A l l data shown are representative of at least three experiments. 69 3.2.12 Role of the R a f / E r k M A P kinase signaling path in c-myc induction It has been shown that phorbol ester compounds, which are similar in structure to the lipid diacylglycerol (DAG), induce the c-myc gene. D A G or phorbol esters bind to and thus activate the conventional protein kinase C (cPKC) members which phosphorylate and activate Raf, eventually resulting in Erk MAP-kinase activation. My observations with IL-4 strongly suggested that the Ras/Erk signaling pathway was not required for c-myc induction. However, it could not be excluded that an alternative mechanism for c-myc regulation also existed. Stimulation of the murine myeloid progenitor cell line FDC-P1 with IL-3 resulted in c-myc and c-fos induction, however, stimulation with tetradecanoyl phorbol-13-acetate (TPA) resulted in c-fos induction but failed to induce c-myc (Figure 3.13). - IL-3 TPA c-myc c-fos GAPDH Figure 3.13 Raf/Erk MAP kinase signaling path is not sufficient for c-myc induction. Murine FDC-P1 myeloid progenitor cells were growth arrested and prepared as in Figure 3.1. Cells were then treated with synthetic IL-3 (IL-3), Tetradecanoyl phorbol-13-acetate (TPA) for 1 hour or, as an unstimulated control an equivalent amount of phosphate buffered saline (-). Cells were lysed and c-myc, c-fos (upper panel) and G A P D H (lower panel) mRNA levels were analyzed by RNase protection assay. A l l data shown are representative of at least three experiments. 70 3.2.13 R o l e o f P I 3 - K i n a s e i n M A P k i n a s e a c t i v a t i o n Stimuli such as IL-3, G M - C S F and S L F stand out in that these growth factors activate the MAP-kinase family members Erk, J N K , and p38. These same cytokines also stimulate c-fos and c-jun transcription, however the degree to which each of the M A P kinases contribute to c-fos and c-jun transcription is unknown. M y observations on c-fos and c-jun induction by these cytokines in the presence of wortmannin provided a clue as to which M A P kinase could the principal regulator of these genes. Recent evidence has implicated PI 3-Kinase in the regulation of the MAP-kinase family members. Wortmannin has been shown to interfere with Erk and J N K M A P kinase activation (Scheid and Duronio, 1996), (Duckworth and Cantley, 1997), (Logan et al., 1997), however it has also been demonstrated that LY294002 does not interfere with Erk (Scheid and Duronio, 1996) activation and the effects of LY294002 on J N K activation are unknown. Since I had observed that wortmannin but not LY294002 inhibited c-fos and c-jun induction I assessed the effects of wortmannin and LY294002 on J N K activation. Pre-treatment of murine M C / 9 mast cells with LY294002, wortmannin or rapamycin had no effect on J N K activation in response to IL-3 or S L F (Figure 3.14). Since wortmannin interferes with Erk but not J N K activation and since it also interferes with c-fos and c-jun induction, this observation suggests that Erk is the principal M A P - K i n a s e family member responsible for c-fos and c-jun induction in response to IL-3 or S L F . 71 Stimulation: - IL-3 SLF Inhibitor: - - L W R - L W R GST/c-jun mm m mm m mm m mm * m\ m..m. wm&z * I IB: oe-JNK1 Figure 3.14 PI 3-Kinase activity is not required for JNK activation in response to IL-3 or SLF. Murine MC/9 mast cells were growth arrested and prepared as in Figure 3.1. Prior to stimulation with the indicated cytokines the cells were treated with 0.05% DMSO (-), 25 uM LY294002 (L), 100 nM wortmannin (W) or 100 nM rapamycin (R) for 10 minutes. Cells were then stimulated with IL-3 (IL-3) or SLF (SLF) for 10 minutes or as an unstimulated control, an equivalent volume of phosphate buffered saline (-). Cells were then lysed and JNK1 was immunoprecipitated with an anti-JNKl antibody. The relative kinase activity was assessed by an immune complex kinase assay using GST/c-jun as the substrate. The reaction products were resolved by SDS-PAGE and transferred to nitrocellulose. Phosphorylation of GST/c-jun was assessed by autoradiography (top panel). The membrane was immunoblotted (IB) with an anti-JNK antibody (a-JNKl) to confirm equivalent amounts of kinase were loaded in each lane (bottom panel). 72 C H A P T E R 4. Discussion While IL-4 is not a true growth factor, due to its inability to activate Ras/Erk M A P kinase pathway (Levings in press), it does activate some of the key molecules required for proliferation, namely PI 3-Kinase (Gold et al., 1994), P K B (Figure 3.8), p 7 0 s 6 K (Figure 3.9) and c-myc (Figure 3.3). These results are consistent with the notion that while PI 3-Kinase activity is required for proliferation, it is not sufficient. Moreover the ability of IL-4 to promote survival correlates with the biological activity of PI 3-Kinase. However there are other molecules that promote survival as well . The biological activity of B-cel l lymphoma/leukemia-2 (bcl-2) has also been described as providing enhanced survival but as not being sufficient for proliferation (Nunez et al., 1990). There is recent evidence that bcl-2 expression is upregulated by PI 3-Kinase (Skorski et al., 1997). However, caution should be exercised in the interpretation of these experiments as constitutively active mutants of PI 3-Kinase and P K B are notorious for inducing autocrine effects, leading to the activation of Ras/Erk M A P kinase and a c-fos reporter (Hu et al., 1995). Clearly increased PI 3-Kinase activity is not sufficient to activate these molecules since IL-4, which does activate PI 3-Kinase and P K B , does not activate the Ras/Erk pathway (Duronio et al., 1992), (Welham et al., 1994) or induce c-fos transcription (Figure 3.3). Additionally PI 3-Kinase has been shown to inactivate the pro-apoptotic bcl-2 member Bad through phosphorylation by P K B (Datta et al., 1997). Once again this mechanism may account for the ability of IL-4 to enhance survival but does not explain how IL-4 enhances proliferation in combination with true growth factors. I have shown that IL-4 also leads to c-myc induction (Figure 3.3) which can be maintained for at least 4 hours (data not shown). These kinetics are in contrast to those induced by true growth factors which induce c-myc maximally at 2 hours with levels declining to basal by 4 hours. This prolonged kinetics of c-myc induction may account for the capacity of IL-4 to 73 enhance the proliferative activity of other growth factors. The aim of this research was to explore the biochemical mechanism by which growth factors regulate c-myc induction. Here I have shown that c-myc induction in response to IL-4 is dependent on PI 3-Kinase activity (Figure 3.4). I reached this conclusion using two mechanistically and structurally distinct inhibitors of PI 3-Kinase, wortmannin or LY294002, and a dominant negative mutant of the regulatory subunit of PI 3-Kinase (Figures 3.4, 3.6 lower and 3.11). I have also shown that the increased expression of c-myc induced by a variety of growth factors also requires PI 3-Kinase activity. This was demonstrated both for growth factors that signal through tyrosine kinase receptors, namely S L F and P D G F (Figures 3.6 and 3.7), and those that signal through receptors of the cytokine receptor superfamily, namely LL-3 and LL-4 (Figures 3.4 and 3.5). The increased expression of c-myc m R N A by growth factors correlated with the activation of P K B (Figure 3.8) but not p 7 0 s 6 K (Figures 3.9) suggesting that the signaling pathway to c-myc regulation does not involve m T O R activation (Figure 3.6 lower) but may involve P K B . IL-4 provided a unique tool by showing a minimal signaling path to c-myc induction. Although clearly PI 3-Kinase was required, we could not exclude contributing roles of other paths. Our experiments with primary mast cells derived from mice lacking S T A T - 6 exclude a role for this transcription factor in IL-4 mediated increases in c-myc m R N A (Figure 3.10). Furthermore, the ability of IL-4 to induce c-myc m R N A expression, despite its inability to activate Ras or the Erk, J N K , or p38 M A P kinases, also excludes a requirement for these paths. Although the Ras/Erk pathway was not required it could however serve as an alternative mechanism for c-myc gene regulation. To examine this possibility, the Raf/Erk pathway was activated using phorbol esters. Contrary to published evidence (Coughlin et al., 1985), (Ning et al., 1996), (Kerkhoff et al., 1998), stimulation of this pathway was not sufficient for c-myc induction (Figure 3.13), suggesting that perhaps Ras/Erk pathway plays a more significant role in adherent cells. Alternatively, this system could be functioning through an autocrine mechanism, 74 when the length of time that was required for enforced expression of a Raf-estrogen receptor fusion to induce c-myc is considered (Kerkhoff et al., 1998). Kerkoff et. al. also showed that in cells expressing Raf/ER, estrogen induced c-myc but not c-fos. This is unusual as T P A is an excellent activator of c-fos and c-jun transcription. Additionally the expression of the Ras binding domain of Raf blocked serum induced c-myc and c-fos induction, suggesting that Raf is required for c-fos transcription but is not sufficient. Again this result disagrees with my results obtained using phorbol esters (Figure 3.13) and results obtained by Roussel et. al. who showed that the Y 8 0 9 F mutant of the CSF-1 receptor still activated Ras/Erk M A P kinase (as evident from c-fos and c-jun transcription) but did not induce c-myc. Toxici ty of LY294002 or wortmannin can be excluded, since these inhibitors did not interfere with c-fos or c-jun expression stimulated by P D G F in NIH-3T3 fibroblasts (Figure 3.7). However wortmannin, but not LY294002 , partially interfered with c-fos and c-jun induction during IL-3 or S L F stimulation of M C / 9 or bone marrow-derived mast cells (Figure 3.6 A ) . Wortmannin has been shown to inhibit the activation of Erk in M C / 9 mast cells (Scheid and Duronio, 1996), and I have shown that wortmannin failed to inhibit Jnk activation in these same cells with the same stimuli. Thus Erk inhibition correlates with inhibition of c-fos and c-jun transcription suggesting that Erk is the primary M A P - K i n a s e family member regulating these genes. The failure of IL-4 to induce the expression of c-fos or c-jun m R N A (Figure 3.3) is also consistent with its inability to activate Ras and the MAP-fami ly of kinases. These kinases have been shown to phosphorylate and activate the eto-family transcription factors E l k - 1 , Sap-1 and Sap-2, which bind to the transcription factor serum response factor (SRF) resulting in c-fos transcription (Janknecht and Hunter, 1997). The most likely mechanism of c-jun transcription in response to IL-3 or S L F probably occurs through Erk activation. Activation of the Raf/Erk path by phorbol esters has been shown to induce dephosphorylation of the carboxy terminal region of 75 Jun allowing it to bind to the AP-1 site found in the c-jun promoter (Boyle et al., 1991). Jnk activation in response to IL-3 or S L F probably does not play a significant role since wortmannin had no effect on Jnk activation (Figure 3.14), yet wortmannin did interfere with c-jun transcription (Figure 3.6 A ) . Although the effects of wortmannin on p38 activation were not examined, it is unlikely that wortmannin would have interfered with p38 activation as Jnk and p38 activation are so far not separable events. Enforced expression of M y c leads to apoptosis, which can be antagonized through increased PI 3-Kinase activity (Kauffmann-Zeh et al., 1997). Our experiments, however, indicate that growth factor stimulated increases in the levels of c-myc m R N A , w i l l necessarily occur in the context of increased PI 3-Kinase activity, providing a mechanism where by physiological increases in M y c expression w i l l invariably be coordinated with increased resistance to apoptosis. Bcl -2 expression also antagonizes c-myc induced apoptosis (Bissonnette et al., 1992). If the observation that PI 3-Kinase induces bcl-2 expression is correct, then once again growth factor stimulated c-myc induction wi l l also result in bcl-2 expression. Apoptosis induced by enforced M y c expression only occurs under low serum conditions, where presumably there is insufficient PI 3-Kinase activity to upregulate bcl-2 expression. This coordinated regulation of c-myc and bcl-2 in vivo by PI 3-Kinase possibly explains why these two molecules cooperate in co-transformation type assays (Fanidi et al., 1992), as co-expression of these two molecules mimics increased PI 3-Kinase activity stimulated by growth factors. Before tumors acquire constitutive c-myc expression they might first have to upregulate bcl-2 expression or develop a homozygous deletion of the tumor suppressor p53. In p53 null fibroblasts, enforced M y c expression does not lead to apoptosis but instead promotes the cell cycle (Hermeking and Eick, 1994), suggesting that p53 functions as a check point in regulating proliferation or apoptosis in response to oncogene activation. However, when cell lines acquire constitutive M y c expression there isn't the same need for other genetic alterations. Cel l lines 76 wi l l exhibit increased PI 3-Kinase activity in response to either growth factors or the serum they are cultured in. Previous work has established a link between the Ras/Erk pathway and c-myc gene regulation as discussed above. However other molecules have been implicated in c-myc regulation as well . The tyrosine kinase c-src is of principal interest as a correlation exists between mutants of src that bind to and increase activity of PI 3-Kinase and those that induce c-myc (L iu et al., 1993). Additionally the CSF-1 receptor mutant (Y809F) that fails to induce c-myc also fails to activate c-src (Courtneidge et al., 1993). However Courtneidge and co-workers also show that this same receptor mutant failed to phosphorylate a polypeptide of 85 kDa. Based on my results I would predict that the primary mechanism for increased PI 3-Kinase activity in response to CSF-1 involves c-src activation. Thus the loss of c-src binding to Y809 resulted in the loss of increased PI 3-Kinase activity and thus c-myc induction. Other tyrosine kinases such as c-abl also bind to and increase PI 3-Kinase activity as well as induce c-myc. L ike c-src these kinases might also regulate the c-myc gene in a PI 3-Kinase dependent manner. Figure 3.1 summarizes my current model for the signal transduction pathway to the c-myc gene. Several different types of receptors including tyrosine kinase and those receptors that contain no intrinsic tyrosine kinase activity all induce c-myc in a PI 3-Kinase dependent manner. M y observation that PI 3-Kinase activity is required for the increased expression of c-myc m R N A induced by growth factors, clearly identifies at least one essential role for PI 3-kinase in mitogenesis. Future work wi l l investigate the theory that membrane-associated or cytosolic tyrosine kinases also induce c-myc in a PI 3-Kinase dependent fashion. This w i l l involve the inducible expression of a constitutively active mutant of c-src (srcY527F) under the control of a tetracycline inducible promoter system. Additionally the possibility that increased PI 3-kinase 77 Growth Factor Receptor Figure 4.1 Model of signal transduction path to the c-myc gene. The above diagram depicts a variety of extracellular stimuli and activated molecules that induce c-myc in a PI 3-Kinase dependent manner. activity alone w i l l be sufficient for the expression of c-myc m R N A w i l l be investigated. The antibiotic coumermycin binds and forms dimers of the bacterial D N A gyrase B molecule. 78 Expression of the drug-binding region of gyrase B fused to Raf resulted in the dimerization and activation of Raf upon the addition of coumermycin (Farrar et al., 1996). 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