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The 70 kDa S6 kinase : enrichment and characterization of sea star homologue, and the role of p70S6K.. Charlton, Lorin Ann 1998-12-31

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The 70 kDa S6 Kinase: Enrichment and Characterization of a Sea Star Homologue, and the Role of p7056/ir in the Transformed Keratinocyte cell line Pam212 in Response to Photodynamic Therapy by LORIN ANN CHARLTON B.Sc. (Biology), Simon Fraser University, 1991 A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Faculty of Graduate Studies, Division of Experimental Medicine Faculty of Medicine We accept this thesis as conforming to the required standard The University of British Columbia 1998 © Lorin Ann Charlton, 1998 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 cSXfer^ >K g^T/VC M & b I C_ 1[ Aj The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The 70 kDa ribosomal protein S6 kinase (p70 ) has been implicated in the control of cell proliferation and the onset of protein synthesis following mitogenic and hormonal stimulation. Due to the importance of this role in signal transduction we undertook to purify and characterize the echinoderm isoform of p70S6/\ and to evaluate the role of p7056^ in a mammalian system. A rapid protocol was developed for the enrichment of a catalytically active isoform (p52S6K) of the S6 kinase from oocytes of the sea star Pisaster ochraceus. The enzyme was purified ~50,000 fold from oocyte cytosol with a specific enzyme activity of 1.6 umol per min per mg. The enriched preparation was characterized with respect to ion dependence, phosphotransferase activity and consensus sequence for substrate phosphorylation, determined to be RXXSXR, which was partially distinct from that reported for mammalian p70s<w\ Phosphoamino acid analysis of the 40S ribosomal protein S6 phosphorylated by partially purified p5256A: yielded incorporation of 32P exclusively on serine residues, while 2-dimensional phosphopeptide analysis revealed phosphorylation on at least 8 distinct tryptic peptides, S6K S6K consistent with previous studies of mammalian p70 . Inhibition of sea star p52 phosphotransferase activity after treatment with protein-serine/threonine phosphatases confirmed that p52S6K was regulated by phosphorylation. The p52S6K co-purified with the regulatory and catalytic subunits of protein phosphatase 2A and the heat shock protein 60. A variety of cellular stresses are known to affect the activity of protein kinases. It was demonstrated that protein levels of sea star S6 kinase decreased in response to the stress of heat shock, in a time-dependent manner. In collaboration with Dr. Jing-Song Tao (Ph.D. candidate), I went on to evaluate how p70S6K was affected in mammalian Pam212 keratinocytes in which stress pathways were activated by treatment with photodynamic therapy (PDT) with benzoporphyrin derivative mono-acid ring A (BPD-MA). We demonstrated that PDT caused a profound suppression of the phosphorylation and ii phosphotransferase activity of pVO^0* in cells maintained in serum-containing medium. In serum-starved Pam212 cells, PDT prevented the insulin-induced activation of phosphatidylinositol 3-kinase (PI 3-kinase), protein kinase B (PKB) and p70S6K. PDT supressed the activation of p70S6K induced in response to anisomycin and UV irradiation. The activation of p70S6K by these stimuli was found to be independent of PI 3-kinase and PKB. Therefore, inhibition of p70S6K may represent a key signaling event mediating PDT-induced apoptosis. The key novel findings of the research presented here include a substantial enrichment of an echinoderm homologue of p70S6K, and analysis of the regulatory properties of the kinase. We also show, for the first time, that p70S6K is profoundly inhibited in response to PDT, although we do not at present understand the mechanism of PDT-induced signaling. 111 Table of Contents Abstract -Table of Contents jv List of Tables List of Figures x-List of Abbreviations jjj List of Amino AcidsAcknowledgements xvDedication xvii Chapter 1: General Introduction 1.1 Signal Transduction 1.1 1 Protein kinase structure and function 3 1.1 2 Receptor-mediated signal transduction 5 1.1 3 MAP kinase pathways 7 1.1 4 PI 3-kinase/PKB/p705<5/: pathways 12 1.2 70 kDa S6 Kinase 1.2 1 Structure of p70S6K- functional domains 20 1.2 2 Regulation and activation of p!0S6K 21 1.2 3 FRAP and 4E-BP1 25 1.2 4 Protein translation and the cell cycle 26 1.3 Photodynamic Therapy and Apoptosis 28 1.4 Rationale and Research Hypotheses 1.4 1 Rationale 31 1.4 2 Objectives 32 iv Chapter 2: Materials and Methods 2.1 Materials 33 2.2 Preparation of mature sea star oocyte cytosol 34 2.3 Enrichment of the 40S ribosomal S6 protein kinase 35 2.4 Kinase assays, autophosphorylation and protein assays 36 2.5 Electrophoresis, silver staining and immunoblotting 37 2.6 Gel filtration chromatography of sea star S6 kinase 38 2.7 Regulation of sea star S6 kinase by ions and inhibitors 39 2.8 Photoaffinity labelling of sea star S6 kinase with [y-32P]azido-ATP 39 2.9 Phosphoamino acid analysis 32.10 Two dimensional phosphopeptide mapping 40 2.11 Substrate specificity of sea star S6 kinase2.12 Protein micro-sequencing, alignment and identification of sequenced 41 fragments 2.13 Inactivation of sea star S6 kinase by phosphatase treatment 42 2.14 Immunoprecipitation 42.15 Immunoprecipitation of S6 phosphotransferase activity by anti-PP2A 43 antibody 2.16 Procedure for heat shock-treatment of sea star oocytes 43 2.17 Expression and purification of GST- S6 kinase from Baculovirus 44 2.18 Stimulation of GST-S6 kinase with PM A 45 2.19 Microinjection studies 42.20 Cell culture, stimulation and preparation of Pam212 cell lysates 47 2.21 In vitro PDT treatment and UV irradiation of Pam212 cell lysates 48 2.22 Insulin, EGF and anisomycin treatment of Pam212 cell lysates 48 2.23 In vitro lipid kinase assay 49 2.24 Statistical analysisv Chapter 3: Results and Discussion: p70s** in Pisaster ochraceus 3.1 Introduction and Model System 50 3.2 Identification and Enrichment of Active S6 Kinase from Mature 51 Sea Star Oocytes 3.2.1 Identification of S6 phosphotransferase activity in mature sea star 52 cytosolic extract 3.2.2 Enrichment of a 40S ribosomal protein S6 kinase from mature seastar 54 oocytes by sequential column chromatography 3.2.3 Immunological identification of sea star S6 kinase as a 63 homologueof pltf6* 3.3 Characterization of Active S6 Kinase from Mature Sea Star Oocyt 3.3.1 Immunological identification of partially pirrified sea star S6 kinase 67 3.3.2 Dose- and time-dependence of sea star S6 kinase against the S6 peptide 69 3.3.3 Regulation of sea star S 6 kinase by a variety of ions and inhibitors 71 3.3.4 Phosphorylation of 40S ribosomal proteins by sea star S6 kinase 73 3.3.5 Photoaffinity labelling of sea star S6 kinase with [732P]azido-ATP 73 3.3.6 Phosphoarriino acid and 2 dimensional phosphopeptide mapping of the 76 40S ribosomal protein S6 phosphorylated by sea star S6 kinase 3.3.7 Phosphotransferase activity of sea star S6 kinase against a variety of 78 exogenous substrates 3.3.8 Substrate specificity of sea star S6 kinase 82 3.3.9 Inactivation of sea star S6 kinase by dephosphorylation 85 vi 3.4 Complex Formation of Active Sea Star S6 Kinase 3.4.1 Identification of co-purifying proteins by micro-sequencing and 87 immunoblotting 3.4.2 Co-immunoprecipitation of sea star S6 kinase, HSP-60 and PP2A 90 3.5 Functional Studies of Sea Star S6 Kinase 3.5.1 Immunoprecipitation of active S6 kinase from immature and mature 94 sea star oocytes 3.5.2 Immunoblot analysis of heat shock-treated immature and mature 97 sea star extracts 3.5.3 Expression of active recombinant S6 kinase from baculovirus 99 3.5.4 Microinjection studies 101 vii Chapter 4: Results and Discussion: Photodynamic Therapy and it's Effect on the Phosphorylation and Activation State of p70 K 4.1 Introduction and Model System 4.2 Initial Observations: PDT Effect on p705<iJf 4.2.1 Effect of PDT on activation of p7056* 105 4.2.2 Dose- and time-dependence of PDT effect on p70w 107 4.3 Analysis of the PI 3-kinase/PKB/ p705<5Ar Pathway in PDT Treatment 4.3.1 Effect of insulin on PI 3-kinase, PKB and p7056* in serum-starved 110 Pam212 cells 4.3.2 Effect of PDT on insulin-induced activation of PI 3-kinase, PKB 112 and plQS6K in serum- starved Pam212 cells 4.3.3 Effect of UV irradiation on insulin-induced activation of PI 3-kinase, 117 PKB and p70S6* in serum-starved Pam212 cells 4.3.4 Effect of PDT on anisomycin-induced activation of p70S6^ 122 viii Chapter 5: General Discussion, Future Directions and Conclusions 5.1 Introduction 5.2 p!0S6K in Pisaster ochraceus 5.1.1 Identification of p52 as a homologue of p70 and regulation of the 126 kinase activity 5.1.2 Multi-protein complex formation 129 5.1.3 Functional aspects of p70S6K signaling 130 5.3 PI 3-kinase/PKB/ p70S6* Signaling in PDT 5.3.1 Effect of PDT 132 5.3.2 Specificity of the PDT effect 137 5.3.3 PDT in apoptosis 138 5.4 Conclusions Bibliography ix List of Tables Table Page 1. Summary of columns tested for S6 kinase purification 55 2. Enrichment of S6 kinase from mature sea star oocytes 60 3. Summary of various inhibitors of sea star S6 kinase 72 4. Summary of substrate specificity of sea star S6 kinase 83 5. Alignment and identification of sequenced fragments 88 6. Microinjection of purified human p70S6* into sea star oocytes 103 x Figure List of Figures Page 1. Essential components of the MAP kinase signaling pathways 8 2. Essential components of the PI 3-kinase/PKB/p7056^ signaling pathway 13 3. Structure and regulation of \)1QS6K 22 4. 1 -methyladenine-induced maturation of Pisaster ochraceus oocytes 51 5. Identification of S6 phosphotransferase activity in mature sea 53 star extract 6. Representative S6 kinase partial purification 57 7. Enrichment of a 40S ribosomal protein S6 kinase 58 8. Phosphorylation of 40S ribosomal proteins by sea star S6 kinase 62 9. Silver stain, immunoblot and autophosphorylation of sea star 64 S6 kinase 10. Gel filtration chromatography of sea star S6 kinase 66 11. Immunological identification of partially purified sea star S6 68 kinase 12. Concentration dependence and time course of the sea star S6 kinase 70 13. Concentration response of sea star S6 kinase with 40S ribosomal 74 proteins 14. Photoaffinity labelling of sea star S6 kinase with [y- P]azido-ATP 75 15. Phosphoamino acid and 2D phosphopeptide mapping of the 40S 77 ribosomal protein S6 phosphorylated by sea star S6 kinase 16. Phosphotransferase activity of sea star S6 kinase against a variety 79 of exogenous substrates 17. Radiography and phosphoamino acid analysis of exogenous substrates 80 18. 2D phosphopeptide map of MBP phosphorylated by sea star 81 S6 kinase xi 19. Inactivation of sea star S6 kinase by dephosphorylation 86 20. Immunological identification of PP2A and HSP-60 in sea star S6 kinase 89 preparation 21. Co-immunoprecipitation of sea star S6 kinase, HSP-60 and PP2 A 91 22. Phosphoamino acid analysis of p55 phosphorylated by sea star 93 V52S6K 23. Immunoprecipitation of active p70 from immature and mature 95 sea star oocytes 24. Immunoblot analysis of heat shock-treated immature and mature 98 sea star extracts 25. Expression of active recombinant S6 kinase 100 26. Microinjection of p7056^ into immature sea star oocytes 102 27. Effect of PDT on activation of p70S6A: 106 28. Dose response of PDT inhibition of p!0S6K activity 108 29. Time course of PDT inhibition of plQS6K activity 109 30. Insulin-induced activation of PI 3-kinase, PKB and p70 111 31. Effect of PDT on insulin-induced activation of PI 3-kinase 113 32. Effect of PDT on insulin-induced activation of PKB 114 33. Effect of PDT on growth factor-induced activation of p!0S6K 115 34. Effect of UV irradiation on insulin-induced activation of PI 3-kinase 118 3 5. Effect of UV irradiation on insulin-induced activation of PKB 119 36. Effect of UV irradiation on insulin-induced activation of p70 121 37. Effect of PDT on anisomycin-induced activation of p70 123 xu List of Abbreviations aa amino acid(s) ATP adenosine triphosphate BCIP 5-bromo-4-chloro-indolyl phosphate BME beta-mercaptoethanol BPD-MA benzoporphyrin derivative mon-acid ring A BSA bovine serum albumin CSAID cytokine-suppressive anti-inflammatory drugs DMEM Dulbecco's minimum essential medium DMF dimethylformamide DMSO dimethylsulfoxide DTT dithiolthreitol EDTA ethylenediaminetetraacetic acid (disodium salt) EGF epidermal growth factor EGTA ethylene bis(oxyethylenenitrilo) tetraacetic acid ERK1/2 extracellular signal regulated protein kinase 1/2 FBS fetal bovine serum FRAP FKBP-rapamycin associated protein GDP guanosine diphosphate GTP guanosine triphosphate Grb-2 growth receptor binding-2 GSK-3 glycogen synthase kinase-3 GST glutathione-S-transferase H2O2 hydrogen peroxide HSP27/60 27/60 kDa heat shock protein HTP hydroxylapatite IRS-1 insulin receptor substrate-1 INK c-jun amino-terminal kinase LPS lipopolysaccharide pg microgram pi microlitre pM micromolar mg milligram ml millilitre M molar MAPK/ERK mitogen-activated protein kinase/extracellularly-regulated kinase MAPKAP mitogen-activated protein kinase activated protein kinase MEK MAPK/ERK kinase MEKK MAPK/ERK kinase kinase MOPS morpholinopropanesulfonic acid NBT nitro blue tetrazolium ng nanogram run nanometer PAK p21 -associated protein kinase Xlll PAP potato acid phosphatase PBS phosphate buffered saline PDGF platelet derived growth factor PDK1 3-phosphoinositide-dependent protein kinase-1 PDT photodynamic therapy PI phosphatidylinositol PI(4,5)P2 phosphatidylinositol-4,5-bisphosphate PH pleckstrin homology PKA protein kinase A PKB protein kinase B PKC protein kinase C PKI peptide inhibitor of protein kinase A PLCy phospholipase C gamma pM picomolar PMA phorbol 12-myristate 13-acetate PMSF phenylmethylsulfonyl fluoride PP2A protein phosphatase 2A ROS reactive oxygen species rpm revolutions per minute SAPK stress-activated protein kinase SDS sodium dodecyl sulphate SH2/SH3 src homology 2/3 domain SOS son-of-sevenless TBS Tris buffered saline TOR target of rapamycin Tris tris(hydroxymethyl)aminomethane TTBS TBS containing 0.05% Tween 20 Tween 20 polyoxyethylene-20-sorbitan monolaurate UV ultraviolet xiv List of Amino Acid Codes Name Three letter code One letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glycine Gly G Glutamic Acid Glu E Glutamine Gin Q Histidine His H Isoleucine He I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Acknowledgements: I would like to thank my supervisor Dr. Steven Pelech for providing such a stimulating environment for the duration of my thesis. In spite of one or two upheavals, we always had access to the best of equipment and reagents, and interactions with top-notch collaborators and postdoctoral fellows in the laboratory. I would also like to thank my committee members, Dr. Frank Jirik, Dr. Roger Brownsey and Dr. Neil Reiner for support and guidance, and for allowing me to make my own decisions. I would like to acknowledge some of the many co-operative education students that have passed through our laboratory over the years for providing fun, laughs and general goofiness. Working with these students has helped me to maintain a sense of balance; their energy, youth and honesty are infectious. Thanks to (now) Dr. Faye Chow, Dr. Stephanie Buchanan, Angela Carter, Mikel Lefler (for the great tattoo experience), Graeme McLean (for egg McMuffms), Kirsteen Robbie (for morning runs). And, special thanks to Mark Sidney who reminded me just how much fun life can be, and for support and friendship. I would also like to thank my co-workers in the lab over the years, for unending support, guidance, technical help and generosity. To Harry Paddon, whose calming influence and ability to fix anything and everything kept the Pelech lab from self-destruction many times, Darren Daley (for giggles and martinis), Dan Leung just for being so Dan, Georgia Tai (for massages), Marketa Kosar (for wings and beer), Glen McNeil, Peggy Irwin and George Gill. Many thanks to Dr. Jasbinder Sanghera for help and support in the early years of my research, and to Dr. Bill Salh for his patience and advice in matters ranging from medical to philosophical to scientific. My fellow graduate students over the years have become like family, complete with squabbles, and eternal affection, Dr. Chrystal Palaty for her humor and creativity, Dr. Donna Morrison for her sanity and reason, Dr. David Charest for calm and balance, Venska Wagey, Sung Kim and Jaspal Girn (Boxer extrordinaire). I would also like to acknowledge and thank my collaborators over the last few years for keeping me going when my own project was floundering. Thanks to Michael Scheid, and Dr. John Jackson. Special thanks and gratitude to Dr. Jing-Song Tao for helping me to enjoy science again. The work we did together represents an important part of my thesis, and I look forward to more productive collaborations in the future. Finally, I'd like to acknowledge all my other friends and family who have put up with so much for so long. Thanks to Rick Kozak, Ron Glen and Claudine Copek for support. Heartfelt thanks to my dad, George Charlton, my father-in-law Tegryd Lloyd, and especially to my wonderful mother-in-law Donalda Lloyd who has given so much unconditional support, both emotional and financial. Lastly, thanks from the bottom of my heart to my husband Robert, the only person in the world who could have made it through the last ten years. See you all at the party xvi For my mom, and Robert, with love xvii Chapter 1: General Introduction The millions of cells that make up all living organisms are derived from a single fertilized cell. The genetic program within our DNA orchestrates the differentiation of cells into tissues and organs that respond to a vast variety of hormones and extracellular signals. All cells contain hundreds of enzymes that play key roles in the transmission and integration of signals from outside the cell. Protein kinases are among these key signaling molecules. These enzymes, along with protein phosphatases, mediate the reversible phosphorylation of target proteins, thus affecting the subsequent activity of these signaling components. The research contained in this thesis examined the role of a specific protein kinase, the 70 kDa S6 kinase (p70S6K). This enzyme mediates the onset of protein synthesis required for cell division to occur and as such has an important role in cell proliferation and cell survival. In the Introduction, I will briefly discuss the structure and function of protein kinases and the current state of knowledge in the field of signal transduction. I will then elaborate on the mechanisms by which p70S6K may be regulated, from receptors on the cell surface, via adaptor proteins and protein phosphorylation/dephosphorylation cascades. I will briefly discuss the models systems used in these studies, and present a rationale and research objectives for the work presented. 1 1.1 Signal Transduction Cells respond to a number of extracellular signals that mediate growth, survival, differentiation, proliferation, homeostasis or apoptosis. The signals are transmitted across the cell membrane, through the cytoplasm and into the nucleus, resulting in regulation of gene expression. Divergent signals are integrated, processed and relayed through the cell by thousands of different proteins. Reversible protein phosphorylation has become widely recognized as a major mechanism of signal transmission within the cell and protein kinase cascades form a highly integrated network through which signals received at the cell surface are transduced through the cytoplasm and into the nucleus where they mediate events required for growth and cell division. At least a third of all cellular proteins undergo phosphorylation, and although some of these phosphorylation events are likely without consequence, this covalent modification affects biological activity in many cases. Many diseases are the result of the disruption of signaling pathways, from mutations in receptors, constitutive activation or inhibition of protein kinases, or deregulation of other downstream signaling molecules and transcription factors. These diseases include proliferative disorders such as cancers (Aaronson 1991), as well as diabetes (Reaven 1988), amyotrophic lateral sclerosis (ALS) (Krieger et al, 1996), Huntington's disease (Tanaka et al, 1993), and Alzheimer's disease (Saitoh et al, 1991). Many mitogen and oncogene-regulated protein-serine/threonine kinases have been identified and characterized in a variety of cell systems, and the roles that these kinases play in signaling pathways are currently an active area of research. 2 1.1.1 Protein kinase structure and function Protein kinases are enzymes that add the gamma phosphate from adenosine triphosphate (ATP) to target proteins and enzymes. The phosphorylation event may activate or inhibit the activity of target proteins. In this way, protein kinases, and protein phosphatases which cleave the phosphate from target proteins, can transduce and integrate signals from outside the cell. All protein kinases share certain features required for their catalytic activity. A readily identifiable catalytic domain comprising about 250-300 amino acid residues, corresponding to about 30 kDa characterizes this family of proteins (Hanks and Quinn 1991). The homologous nature of the catalytic domains implies that they all fold into similar three-dimensional core structures, and catalyze the phosphotransferase reaction according to a common mechanism. Indeed, solution of the crystal structures of several protein kinases has demonstrated that the kinase domain folds into a two-lobed structure with the deep cleft between the lobes acting as the site of catalysis (Knighton et al, 1991A and B; DeBondtera/., 1993; Zhang etal, 1994). Much of the diversity of signaling through protein kinases stems from regulation of the activity and substrate specificity of the catalytic domain by other regions of the protein, or by other enzymes and co-factors. The location of the catalytic domain within the protein is not fixed, but it often lies near the carboxyl terminus while the amino terminus often plays a regulatory role. The two best described classes of protein kinases are those specific for serine/threonine and those that phosphorylate tyrosine residues. There is also evidence for a third class of protein kinases that are specific for histidine residues (Hanks and Quinn 1991). Although both serine/threonine and tyrosine kinase family members have very similar catalytic domain primary structures, certain short amino acid stretches can be used to predict the kinase specificity (Hanks 1987). Comparisons of the primary 3 sequences of kinase catalytic domains has allowed identification of eleven major conserved subdomains, separated by regions less well conserved. The conserved regions have been found to be important in regulating catalysis, either directly, as components of the active site, or indirectly by contributing to secondary structure and the formation of the active site. Non-conserved regions likely mediate secondary and tertiary structure, allowing essential conserved regions to come together. Twelve amino acid residues within the eleven subdomains have been identified that are nearly invariant (conserved in over 95% of known sequences), throughout the family members. According to the numbering used in the primary structure of protein kinase A (PKA), these are equivalent to Gly-50 and Gly-52 in subdomain I, Lys-72 in subdomain II, Gly-91 in subdomain III, Asp-166 and Asn-171 in subdomain VI, Asp-184 and Gly-186 in subdomain VII, Glu-208 in subdomain VIII, Asp-220 and Gly-225 in subdomain IX, and Arg-280 in subdomain XI. The sequences within subdomains VI, VIII and IX have been particularly well conserved among the individual members of the different families, and thus have been targetted most often in cloning strategies aimed at identifying new members. Subdomain I appears to play a role in anchoring the non transferable phosphates of ATP. The invariant Lys-72 in subdomain II is the best characterized cataytic domain residue and is essential for maximum kinase activity. This residue is required to anchor and orient ATP by interacting with the a- and P-phosphates and has been exploited to create catalytically inactive mutants of active kinases by altering this residue, usually to alanine. Subdomains VI and VII also play roles in ATP binding. Subdomain VIII contains the highly conserved Ala-Pro-Glu (APE) sequence that appears to play a major role in substrate recognition. In addition, many protein kinases are known to be activated by phosphorylation of residues in subdomain VIII. Subdomain VIII may maintain the kinase in an inactive state by occluding the substrate binding site, and the inhibition may be reversed by a conformational change in the kinase mediated either by autophosphorylation or phosphorylation by activating upstream 4 kinases. Protein kinases can be regulated by a variety of mechanisms, some of which will be discussed in more detail in the following sections. It is important to keep in mind that the criteria are by no means mutually exclusive, and an individual protein kinase may be regulated by one or more of these mechanisms. First, many protein kinases are regulated by second messengers such as cAMP, phosphatidylinositol (3,4,5) trisphosphate (PIP3), and Ca2+, that are generated in response to extracellular ligands binding to cell surface receptors. A common feature of this class of protein kinase is the presence of autoinhibitory or pseudosubstrate regions that maintain the kinase in an inactive conformation in the absence of second messenger. Second, some protein kinases (for instance, the cyclin-dependent protein kinases) are composed of catalytic and regulatory subunits which must interact for kinase activity. Third, some kinases (notably receptor tyrosine kinases) are activated by ligand-induced dimerization and cross-phosphorylation on tyrosine residues. Fourth, many protein kinases are themselves regulated by phosphorylation by upstream or downstream components of the signaling pathways, or by autophosphorylation. In addition, many kinases now appear to be regulated, at least in part, by subcellular localization, nuclear translocation, or by interaction with other signaling components such as small G-proteins. 1.1.2 Receptor-mediated signal transduction Cell growth and differentiation are regulated by a vast number of extracellular signals including growth factors, hormones, neurotransmitters and cytokines. The ligands interact with high-affinity receptors on the cell surface and the signal is perpetuated via signaling cascades through the cell cytoplasm to the nucleus. Heterotrimeric G-protein coupled receptors, receptor tyrosine kinases and cytokine receptors share the ability to transduce signals from the outside to the inside of the cell. These receptors utilize small guanine nucleotide-binding (G) proteins, cytoplasmic serine/threonine or tyrosine protein 5 kinases, lipid kinases, phospholipases and various adaptor proteins to relay and integrate the signal. Although the targets of the receptors may have quite different biochemical activities and biological functions, they often contain related sequences that mediate protein-protein interactions. These sequences include Src-homology-2 (SH2), Src-homology-3 (SH3), pleckstrin homology (PH) and C2 domains. The domains fold into functional modules independently of the rest of the protein and are found in many different signaling molecules. SH2 domains, first identified as modules contained in the Src tyrosine kinase, recognize short peptide motifs containing phosphotyrosine residues, while SH3 domains, also originally identified in Src, interact with proline-rich regions in target molecules (Anderson etal, 1990; Ren etal, 1993). The conserved features lie within more variable regions, allowing for specificity and the high affinity with which the individual SH2 or SH3 domains bind their target proteins. PH domains, first identified in pleckstrin, are less well-characterized, but interact with phospholipids and may serve to link signaling proteins with membranes (Harlan et al, 1994). The C2 domain is present in conventional and novel PKC isoforms. This domain has been shown to be responsible for binding both Ca2+ and acidic phospholipids (Sutton et al, 1995). As a number of proteins with C2 domains do not bind Ca , notably the novel PKCs, presumably this domain plays a further role in signaling via interaction with lipid molecules (reviewed in Newton 1995). Thus, binding of a growth factor to a specific receptor tyrosine kinase induces receptor dimerization and tyrosine cross-phosphorylation which in turn recruits SH2 and PH domain-containing proteins. Similarly, some members of the cytokine receptor superfamily, while not containing intrinsic tyrosine kinase activity, recruit cytoplasmic tyrosine kinases which relay the signal through many of the same molecules as above. Analogously, binding of ligand to G-protein coupled receptors leads to dissociation of Py subunits from a subunits upon exchange of GTP for GDP. The Py subunits subsequently 6 interact downstream with many of the same intracellular signaling molecules and adapter proteins. Whatever the mechanism of transmembrane signaling, it is becoming increasingly clear that the pathways converge on the same effectors and cytoplasmic signal transduction cascades, and may use functionally analagous mechanisms for the co ordination and integration of signaling. The transmission of signals appears to utilize specific protein-protein interactions to localize key signaling molecules to the appropriate subcellular location where they interact with substrates, effectors or other downstream components. Specifically, post-receptor signaling leads to activation of the small G-protein Ras and phosphatidylinositol 3-kinase (PI 3-kinase) and on to the activation of a number of cytoplasmic protein-serine/threonine kinases. Many of these kinases are arranged in cascades, one of which leads to activation of mitogen-activated protein kinase (MAP kinase) family members and another that results in activation of protein kinase B (PKB) and the 70 kDa S6 kinase ($7QS6K). 1.1.3 MAP kinase pathways The first MAP kinase cascade to be elucidated involves activation of the isoforms known as extracellular signal regulated protein kinase 1 and 2 (ERK1 and ERK2) following stimulation with mitogens such as epidermal growth factor (EGF) or platelet derived growth factor (PDGF) (reviewed by Cobb etal., 1991). One of the proteins that binds to activated receptor tyrosine kinases is the SH2 domain-containing adaptor protein Grb-2 (growth receptor binding-2). Grb-2 acts as a scaffold for binding other proteins through two SH3 domains that flank the SH2 domain (Schlessinger 1994) (Fig. 1). A proline-rich region of the guanine nucleotide exchange protein Son-of sevenless (SOS) associates with the SH3 domains of Grb-2, causing translocation to the membrane, and allowing interaction with Ras (Aronheim et al, 1994). 7 ;on I I I TEY TPY TGY ERK1/2 SAPK p38 Fig. 1. Essential components of the Map kinase signaling pathways: A schematic illustration of some of the key post-receptor signaling molecules. Three well-characterized MAP kinase pathways include activation of extracellular signal-regulated (ERK1/2) protein kinases via growth factor dependent signaling, activation of the stress-activated (SAPK) pathways, or activation of the p38 signaling pathway. The SAPK and p38 pathways are activated by overlapping stress agents such as UV irradiation, toxins, heat shock or oxidative stress. Known links are in solid lines, while possible connections are in dotted lines. See text for details. 8 Although the basic scheme of post-receptor activation of Ras is often depicted as a linear pathway, the reality is much more complex. For instance, although the PDGF and EGF receptors can bind Grb-2 directly, they also bind and phosphorylate other SH2-containing proteins such as She and the Syp tyrosine phosphatase, and Grb-2 itself can directly bind She and Syp. The activation of Ras is accomplished via the exchange of GDP for GTP, and Ras proteins contain an intrinsic GTPase activity that hydrolyzes GTP, thus abrogating the signal (Chardin etal, 1993; Gale et al, 1993). The primary effector of Ras signaling is the protein serine/threonine kinase Rafl. Ras-Raf 1 binding serves to localize Rafl to the plasma membrane where it is activated by as yet unclear events including phosphorylation and interaction with membrane lipids and certain isoforms of the 14-3-3 polypeptides (Marais etal, 1995; Vojtekera/., 1993). A number of other proteins have been shown to interact with Ras in a GTP-dependent manner, including the pi 10 subunit of PI 3-kinase and MEKK1, and it appears that Ras acts as a GTP-regulated switch recruiting a variety of signaling molecules to the membrane depending on the context of signals received at the cell surface. Thus, there is a complex network of interactions that integrate Ras activation with other signaling events and modulate downstream events. The mitogenic activation of Rafl leads to activation of MEK1/2 which in turn activates ERK1 and ERK2 via phosphorylation of tyrosine and threonine residues in a conserved TEY motif (Ann et al, 1992). Activated ERK1/2 translocates to the nucleus where it mediates phosphorylation of various transcription factors such as Elk-1, Etsl/2 and ATF-2 involved in gene transcription leading to proliferation, differentiation or survival (Marais et al, 1993; Coffered/., 1994). The three enzyme signaling module (Rafl—» MEK—> ERK1/2) is considered the archetype of at least three other signaling pathways that are conserved in organisms as diverse as yeast, sea star, amphibians, and mammals. 9 Cells respond not only to physiological cues leading to cell growth, differentiation or proliferation, but also to environmental stresses such as UV irradiation, metabolic toxins, osmotic or heat shock, and DNA damage. Environmental assaults of this type often lead to growth arrest or apoptosis in damaged cells. The signaling cascades that mediate the stress response are related to, but distinct from the ERK1/2 pathway, as these agents stimulate activation of the ERK isoforms only poorly, but potently activate the closely related stress-activated protein kinases (SAPKs, also known as c-Jun amino-terminal kinases (JNKs)). In addition, SAPKs are strongly activated by cytokines such as tumor necrosis factor alpha (TNFa) and interleukin-1 (IL-ip) (Minden et al, 1994; Kallunki et al, 1994; Slussera/., 1994). This pathway is composed of a distinct signaling module comprising MEKK1 phosphorylation of SEK1/MEK4 which activates the SAP kinases via phosphorylation of tyrosine and threonine residues in a TPY motif, analogous to the activation of ERK1/2 (Yan et al, 1994; Lin et al, 1995) . While Ras and Rafl are upstream activators of the ERK 1/2 signaling cascade, these proteins do not activate the SAPKs. The pathway is induced by a wide variety of agents, and it is likely that many of these act through independent targets. For instance, UV irradiation induces cysteine cross-linking leading to dimerization of receptors and other proteins. Inflammatory cytokines like IL-1 act via cell surface receptors that mediate their signals through activation of sphingomyelinase and production of the second messenger ceramide. Two Ras related GTP-binding proteins of the Rho family, Racl and cdc42 have been shown to activate SAPKs (Minden et al, 1995; Coso et al, 1995A; Brown et al, 1996). These two molecules do not activate MEKK1 directly but have been implicated in activation of p21-activated kinase-1, 2 and 65 (PAK1, PAK2 and PAK65) (Martin et al, 1995) leading to induction of the SAPK pathway (Coso et al, 1995B). Members of the PAK family bind GTP-Rac and Cdc42 and are activated by them in a GTP-dependent manner, however the details of this activation remain to be clarified (Martin et al, 1995; Manser et al, 1994). Racl and cdc42 have also been 10 shown to be involved in regulation of the actin cytoskeleton, cell adhesion, morphology, motility and cytokinesis, although the precise signaling pathways remain to be elucidated (reviewed by Amano et al, 1998). Activated SAPKs also translocate to the nucleus where they phosphorylate transcription factors such as c-Jun and ATF-2, leading to the transcription of genes that mediate growth arrest or apoptosis. c-Jun is a component of the AP-1 transcription factor, which is composed of homo- and hetero-dimers of c-Jun and Fos. c-Jun is basally phosphorylated in the carboxyl terminus, and is transcriptionally inactive in resting cells. In response to mitogenic stimulation, c-Jun becomes dephosphorylated on these sites, allowing interaction with Fos and the activation of transcription. A different set of amino terminal residues are the target for phosphorylation by SAPKs. These sites (Ser-63 and Ser-73) lie near the 5 transactivation domain and their phosphorylation by SAPK's greatly enhances the transactivation activity of these proteins in response to stress-related agonists (Franklin et al, 1993). The existence of multiple MAP kinase pathways in yeast led to the search for novel mammalian components of these signaling pathways and a mammalian homologue of a yeast protein involved in the response to high osmolarity (HOG1) was cloned. The mammalian kinase, p38 is relatively poorly characterized but appears to be activated by similar agonists as the SAPKs. However, p38 is not activated by SEK1, but is activated by MEK3/6 phosphorylation on tyrosine and threonine residues in a TGY motif (Han et al, 1996; Raingeaud et al, 1996). Many of the transcriptional events mediated by this pathway appear to be via the phosphorylation and activation of MAPKAP kinase-2 which is a direct target of p38. MAPKAP kinase-2 phosphorylates the same sites in both CREB and ATF-2 that are targeted by protein kinase A (PKA). The p38 family is activated by lipopolysaccharide (LPS), a known cytokine inducer, and is potently inhibited by a series of bio-active, anti-inflairimatory pyridinyl-imidazole compounds called cytokine-suppressive anti-inflammatory drugs (CSAIDs). Since p38 is also activated by 11 inflammatory cytokines, it is likely to be involved in the induction and response to these agonists. The use of these specific inhibitors and activators should lead to a more complete characterization of the p38 pathway. Thus, the MAP kinase isoforms are grouped into subfamilies based on sequence similarity, mechanism of upstream regulation, and activation by different MEKs. Most MAPK family members require phosphorylation on tyrosine and threonine in specific motifs, but ERK3 lacks this dual phosphorylation motif, and lies in an as yet uncharacterized pathway (Cheng et al, 1996). More than a dozen MAPK family members have now been cloned, and the variation in specificity within the signaling modules may result from integration of different extracellular and upstream cues. 1.1.4 PI 3-kinase /PKB/p70 S6 kinase pathways Another major signaling cascade that has been described entails signaling through phosphatidylinositol 3-kinase (PI 3-kinase), and on to the activation of protein kinase B (PKB) and the 70 kDa S6 kinase (p7056*) (Fig. 2). A role for activation of the PI 3-kinase pathway has been established in mitogenic signaling, intracellular vesicle trafficking, and cytoskeletal regulation (Carpenter and Cantley 1996). Activation of PI 3-kinase and PKB inhibits the induction of apoptosis by serum withdrawal (Kennedy et al, 1997), ultraviolet irradiation (Kulik et al, 1997), and the c-myc protein (Kaufmann-Zeh et al, 1997), implying a role in transducing survival signals. Membrane lipids not only play a structural role in maintaining membrane integrity, but are also actively involved in signaling processes as second messengers. One of the best characterized examples is the hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) by phospholipase Cy (PLCy), giving rise to diacylglycerol and inositol (l,4,5)-trisphosphate (IP3), which causes release of calcium from intracellular stores and protein kinase C (PKC) activation (Divecha and Irvine 1995). The PI 3-kinase 12 Fig. 2. Essential components of the PI S-kinase/PKB/pVO^ signaling pathway: A schematic illustration of some of the key post-receptor signaling molecules. PI 3-kinase activates PDK1 which phosphorylates and activates PKB. PI 3-kinase is also upstream of FRAP/TOR, but the interactions are undefined. Known links are in solid lines, while possible connections are in dotted lines. See text for details. 13 family members phosphorylate the hydroxyl group at the 3' position on the inositol ring and convert phosphatidylinositol (PI), PI(4)P, and PI(4,5)P2 to PI(3)P, PI(3,4)P2, and PI(3,4,5)P3, respectively, within seconds to minutes in response to mitogenic stimulation. These products are not substrates for PLCy and are now known to act as second messengers downstream of PI 3-kinase. PI(3,4)P2 and PI(3,4,5)P3 interact with protein targets and modulate their cellular localization and/or activity. Protein modules, such as PH and C2 domains that can bind lipids, are found in many of the downstream molecules that are modulated by the PI 3-kinase lipid products (Shaw 1996; Salim et al, 1996). Functional analysis of the various isoforms of PI 3-kinase has been facilitated by the existence of a number of specific inhibitors, as well as the availability of dominant negative or constitutively active mutants. One of the most commonly used inhibitors is the fungal metabolite, wortmannin, which inhibits PI 3-kinase at nanomolar concentrations by binding covalently to the pi 10 catalytic subunit (Yano et al, 1993). Another inhibitor, LY294002 acts at micromolar concentrations by competitive inhibition of the ATP binding site (Vlahos et al, 1994). These inhibitors and various mutants have helped to clarify not only the activation of the different isoforms of PI 3-kinases, but have proved to be useful reagents for analysis of the downstream signaling components of this family of lipid kinases. There are multiple isoforms of PI 3-kinases, belonging to different families depending on substrate specificity and mechanism of activation. The class I enzymes are heterodimeric proteins composed of separate regulatory and catalytic subunits that utilize PI, PI(4)P and P(4,5)P2 as substrates. The 110-kDa catalytic subunits, pi 10a and pi 1 Op both bind 85-kDa regulatory subunits and are stimulated by both receptor and nonreceptor protein tyrosine kinases. The p85 regulatory subunits contain an amino terminal SH3 domain followed by a proline-rich sequence, a breakpoint cluster region (BCR) homology domain, a second proline-rich domain, and two SH2 domains (Pleiman et al, 1994; Klippel et al, 1993). The regulatory subunits contain putative coiled-coil domains that 14 mediate stable dimerization with the pi 10 catalytic subunit which contains a p85 binding region, a ras-binding domain, and the catalytic core (Dhand et al, 1994). Regulation of the PI 3-kinase is complex, likely involving a number of second messengers and other signaling components. The pi 10 subunit has been shown to bind and be activated by ras-GTP in vitro (Kodaki et al, 1994). The p85 SH3 domain binds to proline-rich peptides while the p85 proline-rich sequences bind to the SH3 domains from Fyn and Lyn (Booker et al, 1993). The BCR homology domain binds to GTP-bound Cdc42, and the SH2 domains bind phosphotyrosine containing proteins (Songyang et al, 1993; Zheng etal, 1994). Each of these binding events activates PI 3-kinase in vitro, but it is unclear whether the interactions are redundant or additive (Pleiman et al, 1994; Zheng et al, 1994). Very little is known about negative regulation of the PI 3-kinase isoforms. The family members, in addition to their lipid kinase activity, possess an intrinsic protein serine/threonine kinase activity. Autophosphorylation of PI 3-kinase on serine has been shown to down-regulate it's lipid kinase activity (Carpenter et al, 1993; Dhand et al, 1994B). A recent report demonstrated that c-Abl, a cytoplasmic tyrosine kinase that plays a role in DNA damage-induced apoptosis (Yuan et al, 1997A), binds the p85 subunit and inhibits PI 3-kinase activity in vitro and in irradiated cells (Yuan et al, 1997B). Since transforming variants of c-Abl activate PI 3-kinase (Varticovski et al, 1991; Skorski et al, 1995), c-Abl may play a role in the regulation of cell fate by down-regulation of PI 3-kinase. There is controversial evidence implicating a role for PI 3-kinases in activation of the various MAP kinase pathways. The class I family of PI 3-kinases (pi 10a-p85a) have been shown to interact with Ras in a GTP-dependent manner (Kodaki et al, 1994; reviewed by Marte and Downward 1997). Several direct targets of the lipid products of PI 3-kinase have been identified that may also link this pathway to MAP kinases. Members of the calcium-independent, novel PKC family, PKCs, PKC8, and PKCr| have 15 been shown to be activated in vitro by PI(3,4)P2 and PI(3,4,5)P3 (Toker et al, 1994), and these PKC isoforms have been implicated in activation of the MAP kinase pathway via phosphorylation of Rafl (Kolch etal, 1993; Troppmair et al, 1994). Another report has shown that a membrane-targeted form of PI 3-kinase can activate SAPK/JNK, and that a dominant negative mutant of PI 3-kinase can block EGF-induced activation of JNK (Logan et al, 1997). Treatment with wortmannin indicates that the effect of PI 3-kinases on the MAP kinase pathway may be cell- and tissue-specific and may depend on the strength of the signal (Duckworth and Cantley 1997). Interpretation of this data requires caution, however, as wortmannin may not be specific for PI 3-kinase and may in fact directly inhibit other enzyme activites including the FKBP binding protein FRAP (also known as target of rapamycin (TOR)), MAP kinase, and p7056A: (Cheatham et al, 1994; Brunn etal, 1996; Brunn etal, 1997). One of the first targets of PI 3-kinase to be identified was the product of the retroviral oncogene v-akt, also known as protein kinase B (PKB). The kinase is activated by mitogens and growth factors in a PI 3-kinase-dependent manner and is inhibited in response to agents that block PI 3-kinase. Nevertheless, PKB can also be activated by PI 3-kinase-independent pathways (Konishi et al, 1996). PKB contains an amino terminal PH domain, a central catalytic core and a carboxyl terminal regulatory domain. The PI(3,4)P2 generated by PI 3-kinase directly binds to the PH domain of PKB and activates its phosphotransferase activity (Freeh et al, 1997). Franke et al (1997A) also demonstrated that binding of the PH domain to PI(3,4)P2 led to dimerization of PKB and subsequent activation of the kinase. PKB activity is also regulated by phosphorylation, and the in vivo phosphorylation sites have been identified as Thr-308 and Ser-473 (Franke et al, 1997B). Thr-308 lies in the activation loop between subdomains VII and VIII of the catalytic domain, nine residues upstream of the APE motif that is conserved in protein kinases. This is a similar position to the activating phosphorylation sites found in many other protein kinases, including the 16 MAP kinase isoforms. Ser-473 is close to the carboxyl terminus in a motif found in other kinases that are activated by second messengers (Pearson et al, 1995). Both sites require phosphorylation for PKB to be fully active (Alessi et al, 1996). A novel PH domain-containing protein, 3-phosphoinositide-dependent protein kinase-1 (PDK1) has recently been detected and purified (Alessi etal, 1997B). PDK1 is activated specifically by PI(3,4)P2 and PI(3,4,5)P3 and activates protein kinase B (PKB) via phosphorylation of Thr-308. It is possible that reports showing that PKB is activated directly by PI(3,4)P2 may have been the result of contamination of PKB preparations with trace PDK1 activity. PDK1 was recently cloned by two groups, and its function has been further clarified (Stephens et al, 1998; Alessi et al, 1997A and B). PDK1 also contains a PH domain that tightly binds PI(3,4,5)P3, although the enzyme is constitutively active, and is not stimulated by insulin nor inhibited by wortmannin. The current model is that the PH domain of PKB normally restricts access of PDK1 to Thr-308, but is displaced when PKB binds PI(3,4,5)P3, allowing phosphorylation to take place. PI 3-kinase is necessary for the translocation of PKB to the membrane and the phosphorylation of Ser-473. A recent study reported that Ser-473 was phosphorylated in vitro by the integrin-linked kinase (ILK), and that a kinase-deficient form of ILK severely inhibited Ser-473 phosphorylation in vivo (Delcommenne et al, 1998). Thus, the regulation of PKB is complex and is mediated by multiple components. It is clear that an intact PH domain is absolutely required for activation and may cause translocation of the kinase to the plasma membrane where it can be further activated by phosphorylation. Indeed, altering the subcellular localization of PKB by attaching the myristoylation/ palmitoylation motif from the Lck tyrosine kinase (m/p-PKB), led to a 60-fold increase in PKB activity that could not be further stimulated by growth factors (Andjelkovic et al, 1997). The m/p-PKB was found to be phosphorylated on Thr-308 and Thr-473 in the absence of mitogenic stimulation, and the phosphorylation and activation did not require the 17 presence of an intact PH domain, supporting the model of the PH domain serving as a membrane-targeting module. PKB can also be activated by cellular stresses such as heat shock and hyperosmolarity, both of which activate the p38/HOGl pathway via a wortmannin-insensitive (ie. PI 3-kinase independent) pathway (Konishi et al, 1996). Heat shock induces association of PKC8 with the PH domain of PKB, and the kinase also associates with PKC<^ (Konishi et al, 1994). PKB is also activated by oxidative and chemical stressors, and associates with a small heat shock protein (HSP-27) in response to these stresses (Konishi et al, 1997). Overexpression of HSP-27 in embryonic stem cells has been shown to lead to differentiation, while underexpression caused apoptosis in these cells (Mehlen et al, 1997). HSP-27 is known to be phosphorylated by MAPKAP kinase-2 in response to cellular stresses (Ciocca et al, 1993), but the relationship between MAPKAP kinase-2, HSP-27, PKB and various PKC isoforms is as yet unclear. Heat shock has also been shown to activate PI 3-kinase, c-Src, and plQS6K in NIH 3T3 fibroblasts (Lin et al, 1997). Until recently, the only known in vivo substrate for PKB was glycogen synthase kinase-3 (GSK-3). GSK-3 phosphorylates and inactivates glycogen synthase (GS) in the absence of insulin stimulation. Upon insulin stimulation, PKB phosphorylates GSK-3 on a single regulatory amino-terminal serine residue in a PI 3-kinase dependent manner (Cross et al, 1995). This phosphorylation inhibits GSK-3 activity, resulting in the dephosphorylation and activation of several metabolic and gene-regulatory proteins including GS, involved in the insulin response (reviewed by Welsh et al, 1996). GSK-3 also phosphorylates and inactivates the initiation factor eIF2B, and this inhibition is abolished with insulin treatment, leading to the onset of protein synthesis (Welsh and Proud 1993). It has long been known that activation of PI 3-kinase and PKB promotes cell survival and blocks apoptosis, but the signals acting downstream have been difficult to 18 characterize. Apoptosis is a highly ordered mechanism by which cell death is induced in development, oncogenesis and the immune response (Gerschenson and Rotello 1992). Two families of proteins appear to play an essential role in the induction of apoptosis. The first is the family of cysteine proteases related to the interleukin lp-converting enzyme (ICE) (Martin and Green 1995). Activation of the ICE protease cascade is a necessary event in the execution of apoptosis. The second family is the Bcl-2 family, members of which can either promote or inhibit apoptosis. The ratio of death-promoting (ie. Bax, Bad) to death-inhibiting (ie. Bcl-2, BC1-XL) factors determines the ultimate fate of the cell (Oltvai and Korsmeyer 1994). The mechanism by which Bcl-2 members function is not well understood, but may include the formation of ion channels and regulation of the ICE protease cascade (Stellar 1995). PKB phosphorylates a member of the Bcl-2 family (Bad), at Ser-136 in vitro and in vivo inhibiting Bad-mediated death induction (Datta et al, 1997; del Peso et al, 1997). Bad resides in the cytosol, and phosphorylation by PKB leads to its sequestration by the tau form of 14-3-3 proteins, inhibiting its interaction with BC1-XL and therefore promoting cell survival (Zha et al, 1996). The ability of PKB to promote cell survival may reflect its oncogenic potential. PKB can synergize with the Rafl/MAP kinase pathway to cause transformation of NIH 3T3 cells (Marte et al, 1997) and may also drive cell-cycle progression through induction of E2F transcriptional activity. In Ras-transformed epithelial cells, the ability of the Ras oncogene to promote cell-survival is mediated through a PI 3-kinase/PKB-dependent pathway (Khwaja et al, 1997), and it is likely that PKB plays a role in mediating survival signals in human cancers resulting from mutation and activation of Ras. Several other proteins are known to lie downstream of PI 3-kinase and PKB though the precise interactions between them are unclear. These include a PI 3-kinase family member, the FKBP binding protein (FRAP, also known as target of rapamycin (TOR)), the elongation initiation factor binding protein, 4E-BP1 (also known as PHAS-19 1), and p70 . Much of the detail regarding these proteins has been established by use of the immunosuppressant drug rapamycin which blocks activation of FRAP, 4E-BP1 and plQS6K by all known stimuli and causes cell cycle arrest by delaying or abrogating the GI phase of the cell cycle. Agents such as wortmannin and LY294002 that inhibit PI 3-kinase and PKB also block activation of p70S6K (King et al., 1997; Cheatham et al., 1994), providing a framework to identify other components of this signaling pathway. 1.2 70 kDa S6 kinase Hyperphosphorylation of the 40S ribosomal protein S6 is one of the most dramatic and consistent responses of many cell types to a variety of mitogenic stimuli. The sites in the S6 protein are phosphorylated sequentially in vivo in the order Ser-236>Ser-235>Ser-240>Ser-244>Ser-247 (Krieg etal., 1988; Martin-Perez and Thomas 1983). The enzyme responsible for phosphorylation of the S6 protein, the mitogen-activated 70-kDa S6 kinase (p70S6K) plays a key role in cellular growth control mechanisms by coordinating protein synthesis via regulation of the S6 protein and the activity of 4E-BP1. The p70S6K also plays an important role in the progression of cells from GI to S phase of the cell cycle (Chung et al, 1992). Due to the role this enzyme plays in cell cycle control and the initiation of protein synthesis, much attention has focused on the key question of how the kinase activity is regulated. 1.2.1 Structure of p70S6K - functional domains The p70S6K has been purified or cloned and sequenced from a variety of sources including rat liver (Price et al, 1989; Kozma et al, 1989; Kozmaera/., 1990), vanadate-stimulated Swiss 3T3 cells (Jeno et al, 1989), and rabbit liver (Harmann and Kilimann 1990). The two isoforms, p70 andp85 are generated from alternative 20 translation sites of the same transcript (Grove et al, 1991; Reinhard et al, 1992) and are S6K identical except for a 23 amino acid extension present at the amino terminus of the p85 isoform. Although most researchers use the p70S6K form when describing the regulatory residues, some investigators use the pS5S6K isoform, adding 23 amino acids to the Of. is numbering system (Alessi et al, 1998). The extension targets the p85 form to the nucleus while the p70S6K form is predominantly cytoplasmic. The nuclear form likely plays a key role in the regulation of cell growth, as the S6 protein is present in free form in the nucleus and becomes phosphorylated at the same sites in response to mitogenic stimulation as the cytoplasmic form (Franco and Rosenfeld 1990). Since protein synthesis does not take place in the nucleus, the function of phosphorylated S6 protein in the nucleus remains obscure. The enzyme contains a single catalytic domain most closely related to the amino terminal catalytic domain in p90rsk and to second-messenger-regulated kinases. In addition, p70 contains an amino terminal acidic domain, and a pseudosubstrate sequence resembling the region in S6 that is phosphorylated in response to mitogenic stimulation (Fig. 3A). The pseudosubstrate sequence lies on the carboxyl side of the catalytic domain and acts as an autoinhibitory domain, possibly by interaction with the amino terminal acidic domain (Banerjee et al, 1990; reviewed by Pullen and Thomas 1997). This region contains four proline-directed mitogen-induced phosphorylation sites. The kinase also contains a number of in vivo phosphorylation sites in the kinase domain and linker region, that are flanked by large, aromatic amino acids. The regulation and activation of p7QS6K is complex, requiring multiple phosphorylation events and interactions with additional proteins. 21 A. T-229 Kinase T-389 S-404 1 1 Pseudosubstrate i\—i—r S-411S-418 T-421S-424 B. Pseudosubstrate Kinase C. Proline-directed kinases Kinase Pseudosubstrate I I I S-411S-418 T-421S-424 P P P P D. PI(3,4,5)P3-dependent kinase (ILK?) P T-389 Kinase | Pseudosubstrate 5-411S-418 T-421S-424 P P P P E. PDK1 P T-229 P I T-389 Kinase 1 Pseudosubstrate I I I r S-411S-418 T-421S-424 P P P P Fig. 3. Structure and regulation of p70S6K: A schematic drawing of the S6 kinase showing regions essential for catalytic activity. The various domains and phosphorylatable sites are shown (A). The kinase is maintained in an inactive form by interaction of the pseudosubstrate region with the kinase domain (B). The kinase is activated by multiple phosphorylation events by more than one kinase, on proline-directed sites (C), and by PI(3,4,5)P3 dependent kinases (D, E). See text for details. 22 1.2.2 Regulation and activation of p70' In all types of quiescent cells p70 is basally phosphorylated and mitogenic stimulation induces increased phosphorylation on multiple sites leading to activation of its phosphotransferase activity. Four of the basally phosphorylated, mitogen-induced sites (Ser-411, Ser-418, Thr-421 and Ser-424) lie in the carboxyl terminus within the pseudosubstrate/autoinhibitory domain (Ferrari et al, 1992). Mutation of these residues to alanine inhibits activation of the kinase (Han et al, 1995). Mutation of these sites to acidic residues increases kinase activity (Ferrari et al, 1993) but not fully, supporting the notion that other phosphorylation events and interactions are required for full activation. Although the mitogen-induced sites contain recognition determinants for proline-directed kinases, MAP kinase fails to activate p7QS6K after phosphorylation of these sites in vitro (Mukhopadhyay et al, 1992), and it is now well established that p70S6K activation does not lie on the Ras->Raf->MEK->-MAP kinase pathway (Ballou et al, 1991). A growing body of evidence has implicated PKC, PI 3-kinase and PKB as upstream signaling molecules in p70S6K activation in insulin-, PDGF-, EGF-, and IL2-treated cells (reviewed by Chou and Blenis 1995). The immunosuppressant drug rapamycin, a bacterial macrolide, has become one of the most important and useful tools for dissecting the pathway leading to p70S6K activation and its regulation. Rapamycin blocks activation of p70S6K by all known mitogens by preventing the phosphorylation of a specific subset of sites including Thr-229, Thr-389, Ser-404 and Ser-411 (Ferrari et al, 1993). The rapamycin-sensitive sites are not basally phosphorylated but become highly phosphorylated in response to mitogenic stimulation. Except for Ser-411, these sites are flanked by large, aromatic residues, and the principal inhibitory site has been identified as Thr-389 in the linker region coupling the catalytic and autoirihibitory domains (Pearson et al, 1995). This residue is highly conserved among kinases of the second messenger family and is 23 analogous to Ser-473 in PKB. Mutation of Thr-389 to an acidic amino acid (T389E) confers high basal activity and rapamycin resistance on the kinase (Pearson et al, 1995). Mutation of this residue to alanine (T389A) fully inactivates the kinase (Pearson et al, 1995). Thr-229 is also a highly conserved residue, analogous to Thr-308 in PKB, that lies within the kinase activation loop and is essential for phosphotransferase activity (Pearson etal, 1995; Wenge/a/., 1995A). Emerging evidence indicates that Thr-389 phosphorylation modulates Thr-229 phosphorylation (Pullen and Thomas 1997). The T389E mutant elevates Thr-229 phosphorylation while T389A blocks it. Mutation of Thr-229 to alanine or glutamic acid ablates kinase activity and does not potentiate Thr-389 phosphorylation (Pearson et al, 1995). Since p70S6* and PKB are distantly related, Alessi et al (1997C) and Pullen et al. (1998) tested whether PDK1 could phosphorylate PJQS6K at Tnr_229 and found that indeed it could. Furthermore, they found that a dominant negative form of PDK1 blocked activation of p70 in cells. The small Rho family G proteins, rac-1 and cdc42 can also bind to the hypophosphorylated form of p70S6K in vitro, in a GTP-dependent fashion, and GTPase deficient forms can activate p70S6K activity in vivo (Chou and Blenis 1996). Nevertheless, it is not yet known if these molecules interact directly with p70S6K or if other signaling intermediates are required. Although there has been considerable difficulty in identifying the upstream kinases that phosphorylate Thr-389 and Thr-229, as well as other interacting proteins, a model of the coordinated and heirarchical activation of p70S6K has been proposed (Pullen and Thomas 1997; Downward 1998). In its hypophosphorylated form, the pseudosubstrate domain is thought to bind to the amino terminal, and occlude the substrate binding site (Fig. 3B). Deletion of the amino terminal prevents kinase activation and also prevents phosphorylation of Thr-229 and Thr-389 in response to mitogenic stimulation (Weng et al, 1995B; Dennis et al, 1996). The deletion can be rescued by removal of the pseudosubstrate region (Pearson et al, 1995; Dennis et al, 24 1996) or by mutation of the proline-directed sites in this region to acidic residues (Pullen and Thomas 1997). In both instances, the rescue is accompanied by phosphorylation of Thr-389 and Thr-229 (Pearson etal, 1995; Dennis etal, 1996). These data indicate that the amino terminal and the pseudosubstrate region cooperate in signaling to Thr-389, and deletion of the amino terminal may prevent binding of a necessary effector molecule such as rac-1 or cdc42. Thus, p!0S6K activation may proceed by binding of an effector molecule at the amino terminal which releases the interaction with the pseudosubstrate region. This conformation would then be stabilized by phosphorylation of the proline-directed sites in this region (Fig. 3C). Subsequent phosphorylation of Thr-389 by an as yet unidentified PI(3,4,5)P3-dependent kinase (possibly PDK2 or ILK) (Fig. 3D) and phosphorylation of Thr-229 by PDK1 (Fig 3E) would lead to full activation of the kinase phosphotransferase activity. The similarities in the regulation of PKB and p!0S6K are striking. In the inactive state, both are maintained in a conformation precluding access by the constitutively active PDK1. Binding of PI(3,4,5)P3 to the PH domain of PKB causes a conformational change, while phosphorylation of the pseudosubstrate region of p70S6/f by proline-directed kinases and subsequent phosphorylation of Thr-389 enable PDK1 access to its target site. 1.2.3 FRAP and 4E-BP1 Rapamycin does not directly interact with p70 , but binds to FK506-binding protein 12 (FKBP12) and the complex interacts with FRAP/TOR upstream of p70S6/r (Brown etal, 1994; Sabatini etal, 1994). FRAP is structurally related to the catalytic subunit of PI 3-kinase and DNA-dependent protein kinases (Keith and Schreiber 1995), and is thought to function as a lipid or protein kinase (Hunter 1995). Mutant FRAP proteins which lack the ability to bind FKBP/rapamycin complexes are resistant to the effects of rapamycin (Stan et al, 1994; Browne/ al, 1995). These mutant forms of 25 FRAP confer at least partial rapamycin resistance to p70 , and a further mutation that inactivates the FRAP catalytic domain abrogates the rescue of p!0S6K (Brown et al, 1995). The same FRAP mutants can also protect 4E-BP1 from rapamycin-induced dephosphorylation and such protection again requires the presence of an active catalytic domain (Hara et al, 1997). Rapamycin-induced p!0S6K dephosphorylation and inactivation are paralleled by dephosphorylation of S6 (Chung et al, 1992) and 4E-BP1 (Beretta et al, 1996). In normal resting cells, 4E-BP1 is tightly bound to the elongation initiation factor eIF4E, inhibiting cap-dependent translation (Pause et al, 1994). In response to mitogenic stimulation, 4E-BP1 becomes phosphorylated on five proline-directed sites in a rapamycin-dependent manner (Fadden et al, 1997). The phosphorylation of 4E-BP1 leads to its release from eIF4E, allowing the initiation factor to then interact with the eIF4G subunit of the mRNA cap-binding protein complex (Mader et al, 1995). The p!0S6K does not phosphorylate either the free or eIF4E-bound form of 4E-BP1 in vitro and is likely not involved in this event in intact cells (Diggle et al, 1996). It has recently been shown that anti-FRAP immunoprecipitates contain kinase activity that can phosphorylate the proline-directed sites in free 4E-BP1, but it is not yet clear whether this activity can also phosphorylate 4E-BP1 bound to eIF4E, as must occur in vivo (Brunn et al, 1997). Other evidence indicates that the rapamycin-dependent signaling events immediately upstream of 4E-BP1 and p!0S6K are distinct, but require a common proximal activator (von Manteuffel et al, 1997). Several studies have reported dissociation of plQS6K from the known components of the PI 3-kinase pathway. PI 3-kinase, but not plbS6K, is required for cytokine-regulated hemopoietic cell survival (Scheid et al, 1996), and PKB, but not p70 , mediates growth-factor induced neuronal cell survival (Dudek et al, 1997). Activation of PKC by phorbol ester treatment leads to activation of p!0S6K in a PI 3-kinase-independent manner (Chung et al, 1994), and in a PKB-independent manner (Reif et al, 1997). 26 1.2.4 Protein translation and the cell cycle The translation of mRNA into protein is a complex process, controlled at multiple levels. Translation initiation, which involves recognition of the capped mRNA, binding of ribosomal subunits, and selection of the start codon, is generally the rate-limiting step (Pain 1996) . The phosphorylation of initiation factors and ribosomal proteins modulates the rate of initiation and plays a central role in the control of protein synthesis (Proud 1992). Rapamycin treatment has been shown to significantly inhibit translation of a select group of genes, including those encoding ribosomal proteins (S3, S6, S14 and S24), translation elongation factors (eEFl A and eEF2), and a secreted peptide growth factor, insulin-like growth factor II (IGF-II) (Terada et al, 1994; Neilsen et al, 1995; Jefferies and Thomas 1994). The inhibition is due to structural elements in the 5' untranslated region (UTR) of certain genes, and reflects the specific nature of the inhibition (Jefferies et al, 1994). This class of mRNA transcripts contains an oligopyrimidine tract at the transcriptional start site (5'-TOP ) which plays a key role in the regulation of their translation (Levy et al, 1991). These mRNAs undergo translational up-regulation in response to stimulation leading to activation of initiation and elongation factors, as well as increasing synthesis of ribosomal proteins and other ribosomal components for enhanced translational activity. The 40S ribosomal protein S6 may play a role as it lies in the mRNA-binding site of the ribosome, and is phosphorylated in a rapamycin-sensitive manner (Jefferies and Thomas 1996). S6 remains the only established physiological substrate for plt)S6K with mitogenic stimulation leading to multiple, ordered phosphorylation of the S6 protein at five serines located in the carboxyl terminus (Bandi et al, 1993; Krieg et al, 1988). Thus, dominant negative and rapamycin-insensitive mutants of p70S6K have been used to examine the role of the kinase in the control of 5'-TOP mRNA translation (Jefferies et al, 1997) . Expression of the dominant negative mutant blocked activation of wild-type plOS6K, as well as the activation of 5'-TOP mRNA translation by serum. The expression of the rapamycin-27 insensitive T389E mutant also caused 5'-TOP mRNA translation to become insensitive to rapamycin treatment. These results support the likelihood of a role for p70S6/f in regulation of 5'-TOP mRNA translation, though the details remain to be clarified. P~JQS6K ajgo piavs an imp0rtant role in cell cycle control but the details are unclear. Rapamycin causes cell cycle delay or arrest by prolonging or abrogating the GI phase of the cell cycle. In spite of the fact that rapamycin totally abolishes p70S6K activity in all cell types examined, its effect on GI progression varies with cell type. Complete blockage occurs in mature T-cells (Calvo et al, 1992), but only some GI delay occurs in NIH 3T3 cells (Morice et al, 1993). The variation in effect on GI progression is parallelled by changes in cyclin E and A expression (Morice et al, 1993) and rapamycin treatment in at least some cell types inhibits the activation of specific cyclin-dependent kinases (Nourse et al, 1994). Thus, the importance of p70 for cell cycle progression may be at least partially cell- and tissue-dependent. p!0S6K function is required for GI progression and the kinase activity remains high throughout GI (Lane et al, 1993). Microinjection of antibodies directed against p70S6K abolished serum-induced entry into S phase of the cell cycle, and abrogated the activation of protein synthesis (Lane et al, 1993). Microinjection of neutralizing antibodies specific for the p85 isoform into the nucleus can block cell cycle progression and this block can be reversed by co-injection of the p70S6K isoform (Reinhard et al, 1994). 1.3 Photodynamic Therapy and Apoptosis Photodynamic therapy (PDT) is a new therapeutic procedure being used to treat human malignancies, macular degeneration and psoriasis. PDT is a combination of light and light sensitive agents (porphyrins) in an oxygen-rich environment and is based on the dye-sensitized photo-oxidation of biological macromolecules. A photosensitive dye or 28 agent is delivered to the target tissue via intravenous injection (IV) or topical application. The porphyrins are selectively retained by rapidly growing tissues (e.g., cancer or psoriasis), and are activated by light. Lasers are used as the primary light source since laser light is monochromatic (one wavelength), coherent (parallel light waves allowing precise focusing), and intense (allowing for shorter treatment times). Activation is followed by the generation of toxic molecules such as singlet oxygen and other oxygen free radicals. The reactive oxygen species (ROS) generated during the excitation of the photosensitizer are thought to be responsible for the toxic effects and tissue destruction (Girotti 1990). The ROS are highly reactive and can interact with proteins, lipids, nucleic acids and other cellular components. Cellular responses to PDT include expression of stress-responsive proteins (Gomer et al, 1991 A; Gomerera/., 1988; Curry and Levy 1993), release of cytokines (Henderson and Donovan 1989; Evans et al, 1990), and induction of apoptosis (Agarwal et al, 1991; Agarwal et al, 1993). Apoptosis is a morphologically and biochemically distinct form of cell death that contributes to cell turnover under normal conditions. The apoptotic process can be divided into at least three functionally distinct phases: initiation, effector and degradation. The initiation phase is heterogeneous and involves different signaling pathways depending on the type of apoptotic stimuli. The effector phase involves activation of cascades of proteases in a mitochondria-dependent manner. The degradation phase is characterized by changes in the cytoplasmic redox state and nuclear apoptosis (Kroemer et al, 1997; Nicholson and Thornberry 1997). Protein kinase pathways are likely to be involved in the initiation phase of apoptosis. The SAPKs have been implicated in apoptosis induced by a variety of agents including FAS-ligation (Juo etal, 1997), environmental stress (Zanke et al, 1996), UV irradiation (Chen et al, 1996) and withdrawal of growth factors (Xia et al, 1995). PI 3-kinase and PKB have also been shown to be involved in apoptosis induced by growth factor withdrawal and UV irradiation (Kulik et al, 1997; Dudek et al, 1997). Treatment of cardiac myocytes with 29 sodium arsenite also induced stress responses and apoptosis. Interestingly, this treatment caused activation of the p38 isoform of MAP kinase, PI 3-kinase, and p7056^, but did not activate PKB, JNK, ERK1 or ERK2 (Wang and Proud 1997). A common feature of cells in the effector phase is the activation of caspases, a family of aspartic acid-directed proteases (Alnemri et al, 1996). Caspase-mediated proteolysis of specific proteins results in an irreversible commitment of cells to undergo apoptosis. Rafl and PKB have been shown to be cleaved in a caspase-dependent manner during the apoptotic response induced by Fas activation and UV irradiation (Widmann et al, 1998). Cleavage of Rafl and PKB inhibited their kinase activity which may partially explain why the ERK kinase and PI 3-kinase pathways become inhibited during the progression of apoptosis. ROS have also been implicated in the induction of apoptosis. For instance, apoptosis may be induced by generation of ROS or by the depletion of endogenous antioxidants (Sandstrom et al, 1994; Ratan et al, 1994), and conversely, can be inhibited by exogenous or endogenous antioxidants (Chang et al, 1992; Schulze-Osthoff et al, 1992). Furthermore, there may be a direct relationship in that apoptosis has been correlated with increases in intracellular ROS levels. As mentioned previously, the Bcl-2 family members play a role in regulation of apoptosis and Bcl-2 itself can block the increase in ROS levels associated with apoptosis (Hockenbery et al, 1993; Veis et al, 1993), and can inhibit ROS-induced apoptosis (Hockenbery et al, 1993). Nevertheless, it seems that ROS are not essential for apoptosis to occur as anaerobically cultured cells deprived of IL-3 undergo apoptosis and Bcl-2 can protect them (Jacobson and Raff 1995). Although it is unclear precisely what role ROS play in apoptosis, PDT treatment generates ROS, leads to apoptosis, and antioxidants can protect keratinocytes against the effects of PDT. Thus, analysis of the molecular role of ROS and apoptosis in PDT will lead to a clearer understanding of the signaling systems involved in this response of cells to stresses of all types. 30 1.4 Rationale and Research Hypotheses 1.4.1 Rationale The phosphorylation of ribosomal protein S6 plays a key role in the onset of specific protein synthesis required for cell growth and proliferation (Jefferies and Thomas 1996). During sea star oocyte maturation, dramatic changes in translation rates occur in preparation for the oocyte to become fertilized, and many protein kinases, including previously undefined S6 kinases, become activated. The p70S6K is intimately connected with the translational requirements through phosphorylation of the S6 protein in response to widely divergent stimuli (Bandi et al, 1993; Kriegetal, 1988). I therefore undertook the purification and biochemical characterization of an S6 protein kinase from mature sea star cytosolic extract in order to identify, and establish the regulatory properties of this kinase and to develop a clearer picture of the signaling processes involved in meiotic maturation. The PI 3-kinase pathway is strongly activated by mitogenic stimulation and has been implicated in suppression of apoptosis (Yao and Cooper 1995; Scheid et al, 1995). Photodynamic therapy with benzoporphyrin derivative mono-acid ring A (BPD-MA) is thought to induce apoptosis via generation of reactive oxygen species (ROS). Therefore, the investigation of the role of p70s<J/r and the PI 3-kinase/PKB signaling components in PDT-treatment in a mammalian model system was undertaken. 31 1.4.2 Objectives 1. To purify the ribosomal S6 kinase that became activated during sea star oocyte maturation. 2. To characterize the purified S6 kinase biochemically. 3. To investigate the regulation of the purified S6 kinase. 4. To investigate the function of the purified S6 kinase. 5. To investigate the effect of PDT treatment on p705(5A: and on the potential upstream regulators, PI 3-kinase and PKB. 32 Chapter 2: Materials and Methods 2.1 Materials Pisaster ochraceus were collected by all members of the laboratory from beaches in the Vancouver area. P-glycerol phosphate, EGTA, EDTA, MOPS, P-methylaspartic acid, sodium orthovanadate, ATP, methylsulfonyl fluoride, aprotinin, leupeptin, benzamidine, dithiothreitol (DTT), Coomassie brilliant blue, L-tyrosine agarose, poly-L-lysine agarose, heparin agarose, Q Sepharose, L-arginine agarose, recombinant human insulin, recombinant human EGF, phorbol 12-myristate 13-acetate (PMA), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and the peptide inhibitor of protein kinase A (PKI) were bought from Sigma. Phosphatidylinositol (PI) was from Avanti Polar Lipids. P-81 phosphocellulose filter paper was from Whatman. [y-32P]ATP, [y-32P]azido-ATP, Tris-HCl and glycine were from ICN. Brij 35, nitro blue tetrazolium (NBT), 5-bromo-4-chloro-indolyl phosphate (BCIP), hydroxylapatite (HTP), P-mercaptoethanol (BME) and affinity-purified goat anti-rabbit IgG alkaline phosphatase conjugates were bought from Bio-Rad. Anisomycin, wortmannin and rapamycin were purchased from Calbiochem. Histones H2B, H2A and HI were from Boehringer Mannheim. Bromophenol blue was from Schwarz-Mann Biotech. Protein A- Sepharose beads were from Pharmacia. Protran™ Pure Nitrocellulose membrane was from Schleicher & Schuell, and Polyscreen® PVDF membrane was from Biotechnology Systems. TEMED, ammonium sulfate, silver nitrate and sodium chloride were from Fisher Scientific. The S6 peptides were synthesized at the Biomedical Research Centre, University of British Columbia. The 40S ribosomal subunits were prepared from rat liver by a procedure modified from that of Kreig et al. (1988). Molecular weight standards for gel electrophoresis and gel filtration were prepared in our laboratory by a procedure modified from that of Griffith (1972). Anti-rabbit polyclonal antibodies produced in our laboratory were affinity-purified on peptide-agarose columns. 33 Potassium dichromate, glycerol and all other routine buffer chemicals were from BDH. Liposomally formulated benzoporphyrin derivative mono-acid ring A (BPD-MA) was supplied by QLT PhotoTherapeutics, Inc. (Vancouver, B.C.). Aliquots were reconstituted with distilled water at 1.5 mg/ml and stored in the dark at 4°C. Further dilutions were carried out immediately prior to experimental use with phosphate-buffered saline (PBS). The S6 peptide column was prepared by swelling 5.0 g CNBr-activated Sepharose 4B beads for 15 min in 1 mM HCI. The beads were washed several times with 200 ml aliquots of the same solution on a sintered glass filter (porosity G3). The gel was washed with coupling buffer (0.1 M NaHC03, pH 8.3, 0.5 M NaCI) and incubated with 48 mg of the S6 peptide (AKRRRLASLPvASTSKSESSQK) in coupling buffer overnight at 4°C. The gel was transferred to buffer to block remaining active groups (0.2 M glycine, pH 8.0) for 2 h at room temperature. The blocking agent was then washed away with coupling buffer and the column was stored at 4°C. 2.2 Preparation of mature sea star oocyte cvtosol Oocytes that were arrested in the G2/M border prior to mitosis were isolated from sea stars as described previously (Meijer et al, 1984). The immature oocytes were induced to undergo maturation by the addition of the hormone 1-methyladenine for ~2 h at 14°C. At the end of this period over 90% of the oocytes had undergone maturation as judged by the onset of germinal vesicle breakdown (GVBD). Subsequently, a 35% (v/v) oocyte homogenate was prepared in chilled homogenization Buffer A (50 mM (3-glycerol phosphate, 20 mM MOPS, 2 mM EDTA, 5 mM EGTA, 1 mM sodium vanadate, 0.25 mM DTT, 1 mM PMSF, 1 mM benzamidine, 0.5 mM P-methyl aspartic acid, pH 7.2) by 2 x 30s bursts at the 18,000 setting using a Brinkmann 3000 Polytron Homogenizer. The homogenate was centrifuged at 12,000 x g for 10 min and the supernatant subsequently centrifuged at 100,000 x g for 30 min. The resulting cytosolic (supernatant) fraction was 34 stable for over one year when stored at -70°C and the frozen extract was used for all subsequent experiments. 2.3 Enrichment of the 40S ribosomal S6 protein kinase Cytosol (270 ml, -15 mg/ml protein) was thawed and diluted 1:1 with chilled 1.6 M (NH4)2S04 in Buffer B (50 mM Tris, 0.5 mM EDTA, 0.1 mM EGTA, 0.25 mM DTT, 1 mM p-methylaspartic acid, pH 7.2). The slurry was gently stirred over ice for 20 min, then centrifuged at 10,000 g for 10 min in a pre-cooled Beckman rotor. The supernatant was applied at 0.5 ml/min to two 25-ml L-tyrosine agarose columns equilibrated in 0.8 M (NFLt^SCV The columns were washed with 50 ml of 0.8 M (NFL^SCu and the proteins were eluted on an FPLC (Fast Protein Liquid Chromatography) system (Pharmacia) with a 400 ml linear 0.8-0.0 M (NH^SC^ gradient at a flow rate of 1 ml/min. Fractions (5 ml) were collected and assayed for phosphotransferase activity towards a synthetic peptide substrate modeled after the C-terminus of S6 (AKRRRLSSLRAGGRR). The peak activity fractions were pooled, diluted 1:10 with Buffer B and applied at 0.5 ml/min to one freshly poured 25-ml hydroxylapatite (HTP) column equilibrated in Buffer B. The loaded HTP column was washed with 50 ml of Buffer B, and eluted with a 400 ml linear gradient 0.0-0.35 M K2HPO4/KH2PO4 in Buffer B at a flow rate of 1 ml/min. Fractions (5 ml) were collected, and assayed for phosphotransferase activity. The peak activity fractions were then pooled and frozen at -70°C for later use. To complete one purification, eight of the pooled HTP fractions (ie. 16 L-tyrosine agarose elutions) were combined for the following steps which were completed over two days. The fractions were thawed slowly over ice, diluted 1:4 with Buffer B and applied to a 25-ml heparin agarose (used as a reverse column as S6 kinase does not bind to this resin) and a 25-ml poly-L-lysine agarose column linked in series. The S6 kinase was eluted from the poly-L-lysine column with a 400 ml linear 0.0-1.0 M NaCl in Buffer B gradient at a flow rate of 35 1 ml/min. Fractions (5 ml) were collected and those containing S6 kinase phosphotransferase activity were then pooled, diluted 1:3 with Buffer B and applied to a 25-ml Q Sepharose anion exchange column equilibrated in Buffer B. The column was washed with 50 ml Buffer B, and the S6 kinase activity was eluted with a 300 ml linear gradient of 0.0-0.80 M NaCI in Buffer B at 1 ml/min. Fractions (4 ml) were collected, peak phosphotransferase activity fractions were pooled, diluted 1:3 with Buffer B and loaded on to a 10-ml L-arginine agarose column equilibrated in Buffer B. The column was eluted with a 120 ml linear gradient of 0.0-0.8 M NaCI in Buffer B, and fractions (2 ml) were collected. The fractions were assayed for kinase activity, peak fractions were pooled, diluted 1:3 with Buffer B, and applied to a 1-ml MonoQ column. The column was eluted with a 20 ml linear gradient of 0.0-0.8 M NaCI in Buffer B, and fractions (250 pi) were collected. The fractions were assayed for S6 kinase activity, pooled and frozen at -70°C for future use. 2.4 Kinase assays, autophosphorylation and protein assays Ribosomal protein S6 kinase phosphotransferase activity was assayed by a procedure described previously (Sanghera et al, 1992). Briefly, 5 pi of extract were assayed in a final volume of 25 ul containing 0.40 mg/ml of the S6 peptide in assay dilution buffer (ADB: 25 mM p-glycerol phosphate, 20 mM MOPS, pH 7.2, 5 mM EGTA, 2 mM EDTA, 20 mM MgCl2, 0.25 mM DTT, 5 pM p-methylaspartic acid and lpM PKI) and 50 pM [y-32P]ATP. The reaction was incubated for 5 min at 30°C, then 20 pi were removed and spotted onto a 1.5 cm2 piece of Whatman P-81 phosphocellulose paper. The papers were washed extensively (15x) in 1% (w/v) H3PO4, transferred into 6-ml plastic vials containing 0.5 ml of Ecolume (ICN) scintillation fluid, and the incorporated radioactivity was measured in a Wallac (LKB) scintillation counter. Autophosphorylation of the S6 kinase was performed in a final volume of 50 pi 36 containing 5 mM MgCl2, 12 mM MOPS, pH 7.2 and 50 pM [y-j2P]ATP. The reaction was incubated for 30 min at 30°C, and terminated by the addition of SDS sample buffer (125 mM-Tris-HCl, pH 6.8, 4% SDS, 0.01% Bromophenol Blue, 10% p-mercaptoethanol and 20% glycerol). Protein was determined by the method of Bradford (1976) using bovine serum albumin as a standard. 2.5 Electrophoresis, silver staining and immunoblotting SDS/PAGE was performed on 1.5 mm thick gels, with acrylamide at 11% (w/v) (unless indicated otherwise) in the separating gel and 4% (w/v) in the stacking gel, in the buffer system described by Laemmli (1970). Samples were boiled for 5 min in the presence of SDS sample buffer and electrophoresed for ~17 h at 10 mA. The separating gels were silver-stained by the method of Merril et al. (1981) or for autoradiography, gels were exposed to Dupont reflection film at room temperature. For immunoblotting, SDS/PAGE gels were soaked in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 5 min, sandwiched with a nitrocellulose membrane and transferred at 300 mA for 3 h. The nitrocellulose membrane was stained for proteins with Ponceau S, and blocked for 2 h at room temperature in Tris-buffered saline (TBS: 150 mM NaCI, 20 mM Tris-HCl, pH 7.5) containing 5% skim milk. The membrane was washed 3x5 min with TBS containing 0.05% Tween 20 (TTBS) before incubation with plQS6K specific antibodies (in TTBS with 0.05% sodium azide 1:500 dilution) overnight at room temperature. The next day, the membrane was washed extensively (5x5 min) with TTBS before incubation with the secondary antibody (goat anti-rabbit IgG coupled to alkaline phosphatase in TTBS with 0.05% sodium azide) for 2 h at room temperature. The membrane was washed 2x5 min with TTBS then briefly with Alkaline Phosphatase Buffer (AP: 0.1 M NaHC03 10 mM MgCl2, pH 9.8) before incubation with BCIP/NBT color development solution (3% NBT in 70% DMF and 1.5% BCIP in 100% DMF in 100 37 ml AP buffer). The color reaction was terminated by successive washes in distilled water. Antibodies used for the experiments described included anti-peptide antibodies prepared in our laboratory based on the amino terminal (residues 24-40), subdomain three (residues 127-153) and carboxyl terminal (residues 509-525) of human p70 . In addition, an anti-peptide antibody based on the carboxyl terminal (residues 483-502) of rat p70S6K was purchased from Santa Cruz. Anti-peptide antibodies against HSP-60 were from StressGen. Anti-peptide antibodies to the regulatory subunit of PP2A, anti-PI-3 kinase (N-SH2) monoclonal antibody and anti-rat PI-3 kinase (p85 subunit) polyclonal antibodies were from Upstate Biotechnology Inc. (Lake Placid, NY). 2.6 Gel filtration chromatography of sea star S6 kinase A pre-packed Superose 12 or Superdex 200 (Pharmacia) gel filtration column was equilibrated overnight with pre-filtered, 150 mM NaCl in Buffer A at a flow rate of 0.2 ml/min. Two hundred ug each of BSA (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen (25 kDa) were loaded on to the column in 200 ul of 150 mM NaCl in Buffer A via a 200 pi injection loop. The standards were eluted isocratically with the same buffer at a flow rate of 0.2 ml/min. Fractions (250 pi) were collected and protein concentrations were measured by Bradford assay as described above. An aliquot of the purified S6 kinase (250 u.1) was loaded and eluted as above, and assayed by filter paper with the S6 peptide as a substrate. The size of the purified kinase was estimated by linear regression based on the elution of the standards. 2.7 Regulation of sea star S6 kinase by ions and inhibitors Purified S6 kinase was assayed as described above in a final volume of 30 pi containing 5 ul enzyme, 5 ul compound, 15 ul cocktail and 5 ul ATP. The ions and 38 inhibitors were titrated, and the concentration that caused half-maximal inhibition (IC50) was determined. 2.8 Photoaffinity labelling of sea star S6 kinase with ry-32P1azido-ATP The purified S6 kinase (150 pi) was incubated with 10 mM MgCl2 and 100 uM [y-32P]azido-ATP (20,000 cpm/pmol) at 0°C. The sample was irradiated with UV light (254 nM) at a path length of 2 cm for 5 min. The reaction was terminated by addition of SDS-sample buffer, the sample was electrophoresed and the gel was autoradiographed. The control mixture was incubated under similar conditions in the absence of photoactivation. 2.9 Phosphoamino acid analysis The purified p52S6K was incubated with purified 40S ribosomes (or other substrates), electrophoresed on SDS/PAGE (as described above), and transferred to PVDF membrane for 3 h at 300 mA. The radio-labelled ribosomal protein S6 band (or other protein band) was excised from the membrane and digested in 300 ul of constant-boiling HC1 at 105°C for lh. The acid-hydrolyzed sample was dried under vacuum (Speed Vac), washed with water, re-dried, and then resuspended in 5 ul of electrophoresis buffer containing pyridine/acetic acid/water (1:10:189 by vol.). The sample was spotted on to a cellulose thin layer chromatography (tlx.) plate next to phosphoserine, phosphothreonine and phosphotyrosine standards (1 mg/ml each), and the plate was electrophoresed for 45-60 min at 1000 V with cooling. The plate was dried, sprayed with ninhydrin solution (1%) and baked in a 90°C oven for 10 min. The plate was then exposed to x-ray film. 39 2.10 Two dimensional phosphopeptide mapping Two dimensional tryptic-phosphopeptide map analysis of the phosphorylated S6 protein or MBP was performed by excision of the radio-labelled protein band from SDS/PAGE gels, digestion of the protein with TPCK-treated trypsin in 1 ml of 50 mM NH4HCO3, pH 8.0 at 37°C for 24 h. The samples were then dried under vacuum (Speed Vac), washed with 4 x 200 pi of water, re-dried, and then resuspended in 5 pi of electrophoresis buffer (1% NH4HCO3, pH8.9). The sample was spotted in the center of a cellulose t.l.c. plate 1.5 cm from the bottom. Electrophoresis was performed in the long dimension at 600 V for 40 min. After drying, the plate was chromatographed in the second dimension in butanol/pyridine/acetic acid/water (13:10:2:8 by vol). The plate was dried overnight and exposed to x-ray film. 2.11 Substrate specificity of sea star S6 kinase Synthetic peptides were patterned after the C-terminal sequence in the S6 protein known to be phosphorylated by p!0S6K (Bandi et al, 1993). The peptides were synthesized on an A.B.I. 430A peptide synthesizer and cleaved from the resin by hydrofluoric acid. Purity of the peptides was demonstrated by reverse-phase HPLC, and the identity confirmed by ion spray mass spectrophotometry analysis (model API-Ill). Due to the small size of the peptides and the high purity as confirmed by reverse-phase HPLC, they did not require further purification after lyophilization. The peptides were carefully dried before weighing on an analytical balance. In the experiments shown here, the peptides were solubilized as stock solutions of 2.0 mM in ADB, and serial dilutions from 1 mM to 2 pM final peptide concentrations in a final volume of 25 pi were assayed at least three times in the presence of enriched kinase and 50 pM [y- PJATP. For each 40 peptide, Km was determined using the Michaelis-Menton rate equation, and Vmax was estimated using Lineweaver-Burke double-reciprocal plots of dose-response data. 2.12 Protein micro-sequencing, alignment and identification of sequenced fragments Approximately 2.1 ml of purified MonoQ p52 were concentrated by centrifugation in a Centricon 10 tube to a final volume of ~500 pi (~2 h at 4000 rpm). This was further centrifuged in a microfuge Centricon 10 tube to a final volume of ~ 150 pi (~15 min at 12,000 rpm). Fifty pi of SDS sample buffer were added, the sample was boiled 5 min and electrophoresed on 11% PAGE. A CD Immobilon membrane was pre-equilibrated in methanol for 15 min followed by equilibration in transfer buffer for 15 min. The proteins were transferred in transfer buffer containing 10% methanol for 3 h at 300 mA, and then for a further hour at 200 mA. The CD membrane was stained with 10 ml of quick stain solution A for 1 min, and then with 25 ml of quick stain solution B for 3 min (Immobilon-CD, Millipore). This negative stain is compatable with sequencing techniques. The membrane was washed for 1 min in distilled water, and the protein bands were excised, cut into small pieces and placed in 500 pi Eppendorf tubes containing tryptic cleavage buffer (0.1 M Tris, 1 M NaCI, 2 mM CaCb, 5% acetonitrile (ACN) pH 8.2 containing 0.25 pg bovine trypsin tosylphenylalanylchloromethane (TPCK) treated XII, Sigma/20ul) for 5 h at 37°C. After 5 h, 0.25 pg trypsin in 20 pi cleavage buffer containing 2 M urea were added, and the samples were incubated a further 15 h at 37°C. The supernatant was removed, the samples were washed with 20 p.1 1% TFA, which was pooled with the supernatant and injected directly onto HPLC. Peaks were collected directly from the HPLC elution, and were analyzed by mass and sequenced in the laboratory of Dr. Ruedi Aebersold (University of Washington, Seattle) according to previously published protocols (Patterson et al, 1992; Hess et al, 1993). 41 2.13 Inactivation of sea star S6 kinase by phosphatase treatment Purified protein phosphatase 2 A (PP2A) was the generous gift of Drs. David Brautigan and Jian Chen (University of Virginia, Charlottesville). The enzyme activity was ~1.4 units per ml (1 unit will release 1 nmol of Pj per min from phosphate-labelled phosphorylase). Potato acid phosphatase (PAP) was prepared in our laboratory as active enzyme linked to protein A-Sepharose beads. The purified p52S6K was pre-incubated in the presence of active PP2A or PAP. The PP2A reaction was terminated by the addition of okadaic acid, while the PAP reaction was terminated by centrifugation of the beads and removal of the supernatant for assay. Phosphotransferase activity of the dephosphorylated purified p52S6K was assayed with the S6 peptide substrate as described above. 2.14 Immunoprecipitation Unless indicated otherwise, immunoprecipitations were carried out with 500 pg protein to which 500 pi NEFT buffer (1.0 M NaCI, 5 mM EDTA, 50 mM Tris HC1, 50 mM NaF pH 7.4) containing 6% NP40 (6%NEFT) was added. The PI 3-kinase immunoprecipitations were pre-cleared with 30 pi protein A-Sepharose beads for 30 min prior to immunoprecipitation. The samples were incubated with 5 pi of antibody for 4 h, after which 30 pi of protein A-Sepharose beads were added to precipitate the adsorbed proteins. The immunoprecipitates were washed twice with 6%NEFT buffer, once with NEFT buffer and once with ADB. Immune complex assays were carried out by addition of 30 pi substrate in ADB, 10 pi MgCl2 and 50 uM [y-32P]ATP, followed by incubation at 30°C for 30 min. Aliquots (20 pi) were spotted onto filter papers, washed and counted as described above. SDS-sample buffer (30 pi) were then added to the protein A-42 Sepharose beads and the remaining sample was electrophoresed, transferred and immunoblotted as described above. 2.15 Immunoprecipitation of S6 phosphotransferase activity by anti-PP2A antibody S6 kinase was partially purified via the tyrosine agarose/HTP/polylysine/Q Sepharose steps, and a 1 ml aliquot was pre-cleared with 100 pi of 1:1 slurry of protein A Sepharose beads for 15 min at 4°C. The beads were pelleted by centrifugation for 2 min at 12,000 and the supernatant was incubated with 10 pi of anti-PP2A A subunit antibody overnight at 4°C. A 1:1 slurry of protein A Sepharose beads (40 pi) were added and the mixture incubated for a further 1.5 h at 4°C. The samples were washed once with 6%NEFT buffer, once with NEFT buffer and once with Buffer B. To the precipitated proteins were added 30 pi ADB, 4 ul purified 40S ribosomes and 50 uM [y-32P]ATP, and the samples were incubated at 30°C for 35 min. The reaction was terminated by addition of 40 pi SDS sample buffer, the samples were boiled for 5 min and electrophoresed on a 12.5 % PAGE gel. The gel was transferred to a nitrocellulose membrane and exposed to film overnight. 2.16 Procedure for heat shock-treatment of sea star oocytes Oocytes from individual P. ochraceous were suspended in natural sea water preheated to 35°C. Samples of the oocyte suspension were removed immediately (zero timepoint) and at various indicated times, pelleted by centrifugation, the sea water removed, and the oocytes quick frozen in a dry ice/ethanol bath. Frozen oocytes were stored at -70°C until processing. Due to the treatment, cell volumes changed during the time courses. To maintain equivalent protein concentrations, the oocytes were homogenized in a standard total volume. To each aliquot of packed cells were added 5X 43 homogenization buffer (900 \x\). The samples were diluted to a final volume of 6 ml and homogenized by 2 x 30 s bursts with a sonicator. The extracts were centrifuged at 40,000 rpm for 30 min, the supernatants divided into aliquots (1 ml) and frozen at -70°C. 2.17 Expression and purification of GST-S6 kinase from baculovirus The baculoviral expression and purification of the GST-S6 kinase was carried out by Jun Yan (B.Sc, Research technician) following a protocol developed by PharMingen Canada (Instruction Manual for Baculovirus Expression Vector System, Section 4.6). Spodoptera frugiperda cells (sf9) (Invitrogen) cells were grown in Hink's TNM-FH insect medium supplemented with 10% FBS at 27°C. Three pg of prepared transfection plasmid pAcG2T-S6K were co-transfected with 0.5 pg of linear AcNPV BaculoGold DNA (PharMingen) into sf9 cells using the cationic liposome CellFectin mediated procedure, as described (GibcoBRL). Sf9 cells were incubated with the media from the four days post-transfected plates to amplify recombinant virus. The products from recombinant virus-infected sf9 cells were identified by immunoblotting with the anti-p7Qs6K C/T antibody Approximately 500 ml of High Five cells (Invitrogen, ~5 x 106 cells/ml) in a IL spinner flask were infected with the baculovirus constructs with an MOI of 3~10. After 3 days, the cells were harvested by centrifugation at 1,000 x g for 15 min. The cell pellet was washed once, resuspended in 20 ml ice-cold lysis buffer, and disrupted with a Dounce homogenizer. Debris was pelleted at 20,000 x g for 30 min and 100 pg DNAse I was added to the decanted supernatant. The cell supernatant was loaded onto a glutathione-agarose column (5 ml bed volume) at 4°C. The column was washed with 3 column volumes of STE-500 buffer (50 mM Tris pH 7.4, 500 mM NaCI and 1 mM EDTA) followed by 4 column volumes of STE-50 buffer (50 mM Tris pH 7.4, 50 mM 44 NaCl and 1 mM EDTA). The beads were transferred into Eppendorf tubes, adjusted to 50% concentration with PBS and stored at -70°C. 2.18 Stimulation of GST-S6 kinase with PMA The stimulation experiments were performed by Jun Yan (B.Sc, Research technician). About 350 ml of infected cells were incubated as above for 3 days at 27°C. The cells were pelleted in 50 ml Falcon tubes by spinning at 2,000 rpm at room temperature for 5 min, and resuspended in Excell-400 media to a total volume of 20 ml. The cells were treated with 25 or 50 nM PMA for 15 or 25 min. After stimulation, the cells were placed on ice and pelleted at 2,000 rpm at 2°C for 5 min, washed 2 x with PBS and frozen at -70°C. The fusion proteins were purified on GST-bead columns as described above. 2.19 Microinjection studies The microinjection experiments were performed by Harry Paddon (B.Sc, Research technician). Sea stars, Pisaster ochraceus, were kept in the laboratory in a 100 gallon refrigerated aquarium at a constant temperature of 10°C. They were fed mussels and barnacles collected locally at low tide. For microinjection, oocytes were removed fresh from the gonad by insertion of a 16 guage needle into the arm of a sea star and gently aspirating a small volume which typically contained a few thousand oocytes. The oocytes were placed in 10 ml of filtered normal sea water (NSW) and kept on ice. Oocytes were sorted visually with a dissecting microscope (WILD M3B) at 16x power. At this magnification oocytes (~150 microns diameter) were inspected and healthy, immature oocytes were picked up individually by hand with a pulled capillary pipette and transferred to the microinjection slide. Normally, about 20 oocytes were in the 100 pi 45 NSW on each slide. The NSW was localized within a ring of rubber cement ~1 cm diameter made with a PAP pen (The Binding Site). The glass slides were kept at 12-13°C on a 3/8" thick aluminum plate on a thermoelectric cold plate from Thermoelectrics Unlimited Inc. Evaporation was minimized by using blue plastic Falcon caps to cover the seawater. Microinjection of the oocytes was done using a Leitz Labovert F5 inverted microscope with differential interference contrast optics. The microscope was flanked by right and left Leitz micromanipulators which held the injection needles and holding pipettes respectively. During the microinjection the slide was kept at 10-12°C using a refrigerated microscope stage based on a Peltier element receiving variable direct current 12 volts, 0-2 amps. The microinjection needles were made on a needle puller (model P-80 Brown-Flaming micropipet puller supplied by Sutter Instrument Co., U.S.A.) set at 335°C, Gas 75, velocity 10, pull 7 from 1.0 mm glass capillaries with filament, from World Precision Instruments Inc. The holding pipettes were made from the same pulled needles which were broken, heated and manipulated with a microforge to produce a blunt tip with an inside diameter of 10-20 microns. The microinjection needles were backfilled using pulled capillary pipettes into which approximately 1-2 pi of sample was aspirated. Once the pulled capillary was threaded down the back of the injection needle, the sample was expelled into the needle tip. The capillary was withdrawn from the needle and the needle was inserted into an Eppendorf needle holder connected by Tygon tubing to an Eppendorf 5242 microinjector. This allowed control of pressurized nitrogen to cause expulsion of the sample from the needle into the oocyte. The volume of the oocytes was approximately 2500 picolitres and 50-200 picolitres of sample was injected. The volume was judged by eye and the instrument settings to produce an injection of a certain volume that varied with each needle and during the course of the injections as the needle frequently became plugged with cell debris and needed to be changed. 46 2.20 Cell culture, stimulation and preparation of Pam212 cell lysates All the tissue culture, stimulation and preparation of Pam212 keratinocyte cell lysates was performed by Dr. Jing-Song Tao (Ph.D. candidate) at QLT PhotoTherapeutics Inc. (Vancouver, B.C.). All of the subsequent experiments involving Pam212 keratinocytes were carried out as a collaboration between myself and Dr. Jing-Song Tao (Ph.D. candidate). All the immunoprecipitations, enzyme assays, lipid kinase assays and immunoblots were performed at the Kinetek site, with each of us contributing equally. The spontaneous transformed murine keratinocyte cell line Pam212, was kindly provided by Dr. Stephen E. Ullrich (University of Texas, Houston, TX). The cell line was maintained in Dulbecco's minimum essential medium (DMEM) (Gibco) supplemented with 5% fetal bovine serum (FBS, Sigma), and antibiotics (100 U/ml penicillin, 100 U/ml streptomycin). Cultures were incubated at 37°C in 5% CO2. For stimulation, 5 x 105 Pam212 cells were seeded to 100 mm tissue-culture dishes in 22 ml of medium and incubated for 72 h without further manipulation. After 72 h, the volume of medium was adjusted to 20 ml, and the cells were stimulated by the indicated treatment and incubated for appropriate time periods. The medium was aspirated, and the cells were washed twice with ice-cold PBS containing 1 mM Na3V04. The cells were lysed by scraping them off in 800 pi of homogenizing buffer (20 mM MOPS, pH 7.2, 5 mM EGTA, 1% (W/V) Nonidet P-40, 1 mM DTT, 75 mM P-glycerol phosphate, 1 mM Na3V04 and 1 mM PMSF) and sonication for 30 s. The insoluble material was pelleted by centrifugation (10,000 x g) for 15 min at 4°C and the supernatant removed, divided into aliquots, quickly frozen in liquid nitrogen and stored at -80°C. The protein concentration of the keratinocyte lysate was determined using the bicinchoninic acid assay (Pierce, IL). The assays were performed on flat bottom 96-well tissue culture plates (Falcon) by pipetting 5 pi of variously diluted cell extracts into a 47 well. Protein standards were serial dilutions of bovine serum albumin (BSA). A 100 jal BCA solution was added to individual wells containing test samples and the protein standard. The plate was then incubated at 37°C for 3 h. The optical density was determined in a Dynatech MR 5000 microtiter plate reader at 565 nm (Dynatech Laboratories). For serum starvation, a culture of 75% subconfluently growing Pam212 cells were transferred to DMEM supplemented with 0.5% FCS. The cultures were incubated at 37°C in 5% CO2 for 36 h. The cells were then transferred to serum-free DMEM and incubated for an additional 12 h before treatment. 2.21 In vitro PDT treatment and UV irradiation of Pam212 cell lysates BPD-MA at indicated concentrations was added to the medium for 12 h prior to the light treatment. The cells were then exposed to red light at a fluence of 2 J/cm . The light source was a light box equipped with 2 panels of General Electric red fluorescent tubes with an emission spectrum between 620 to 700 nm. UV irradiation was performed by exposing cells in 100 mm tissue culture dishes with 5 ml of medium to a General Electric germicidal lamp in a biosafety cabinet at a distance of 50 cm from the UV source for 4 min. After PDT or UV treatment, cells were incubated at 37°C in the dark for various periods of time prior to cell lysis. The entire procedure was carried out in a semi-dark room. 2.22 Insulin, EGF and anisomvcin treatment of Pam212 cell lysates Following 12 h serum-starvation, 100 nM insulin, 100 ng/ml EGF or 40 pg/ml anisomycin was added to the culture medium. The cells were returned to the 37°C incubator for an additional period of time as indicated before the cell extracts were 48 prepared. For inhibitor treatment, serum-starved Pam212 cells were pretreated with wortmannin or rapamycin at the indicated concentrations for 15 min prior to stimulation. 2.23 In vitro lipid kinase assay Anti-PI-3 kinase-p85 (N-SH2) immune complexes were resuspended in 50 pi of reaction buffer containing 30 mM HEPES, pH 7.4, 30 mM MgCl2, 50 uM ATP, 0.2 mM adenosine, 10 pCi [y-32P]ATP, and 10 pg PI or a mixture of lipids including 2.5 pg PI, 2.5 pg PI(4,5)P2 and 5 pg phosphatidylserine. The lipids were dispersed by sonication in 30 mM HEPES. The reactions were carried out at room temperature for 15 min and terminated by addition of 100 pi 1 N HC1 and 200 pi chloroform/methanol (1:1, v/v). Lipids were extracted from the chloroform layer and separated by thin-layer chromatography on silica gel G plates impregnated with 1% potassium oxalate as previously described (Matter et al, 1992). PI(3)P and PI(3,4,5)P3 were visualized by autoradiography and quantitated by determining 32P activity in the spots in a liquid scintillation counter. 2.24 Statistical analysis As indicated in the results section, some data were expressed as arithmetic mean±standard error of the mean (S.E.). Bars refer to standard deviation (S.D.). Data from 3 to 5 independent experiments (assuming normal distribution) were pooled and subjected to statistical analysis using analysis of variance followed by Bonferroni multiple comparison test (a=0.05). statistically significant difference between samples derived from variously treated cells and that from untreated control cells. *k , statistically significant difference between samples derived from variously treated cells and that from insulin- or growth factor-treated cells. 49 Chapter 3: Results and Discussion: p70 S6 kinase in Pisaster ochraceus 3.1 Introduction and Model System The meiotic maturation of sea star oocytes is a useful system for investigation of cellular regulatory mechanisms linked to protein synthesis, since during this period dramatic quantitative and qualitative changes in the synthesis of protein occurs. Isolated sea star oocytes are arrested at meiotic prophase I, and can be induced to resume meiosis when exposed to the natural hormone 1-methyladenine at a concentration of 1 uM or more for a cumulative period of time. A large germinal vesicle and its prominent nucleolus are visible in the immature oocyte, and maturation can be followed and correlated to the breakdown of the germinal vesicle (GVBD) (Fig. 4), normally within 2 hours. There are many advantages associated with the oocyte system, particularly the ability to obtain a large amount of an essentially pure population of oocytes at the same developmental stage. Induction of synchronous maturation by the simple addition of hormone allows large amounts of mature sea star extracts to be obtained and used subsequently for analysis and protein purification. The culture and treatment of the oocytes can be performed under non-sterile conditions in the absence of expensive media. The large size of the oocytes (~150 microns diameter) allows micromanipulation such as microinjection to be carried out routinely. The activation of undefined S6 kinases has been previously described during meiotic maturation of frog and sea star oocytes (Cicirelli et al, 1988; Pelech etal, 1987; Meijer et al, 1987) and this system was used herein to attempt to identify, purify and characterize maturation-induced S6 kinase activity. 50 o d 1 m 5 -° in CN C3 S 3 .a d £ .2 d • I-H E o —, d d o 51 3.2 Identification and Enrichment of Active S6 Kinase from Mature Sea Star Oocytes 3.2.1 Identification of S6 phosphotransferase activity in mature sea star cytosolic extract Cytosolic extracts were prepared from mature sea star oocytes treated for 90 min with 1 -methyladenine or from immature oocytes that were arrested at the beginning of prophase. The extracts were fractionated by anion exchange chromatography on a MonoQ (Pharmacia) column and screened with a panel of commonly used substrates of serine/threonine protein kinases. Using a 40S ribosomal S6 protein C-terminal peptide, we detected a phosphotransferase activity that was markedly stimulated in cytosolic extracts as compared to extracts from immature oocytes (Fig. 5A). The MonoQ fractions containing S6 kinase phosphotransferase activity were probed with anti-peptide fC ^/i fC antibodies against the p70 amino terminal (Fig. 5B), the p70 carboxyl terminal (Fig. 5C) and subdomain III of p705W\ All three antibodies recognized a protein at ~70 kDa, the characteristic size of the p7056A:, notably in fraction 26 which contained the peak phosphotransferase activity. The three antibodies also immunoreacted with a protein at ~52 kDa. MonoQ fractionation provided a partial purification, but there were certainly more than one kinase present in the fractions. Therefore, to further identify and characterize the kinase that was responsible for the S6 peptide phosphotransferase activity, I undertook to attempt its purification. 52 H VO H U <o CZ2 i o ON 90 VO ir, 00 vo o 00 vo s e c _o c o e o 03 CQ U ([ui/uiui jad puid) XjiApac asBjajsuBJjoudsoqd 9s in <U 03 0> £ -O ft 53 3.2.2 Enrichment of a 40S ribosomal protein S6 kinase from mature sea star oocytes by sequential column chromatography Column chromatography matrices with a vast array of biochemical properties are commercially available. The columns allow for protein separation and purification based on the particular biochemical characteristics of the protein of interest. Some of the more common resins include ion exchangers, hydrophobic interaction, affinity, and size exclusion or gel filtration. During the course of the purification attempted here, many columns with differing biochemical properties and affinities were tested for retention of kinase activity, separation of contaminating proteins, and binding capacity. For each column tested, eluants were assayed for enzyme activity as well as protein concentration to ensure that separation of the enzyme of interest from contaminating proteins was accomplished. Table 1 lists the columns assayed, the elution buffers and retention of activity, as well as the relative purification attributed to the column. The reactive dye columns, which basically act as affinity matrices, were tested many times, both as early and late steps in the purification protocol. However, the columns that bound the S6 peptide phosphotransferase activity and provided adequate separation were not useful for purification at early stages due to low capacity, or at later stages due to the fact that nearly all contaminating proteins co-eluted. Some of the columns demonstrated differential binding depending on the point in the purification they were tested, presumably due to changes in protein concentrations. For instance, the heparin agarose column bound S6 peptide phosphotransferase activity when used early on in the procedure but did not bind this activity when used at later stages. This could be due to the presence of more than one kinase in the fractionations from earlier on in the purification. ATP-agarose was thought to be useful for purification, as this resin binds ATP-binding proteins. Unfortunately, similarly to the reactive dye columns, this column 54 Table 1 Summary of columns tested for S6 kinase purification Column tested Eluted with S6K bound Relative Relative activity purification Arginine-agarose 0-1.0 M NaCI yes +++ + ATP-agarose 0-1.0 M NaCI yes + NA Butyl-Sepharose 0-0.4 M NaCI no NA Cellex P 0-0.8 M NaCI no NA ++ Cibacron blue 3GA 0-2.0 M NaCI no NA Heparin agarose 0-0.8 M NaCI early-yes + ++ Heptyl-Sepharose 0-0.4 M NaCI no NA Histidine-L-agarose 0-1.0 M NaCI no NA Hydroxylapatite (HTP) 0-0.35 M KHP04 yes +++ +++ MonoS (pH 6.2) 0-0.8 M NaCI yes + + MonoS (pH6.8) 0-0.8 M NaCI yes + + Octyl-Sepharose 0-0.4 M NaCI no NA pll phosphocellulose 0-0.8 M NaCI no NA Phenyl-L-Sepharose 0-3% Brij no NA Polylysine (pH 6.2) 0-0.8 M NaCI yes +++ ++ Polylysine (pH 7.2) 0-1.0 M NaCI yes +++ +++ Q Sepharose (pH 6.5) 0-1.0 M NaCI yes + + Q Sepharose (pH 7.2) 0-0.8 M NaCI yes +++ ++ Q Sepharose (pH 8.0) 0-1.0 M NaCI yes +++ -Reactive blue 4 0-2.0 M NaCI yes + + Reactive blue 72 0-2.0 M NaCI yes + + Reactive brown 10 0-2.0 M NaCI no NA Reactive green 19 0-2.0 M NaCI no NA Reactive green 5 0-2.0 M NaCI no NA Reactive red 120 0-2.0 M NaCI no NA Reactive yellow 3 0-2.0 M NaCI yes ++ + Reactive yellow 86 0-2.0 M NaCI no NA S6 peptide column 0-1.0 M NaCI yes ++ ++ Superose 12 150 mM NaCI ++ + Tyrosine-L-agarose 0.8-0 M NH4SO4 yes ++ ++ Yellow Sepharose 0-1.0 M NaCI no NA 55 had low capacity, and when used as a later step in the purification, S6 peptide phosphotransferase activity co-eluted with many contaminating proteins. A column that did not bind the protein of interest but did bind other contaminating proteins could be used as a negative column to effect a substantial purification. I initially used a Cellex P phosphocellulose exchanger as a negative column as it bound many other proteins but the S6 kinase activity washed through the column. However, this matrix is no longer produced and the substitute, pi 1 phosphocellulose exchanger, exhibited different properties when used during the purification. The pi 1 column did not bind S6 kinase (similarly to Cellex P), but bound far fewer contaminants, and was therefore not an efficient purification step. Representative purifications are shown in Fig. 6 and Fig. 7. One partial purification consisted of six chromatography steps (Fig. 6A) and the peak MonoQ fractions were stained with silver (Fig. 6B) and autophosphorylated (Fig. 6C) as part of the characterization of the phosphotransferase activity. Hydroxylapatite, the initial column used, has a high capacity and is often used as an early step in protein purification protocols. The resin is a crystallized form of calcium phosphate and the biochemical interaction with proteins is an electrostatic binding to calcium and phosphate sites on the matrix. The column is normally eluted with an increasing phosphate gradient, in this case potassium phosphate. The peak phosphotransferase fractions were pooled, diluted to reduce the conductivity of the sample, and applied to a pi 1 phosphocellulose exchanger linked in series with a poly-L-lysine column. As mentioned above, the S6 kinase activity did not bind to the pi 1 phosphocellulose column and the activity washed through on to the poly-L-lysine column. As lysine is a positively charged amino acid, the column primarily acts as an anion exchanger, although it also exhibits some affinity characteristics. The peak phosphotransferase activity fractions eluted from the poly-L-lysine were pooled, diluted with buffer and applied to another anion exchanger, Q Sepharose. The peak fractions 56 A. Purification procedure Hydroxylapatite B. Silver stain i pll I Polylysine Q Sepharose J 106 k— 80 k— 49 k-36 38 40 42 44* 46 48 50 52 54 MonoQ fraction number C. Autophosphorylation S6 peptide column MonoQ 36 38 40 42 44* 46 48 50 52 54 MonoQ fraction number Fig. 6. Representative S6 kinase partial purification: The kinase was chromatographed over several steps (A) and the final MonoQ fractions were subjected to staining with silver (B) and autophosphorylation (C). The peak activity fraction was fraction number 44 (*) (See Fig. 7F), and the phosphotransferase activity was over 8000 pmol per minute per ml in a five minute assay. 57 0 10 20 30 40 50 60 70 80 30 35 40 45 50 55 60 poly-L-lysine agarose fraction number O MonoQ fraction number Fig. 7. Enrichment of a 40S ribosomal Protein S6 kinase: The S6 kinase activity ($) was purified to near homogeneity over a series of chromatography columns. The tyrosine agarose (A), hydroxylapatite (B) and polylysine agarose (C) fractionations include protein concentrations measured by Bradford assay (O). The protein levels were too low to measure in the final three purification steps: Q Sepharose (D), arginine agarose (E) and MonoQ (F). See Materials and Methods for the complete purification protocol. 58 from this column were loaded on to an affinity column prepared by linking the S6 peptide substrate to agarose beads (see Materials and Methods). This column, while exhibiting strong binding, co-eluted many of the contaminating proteins with the S6 kinase activity, and was not effective for purification. The fractions containing S6 peptide kinase were pooled and concentrated on a MonoQ anion exchange column. The peak activity fractions were electrophoresed on 11% SDS-PAGE and either stained with silver (Fig. 6B) or subjected to autophosphorylation (Fig. 6C). Many protein kinases undergo autophosphorylation, and often this intramolecular modification has an effect on kinase phosphotransferase activity. It can be seen in Fig. 6C that proteins at around 50-55 kDa in fractions 42 and 44 underwent strong phosphorylation. It is evident from Fig. 6B that there were still many contaminating proteins in the peak fractions, and it was impossible to unambiguously determine which protein corresponded to the S6 peptide kinase. The final enrichment of the S6 kinase phosphotransferase activity is outlined in Fig. 7 and the purification table is presented in Table 2. The optimized procedure (detailed in Materials and Methods) was completed over 2 days. As the S6 kinase activity was not labile, it was not significantly decreased by the length of purification treatment which ranged up to 4 days. The initial ammonium sulfate precipitation produced a substantial purification as approximately 20% of the total protein was precipitated at 0.8 M (NH4)2SC»4, while the S6 kinase remained in the soluble fraction. This treatment raised the ionic strength of the cytosolic fraction sufficiently to allow the kinase to bind to the hydrophobic L-tyrosine agarose resin (Fig. 7A). Hydroxylapatite (HTP) fractionation also provided significant purification as the kinase activity eluted from the column at ~0.10-0.21 M K2HPO4/KH2PO4 (Fig. 7B). Heparin-agarose was used to bind contaminating proteins, and the wash-through material containing the S6 kinase activity was directly applied to poly-L-lysine agarose. The phosphotransferase activity eluted from the poly-L-lysine agarose column at approximately 0.46 - 0.54 M NaCl (Fig. 7C). The S6 kinase activity was loaded on to an anionic Q Sepharose column, and eluted between 0.33 and 59 ation Id) o l/"> cn CN 1 ILL o o VO^ o o v© o o CD Purific CN cn ©" in Recovery (%) o o CN OO VO o IT) cn VO CN Activity riin/mg) 0.032 0.048 0.75 CN 00 1120 1600 Specific (nmol/ri Protein (mg) 10800 0009 o CN cn in 0.05 0.005 Activity lol/min) o cn VO 00 CN o CN r-- o cn VO oo Total (nrr o CN o VO O CN cn cn 00 CN o 1.25 11 > Column Step homogenate | L-tyrosine agarose | hydroxy lapatite | poly-L-lysine agarose | Q Sepharose |  L-arginine agarose MonoQ 0.40 M NaCl (Fig. 7D). Prior to this step, protein concentrations had been measured by Bradford (1976) assay, but due to low levels of protein present for the latter part of the purification, protein concentrations were estimated from pooled fractions. As the consensus sequence for phosphorylation of the S6 protein by known p70 isoforms contains several arginine residues, it was thought that an L-arginine agarose column might effectively act as an affinity matrix. The S6 kinase activity eluted from this column between 0.26 and 0.32 M NaCl (Fig. 7E). Finally, pooled fractions from the L-arginine agarose column were concentrated on a MonoQ column from which the S6 kinase phosphotransferase activity eluted between 0.30 and 0.35 M NaCl (Fig. 7F). Five MonoQ fractions (250 pi) containing the peak of S6 kinase phosphotransferase activity were pooled, aliquoted into 100 pi samples and immediately frozen at -70°C. The averaged results of three separate purifications are shown in Table 2. Beginning with ~11 g of cytosolic protein, ~5 pg of the enzyme was enriched over 50,000 fold with a final recovery of ~2% of the starting activity. For rapid and convenient screening, column fractions were assayed with a cocktail containing a peptide based on the known p70S6K phosphorylation sites in the S6 protein. The specificity of phosphorylation was enhanced in the kinase assays by the addition of a variety of inhibitors and activators. The peptide inhibitor (PKI) of protein kinase A (PKA) was routinely added to all assays. In addition, assays were performed in the presence or absence of calcium and lipid co-factors to ensure that protein kinase C (PKC) 32 * * isoforms were not present in the fractions. The concentration of [y- PJATP in the kinase assays was assessed, with 50 pM [y-32P]ATP final concentration found to be optimum (Data not shown). The peptide substrate is able to be phosphorylated by other kinases, while the 40S ribosomal protein S6 is selective for S6 kinases. Therefore, to ensure that the kinase activity being enriched was due to S6 kinase activity, the sea star enzyme preparation was assayed in the presence of purified 40S ribosomes. The S6 kinase was purified by a slightly different protocol (Fig. 8A), and assayed in the presence of 5 pg of 61 A. Purification procedure B. Phosphorylation of ribosomal protein S6 Hydroxylapatite Ammonium sulfate ppt. 1 Tyrosine agarose Polylysine \ Q Sepharose i S6 peptide column Superdex 200 MonoQ 50 k — 33 k — 28 k-S6 45 46 47 48 49 50 51 52 53 54 MonoQ fraction number C. Silver stain of ribosomal protein S6 18 k — 4. 45 46 47 48 49 50 51 52 53 54 MonoQ fraction number S6 Fig. 8. Phosphorylation of 40S ribosomal proteins by the sea star S6 kinase: The S6 kinase was enriched over several column chromatography steps (A). The kinase was incubated in the presence of 5 mg purified 40S ribosomes, and ADB for 30 min at 30°C. The samples were electrophoresed on 11% SDS-PAGE, transferred and exposed to film (B). The same fractions were stained with silver (C). The data shown was representative of three separate experiments. 62 purified 40S ribosomes, electrophoresed and the gel exposed to film (Fig. 8B). The same fractions were stained with silver (Fig. 8C), demonstrating similar amounts of total protein in each fraction. The fractions containing the peak phosphotransferase activity in filter paper assays with the peptide substrate (fractions 49 and 50) also contained the peak phosphotransferase activity against the S6 protein (-33 kDa) (Fig. 8B). Additional lower molecular weight protein bands were likely degradation products of the purified 40S ribosomes. A phosphorylated protein at about 50 kDa can also be seen in panel B, though the incorporation of phosphate was significantly less than that incorporated into the ribosomal proteins. This phosphorylation could be due to autophosphorylation of the kinase, or it could be due to phosphorylation of a contaminating protein present in the enriched preparation. 3.2.3 Immunological identification of sea star S6 kinase as homologue of p!0S6K The MonoQ fractions containing the enriched enzyme were electrophoresed on SDS-PAGE (11%) and the proteins were visualized by staining with silver (Fig. 9A). Several distinct protein bands coincided with the peak of S6 kinase activity, and these had apparent molecular masses of -60 kDa, 55 kDa and 52 kDa. The major protein at -52-55 kDa in the enriched preparation was identified as p70S6K by immunoblotting with a polyclonal antibody directed against the amino terminus of the mammalian enzyme (Fig. 9B). Several immunoreactive protein bands ranging in size from -50 to 55 kDa were apparent, perhaps reflecting differences in phosphorylation state or possibly, proteolyzed fragments of the same fractions did not immunoreact with the carboxyl terminal or subdomain III antibodies (data not shown). The enriched preparation was probed with a number of antibodies directed against a variety of protein kinases including casein kinase 2 (CK2), ribosomal S6 kinase (Rsk), ERK 1/2, p38 HOG1, SAPK, and PKCa, p and y. None of these antibodies demonstrated any immunoreactivity with the enriched fractions (Data not shown). 63 ir, ON ON NO © oo * NO 00 NO -H >r, ON ON NO © i-C3 ey T3 d ra CD ra CO T3 d ra o g 0 g +2 t>. Q C3 a a> s-a ^3 CQ -a o jS '-4—» d ra o ra u co ra IH *n d IT H ey +-> OH IZ CD SO 2 o GH u 43 < o 43 OH CO d £ o 43 CA u ra 60 T3 4-1 ra •a d ra 43 bp '53 jra o d CD CD O, S 6 U CD O <D o. co OH CD T3 OH <P O d 3 O © NO .S c 3 eg T3 d CS o 3 o d 3 c/3 ra CD bo w d -o ' d ra ra 4-H d o o ir. d _o *H—' CD ra CD ra iz ra OH co CD d 1/3 O CD '55 _9 0 -S OH H-> d ra O CD 1 "5 (90 <H-H a o CD > w a < PH o Q GO d o -a CD CO CD d o E CD 43 E— CD s ra co CD E— o fc ra 4= O 3 w d >> u H CO -9 CD QH IH ra rv (90 2 .2 CD Tl S-H « s 4H < 50 ON ey co es d 12 o -g jd ? P CD ra s> P J CO "8 3 t- co <D a S H .SPso ra -H _H © «r> <s NO «/i IT) 64 Many kinases have been shown to undergo autophosphorylation and this intramolecular event often correlates with changes in phosphotransferase activity (Ahn et al, 1992; Cobb et al, 1991). An autophosphorylation reaction of the purified kinase showed that protein bands at about 52-55 kDa became strongly phosphorylated as did several other, higher molecular weight proteins (Fig. 9C). Again, this phosphorylation could be due to autophosphorylation of the kinase, but could also be due to cross-phosphorylation of, or by, contaminating proteins. Size exclusion, or gel filtration chromatography is a form of separation based on the molecular size of proteins. The matrix contains pores of specific sizes that exclude proteins larger than that size. The elution is performed isocratically, usually in a salt buffer, and is thought to maintain protein complexes. Gel filtration analysis of the purified kinase yielded a single peak of phosphotransferase activity with an apparent molecular size of 50-55 kDa (Fig. 10). 65 80000 Superose 12 fraction number Fig. 10. Gel filtration chromatography of the enriched sea star S6 kinase: The purified kinase was chromatographed over a Superose 12 gel filtration column. The column was eluted with 150 mM NaCl, and the fractions were assayed for phosphotransferase activity with the peptide substrate. Standards applied to the column (200 pg each) were BSA (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen (25 kDa). The data shown was representative of three separate experiments. 66 3.3 Characterization of Active S6 Kinase from Mature Sea Star Oocytes 3.3.1 Immunological identification of partially purified sea star S6 kinase The -52-55 kDa enriched sea star S6 kinase immunoreacted with an amino terminal antibody, but was not recognized by antibodies generated against other regions of the kinase (Fig. 9). Further, antibodies directed against carboxyl and internal sequences of plQS6K, as well as the amino terminal antibody recognized -52-55 kDa proteins in MonoQ fractionated sea star extracts (Fig. 5). Therefore, immunoblot analysis of a partially purified form of the kinase was carried out. After two column steps (tyrosine agarose and hydroxylapatite) proteins were visualized by staining with Coomassie brilliant blue (Fig. 11 A). Several protein bands were apparent, especially a major protein at -75 kDa and another at -60 kDa. Polyclonal antibodies against the amino terminal (Fig. 1 IB), carboxyl terminal (Fig. 11C) as well as subdomain III (Fig. 1 ID) of the p7056/: recognized -75 kDa and -60 kDa proteins at this earlier stage of the purification. An -75 kDa protein was also recognized by the carboxyl terminal antibody in the MonoQ fractionated sea star extract (Fig. 5). Slight immunoreactivity was also seen at -52 kDa with the antibodies against the carboxyl terminal (Fig 11C) and subdomain III (Fig. 1 ID). Thus, it was possible that several distinct, homologous proteins were present in sea star, and that the purification procedure had effectively purified the -52-55 kDa form. 67 © r-c s 03 T r-VO c '« 41 3 "5 03 E o o U -r ON vo 41 se 3 fl w -* -a 53 vO la S3 03 4> oo T3 4* |B *C 3 O. Cl •rf ' oo CO 03 u H O it O PH o O U xi CO fl 03 .«i I- .3 a .N S 2 £ H a. o it- -fl O (X -a it e © es 41 s "•0 c 4> s u '3D o u -a <+-. o 3 o CO o ft 00 O -5 c -O 3 03 00 PH oo 3 w 2 -3 3 J HO -3 O o3 3 3 PH -a >^ 3 X 3 u p .5 s & c s -fl PH ^ * oi 1> 00 o In 03 bO 03 O -a 3 00 ii 03 oo O PH 00 -3 3 3 •a w -o 3 03 03 68 3.3.2 Dose- and time-dependence of sea star S6 kinase against the S6 peptide To optimize assay conditions for further analyses of the enriched kinase, its phosphotransferase activity with respect to concentration of peptide substrate and length of incubation was investigated. The phosphotransferase activity of the purified kinase was tested in filter paper assays with the S6 peptide at varying concentrations (Fig. 12A). At final concentrations below about 0.06 mg/ml, the phosphotransferase activity of the purified kinase was decreased, while concentrations over about 1.0 mg/ml were found to inhibit the kinase phosphotransferase activity. This inhibition could be due to steric hindrance caused by high peptide concentrations. It could also be due to the presence of contaminating kinases or phosphatases in the enriched preparation. The same phenomenon was also noted during the substrate specificity studies outlined in Section 3.3.8. The optimum concentration for activity was around 0.40 mg/ml, and this concentration of peptide was used for the following experiments unless noted otherwise. Dose response assays were carried out multiple times to obtain the data detailed in Section 3.3.8 characterizing the substrate specificity of the enriched kinase preparation. The time course of incubation with substrate was plotted as counts per min as well as pmol/min per mg (Fig. 12B). The optimum incubation time was from 5 to 10 min, and a 10 min incubation time was used for subsequent experiments. 69 A. S 1500 Peptide concentration (mg/ml) B. g 800000 a o -| • • • • i • • • • i • • • • i • • • • i • • • • i • • - - i o 0 5 10 15 20 25 30 PH Incubation time (min) Fig. 12. Concentration dependence and time course of the purified sea star S6 kinase: S6 kinase was incubated for 5 min in the presence of various concentrations of S6 peptide substrate (A), and with 0.40 mg/ml S6 peptide substrate for different incubation times (B). 70 3.3.3 Regulation of sea star S6 kinase by a variety of ions and inhibitors All protein kinases require metal co-factors, usually magnesium, for phosphotransferase activity. The enriched p52S6K utilized either magnesium or manganese for the phosphotransferase reaction. To further characterize the requirement ofp5256*for various ions and the effect of inhibitors on kinase activity, the enriched kinase was incubated in the presence of a variety of molecules, and the concentration that caused 50% maximal inhibition (IC50) was determined (Table 3). p52S6K was inhibited by sodium fluoride but was relatively insensitive to high concentrations of P-glycerol 2+ phosphate, EGTA, dithiothreitol, spermine, heparin, NaCI and metal ions such as Mn , Zn2+ and Ca2+. p70S6K was first purified from the livers of cycloheximide-treated rats (Price et al., 1989; Kozma et al, 1989) and from vanadate-stimulated Swiss 3T3 cells (Jeno et al, 1989). Both purified enzymes had a molecular size of-70 kDa. Ferrari et al. (1991) have characterized the p7056Ar that was purified and cloned from the livers of rats treated with cycloheximide. They found that the requirement for Mg2+ could not be substituted by Mn2+, and that Mn2+ actually inhibited the plQS6K activity at an IC50 of 100 pM. In addition, they found that Zn2+ was a potent inhibitor of plQS6K activity, with an IC50 of 2 pM. The enzyme was inhibited by protamine, and sodium fluoride, similarly to the enriched p52S6K (see Table 3 and Fig. 16). The S6 kinase purified from Swiss 3T3 cells was found to be inhibited by Mn2+ (Jeno et al, 1989). 71 Table 3 Summary of various inhibitors of purified sea star S6 kinase Compound Concentrations tested IC50 Zinc 2.5 - 150 mM 15 mM Calcium 2.5 - 250 mM 40 mM Manganese 2.5 - 250 mM 15 mM DTT 5.0 - 100 mM >100 mM EGTA 35 - 485 mM 300 mM Spermine 0.5 - 40 mM >40 mM NaCI 0.2 - 5.0 M >5 M NaF 15 - 300 mM 60 mM P-Glycerophosphate 60 - 800 mM >800 mM Heparin 2 - 200 mg/ml >200 mg/ml cAMP 1 - 20 pM >20 pM 72 3.3.4 Phosphorylation of 40S ribosomal proteins by sea star S6 kinase Although the S6 peptide substrate was very useful for quickly and efficiently assaying the S6 kinase phosphotransferase activity, the physiological substrate is the intact S6 protein. Therefore, the enriched sea star kinase was further characterized with respect to phosphotransferase activity against the 40S ribosomal protein S6. The kinase was incubated in the presence of varying amounts of purified 40S ribosomes, electrophoresed on 10% SDS-PAGE, transferred to nitrocellulose and exposed to film (Fig. 13). A protein evident at ~31 kDa corresponded to the predicted size of the intact S6 protein. The incorporation of phosphate into the S6 protein correlated with increasing quantity of purified 40S ribosomes added to the incubation mixture. The autoradiograph of the ribosomal proteins clearly showed phosphorylation of a protein at ~50 kDa similar to that seen in Fig. 8. 3.3.5 Photoaffinity labelling of sea star S6 kinase with ry-32P1azido-ATP Autophosphorylation reactions were carried out as part of the general characterization of the enriched protein kinase. Several proteins became phosphorylated during an autophosphorylation reaction (Fig. 9), and a protein at ~50 kDa became phosphorylated during incubation of the purified kinase with purified 40S ribosomal proteins (Fig. 8 and Fig. 13). It was unclear whether the kinase itself became phosphorylated, or if the signal seen was due to cross-phosphorylation of contaminating proteins in the preparation. To distinguish between these possibilities, photolabelling was carried out in the presence of [y-32P]azido-ATP. [y-32P]azido-ATP cross-links to ATP-binding proteins such as protein kinases so that the kinase cannot release the ATP. The enriched sea star S6 kinase was incubated in the presence of [y-32P]azido-ATP at 0°C (Fig. 14). The mixture was kept in the dark, and the [y-32P]azido-ATP was 73 40S ribosome concentration (units) Fig. 13. Concentration response of enriched S6 kinase with 40S ribosomal proteins: Varying amounts of purified 40S ribosomal proteins were incubated with the enriched kinase. The proteins were subsequently separated by SDS-PAGE (10%), transferred to nitrocellulose, and exposed to film. The 40S ribosomal subunits were standardized and measured as 100 U/ml reading the absorbance at 260 nM, with 1 unit equivalent to 5 mg of protein. 74 1 2 55 k 52 k Fig. 14. Photoaffinity labelling of enriched S6 kinase with [y-32P]azido-ATP: The enriched sea star kinase was incubated at 0°C with lOmM MgCl2 and lOOpM [y-32P]azido-ATP in the absence (lane 1) or presence (lane 2) of UV light at 254 nm at a distance of 2 cm. The reaction was terminated after 5 min by the addition of SDS sample buffer, and the samples were electrophoresed on 11% SDS-PAGE, and exposed to film. The data shown was representative of two separate experiments. 75 activated by a 5 min exposure to UV radiation. The reaction was terminated with SDS sample buffer, and the proteins were electrophoresed, and the gel exposed to film. A protein of about 52 kDa could be seen in the [y-32P]azido-ATP-treated sample, that was not present in the control sample. Based on immunoblot results, the ~52 kDa protein was identified as p52S6K, while the phosphorylated protein at -55 kDa was identified as protein phosphatase 2A (PP2A). The data regarding identification of PP2A will be discussed in detail in Section 3.4. 3.3.6 Phosphoamino acid and 2 dimensional phosphopeptide mapping of the 40S ribosomal protein S6 phosphorylated by sea star S6 kinase The S6 protein is known to become hyperphosphorylated in reponse to many various types of stimuli. The S6 phosphorylation sites consist of a series of five serine residues in a short, carboxyl region of the protein that are phosphorylated in a sequential manner (Bandi et al, 1993; Kreigera/., 1988; Martin-Perez and Thomas, 1983). Although other kinases can phosphorylate some of the S6 sites in vitro, only plOS6K has been shown to phosphorylate all five sites in the order that they are phosphorylated in vivo (Franco and Rosenfeld 1990). I therefore used the enriched sea star S6 kinase to phosphorylate a preparation of purified 40S ribosomes and the S6 protein was isolated prior to phosphoamino acid and phosphopeptide analysis. Purified 40S ribosomes (50 pg) were incubated with 50 uM [y-32P]ATP and enriched p5256* at 30°C for 30 min. The proteins were separated by SDS-PAGE, transferred to PVDF membranes, and subjected to phosphoamino acid (Fig. 15A) and 2D phosphopeptide analysis (Fig. 15B). The S6 protein was phosphorylated exclusively on serine residues, consistent with other reports for mammalian p7056A: (Bandi et al, 1993; Kreig et al, 1988). The 2D phosphopeptide map generated by the tryptic digest of the phosphorylated S6 protein produced at least 8 distinct radio-labeled spots (Fig. 15B) 76 A. Phosphoaminoacid analysis of the S6 protein phosphorylated by enriched p52 S6K B. Two dimensional tryptic map of the S6 protein phosphorylated by enriched p52S6K phosphate P-Ser P-Thr P-Tyr TLC electrophoresis -• + Fig. 15. Phophosphoamino acid and 2D phosphopeptide mapping of the 40S ribosomal protein S6 phosphorylated by enrched sea star S6 kinase: Fifty ug of 40S ribosomal proteins were incubated with the enriched kinase. The proteins were separated by electrophoresis and were subjected to phosphoamino acid analysis (A) and 2-dimensional phosphopeptide analysis (B). The data shown were representative of two separate experiments. 77 indicating several phosphorylation sites. Treatment of the most highly phosphorylated form of S6 from serum- or growth factor-stimulated cells with trypsin has been shown to generate up to 10-11 major phosphopeptides (Martin-Perez and Thomas, 1983; Martin-Perez et al., 1984), so it is possible that I have not induced full phosphorylation of the S6 protein with the sea star enzyme. The incubation time was 30 min and it is possible that further incubation was required for full phosphorylation to occur. 3.3.7 Phosphotransferase activity of sea star S6 kinase against a variety of exogenous substrates The enriched sea star S6 kinase was analyzed by in vitro filter paper assays with respect to its phosphotransferase activity against a variety of common exogenous serine/threonine kinase substrates (Fig. 16). Two separate preparations were assayed and the kinase activity was measured as a percentage of the phosphotransferase activity of the enriched enzyme against the S6 peptide substrate. Myelin basic protein (MBP) (-85%) and histone H2A (HH2A) (-45%) were both efficient substrates for the kinase while histone H2B (-10%), histone HI (-15%), casein (-5%) and protamine (-15%) were not efficient substrates. The p70S6K purified from cycloheximide-treated rats was found to phosphorylate histone H2B, glycogen synthase, and a number of hepatic nonhistone nuclear proteins in addition to efficiently phosphorylating the S6 protein (Price et al, 1989). The enzyme that was purified from Swiss 3T3 cells was found to phosphorylate the S6 protein, but did not phosphorylate casein, histones 2A and 3S, or phosvitin. To further analyze the phosphotransferase activity, MBP, HH2A and HH1 were phosphorylated by the enriched kinase, electrophoresed, and the gel was exposed to film (Fig. 17A). The phosphate-containing protein bands were excised, and subjected to phosphoamino acid analysis (Fig. 17B). MBP and H2A were phosphorylated exclusively on serine residues, while HH1 was phosphorylated on both serine and threonine residues. 78 Substrate (1.0 mg/ml) Fig. 16. Phosphotransferase activity of enriched sea star S6 kinase against a variety of exogenous substrates: The enriched kinase was incubated in the presence of a series of common protein kinase substrates, and the phosphotransferase activity was quantitated as a percentage of that of the S6 peptide. All the proteins were assayed at a final concentration of 1.0 mg/ml except the S6 peptide which was assayed at a final concentration of 0.4 mg/ml. The open and solid bars represent results from two separate purifications. 79 00 >> (3 s C3 !2 S3 03 e •s a 00 O 0. PQ Vi |-< n 3-X ft. CQ ffl CQ s o CJ C3 3 -2 S «> — H 03 oo 03 U oo S3 O CU H—» 00 03 2 vO •-C3 00 33 i> 00 T3 4> S I £ | 5 -a H § « 3 f T3 cu -3 O u -5 <+-o CU u c CO % CO oo <w 03 i-00 Xi 3 ag 00 3 © 3 cv cr. o X 41 PH u .3 -o to •»-» 03 -O 3 O 3 CO 0 — B a 1 § es o >-> 00 £ 3 JS ~o a. 3 SB 03 xa < a, CN a t~ cu 3 • 3. s "O 03 co x • .£ cu ^ oo «» 2 03 w b £ .2 JI Lo O cu >^ -3 ""a & 2 £ o T3 PH o3 3 . O S T3 3 <U «1 g 2 3 o 2 ^ _C H3 3 PH ^ •3 " co - O O ' .3 T3 3 03 U r-H P, SH O 13 -3 cu 2 8-S cu 3 00 >» -O "O CO <—' 03 H c3 oo T3 3 03 a x> U E3 00 .S CU oo C XS PH CO 00 PH 3 to S CU u CU P U P. u c PH PH O 80 Fig. 18. 2 dimensional tryptic phosphopeptide map of MBP phosphorylated by enriched sea star S6 kinase: Myelin basic protein was incubated in the presence of enriched S6 kinase. The proteins were separated by electrophoresis, transferred and the protein bands were subjected to 2 dimensional tryptic phosphopeptide analysis. 81 Phosphorylated MBP was further analyzed by 2-dimensional phosphopeptide digestion, and at least ten distinct phosphorylated spots were evident (Fig. 18). This was the first demonstration that MBP is a reasonable substrate for S6 kinase in vitro. 3.3.8 Substrate specificity of sea star S6 kinase The consensus sequence of optimal peptide substrates for mammalian p70S6A: has been determined (Flotow and Thomas, 1992) and the minimal sequence required for recognition was deduced to be R(R)RXXSX, where X could be any amino acid. The purified mammalian kinase had an apparent Km for 40S ribosomal subunits of 0.25 pM, and the apparent Km for a 32 amino acid synthetic peptide substrate was 2.5-fold higher. The enriched sea star p52S6K was therefore analyzed using synthetic peptides based on the C-terminus of the S6 protein. The peptides were constructed by step-wise alteration of specific amino acid residues surrounding the phosphorylation site. Each peptide was assayed a minimum of three times as described in the Material and Methods Section, the outlying values were discarded, and the Km values were estimated from linear Lineweaver-Burke plots (Table 4). As mentioned in Section 3.3.2, several of the peptide substrates actually inhibited the phosphotransferase activity at higher concentrations. It is possible that this observation was due to the presence of other kinases or phosphatases (see Section 3.4.1) in the enriched preparation. Kinetic analyses of the various peptides provided some intriguing differences from the previously reported consensus sequence for p70S6K isoforms. Flotow and Thomas (1992) found that the arginine residues in the -3 and -5 positions were essential for activity, while arginine in the -4 position was not as important. In our analyses, we found that an arginine residue in the -3 position was critical for kinase activity (Km and Vmax of 0.30 uM and 3.7 nmol/min per mg), but that arginine in the -4 position was dispensible and an additional arginine at the -5 position was actually inhibitory (Km and 82 Table 4 Summary of substrate specificity of purified sea star S6 kinase Peptide Sequence Km(pM) (apparent) v v max (nmol/min per mg) 1. AKRRRLSSLRASTSKSESSQK 0.22 7.3 33 2. RRRLSSLPvASTSKSESSQK 0.19 5.7 30 3. RRLSSLRAGGRR 0.27 3.6 13 4. RLASLRAGGRR 0.30 3.7 12 5. RRLASLRAGGRR 0.31 3.5 11 6. AAQKRPSQRTKYLA 0.34 3.6 11 7. RRLATLRAGGRR 3.91 23.8 6.1 8. RRLASERAGGRR 0.40 1.4 3.6 9. RRRLASLRAGGRR 0.77 1.8 2.4 10. AKRRRASA 0.79 0.9 1.1 11. AKRRRLS-amide 2.07 1.0 0.5 12. RRLASRAGGRR 8.40 0.7 0.08 83 Vmax of 0.77 uM and 1.8 nmol/min per mg) (compare peptide number 4, 5 and 9 in Table 4). In addition, it was found that an arginine residue at the +2 position enhanced kinase activity (compare peptide number 5 with 12). Replacement of the hydrophobic leucine at the +1 position with an acidic residue (compare peptide number 5 with 8) was also inhibitory to kinase activity. The sites in the S6 protein are phosphorylated sequentially in vivo in the order Ser-236>Ser-235>Ser-240>Ser-244>Ser-247 (Krieg et al, 1988; Martin-Perez and Thomas, 1983), so peptides with Ser-235 changed to alanine were tested (compare peptide number 3 with 5), and it was confirmed that Ser-236 was also preferentially phosphorylated by p52S6K. Another interesting observation was that changing Ser-236 to threonine (compare peptide number 5 with 7) resulted in Km and Vmax being affected in opposite directions. The S6 protein becomes phosphorylated strictly on serine residues in vivo, and clearly threonine was not efficiently phosphorylated by p52S6K in our assay. The consensus sequence for phosphotransferase activity of the enriched preparation was therefore found to be RXXSXR where the amino acid in the +1 position cannot be an acidic residue. The apparent Km and Vmax values for the best peptide substrates were approximately 0.2 pM and 6-7 nmol/min per mg respectively. It is certainly possible that substrate phosphorylation by S6 kinase is synergistic such that phosphorylation of one serine leads to increased phosphorylation of subsequent sites. This effect could explain why some serine residues that do not conform to the ideal consensus sequence can be phosphorylated in the S6 protein. 84 3.3.9 Inactivation of sea star S6 kinase by dephosphorylation The enriched p52MA was preincubated in the presence of protein phosphatase 2A (PP2A) or potato acid phosphatase (PAP) on protein A-Sepharose beads. The phosphatase reactions were inactivated by the addition of okadaic acid (PP2A) or by pelleting the beads by centrifugation (PAP), and kinase activity was measured in the presence of the peptide substrate (Fig. 19). Control samples were assayed in the presence of p-nitrophenylphosphate (p-NPP) to ensure that no residual phosphatase activity remained after addition of the inhibitor or removal of the PAP beads. The kinase became dephosphorylated and inactivated by pretreatment with either PP2A and PAP. The S6 kinase purified from liver of cycloheximide-treated rats was also completely deactivated by incubation with PP2A in both autophosphorylation and 40S S6 protein phosphorylating activities (Price et al., 1990). 85 Control PAP PP2A Fig. 19. Enriched S6 kinase is dephosphorylated and inactivated by potato acid phosphatase and protein phosphatase 2A: Enriched S6 kinase was pre-incubated in the presence of PAP or PP2A, then assayed for phosphotransferase activity against the S6 peptide substrate. The experiment shown here was the average of three separate assays (+/-SEM) 86 3.4 Complex Formation of Active Sea Star S6 Kinase 3.4.1 Identification of co-purifying proteins by micro-sequencing and immunoblotting As previously shown, several distinct protein bands were apparent in the enriched enzyme preparation by silver staining. Three of these had apparent molecular masses of -60 kDa, 55 kDa and 52 kDa. To produce enough material to allow micro-sequencing of the proteins, a large-scale purification was completed. An equivalent of five complete purifications (-3.5 liters) of mature cytosolic sea star extracts were used to generate approximately 35 pg of protein. The proteins were separated by electrophoresis, transferred to CD membrane and treated as outlined in the Materials and Methods section. The micro-sequencing was carried out in the laboratory of Dr. Ruedi Aebersold, first at the Biomedical Research Center (Vancouver, B.C.) and later at the University of Washington (Seattle). Micro-sequence analysis identified the -60 kDa protein as HSP-60, an abundant heat shock protein that co-purified with the kinase (Table 5). Four separate peptides were sequenced (T4, T5, T6 and T7). Of these, T4 was identical in 10 of 12 amino acids to the published sequence of human HSP-60. T5 was identical in 3 of the 3 amino acids that were unambiguous, T6 was identical in 8 of 8 residues, and T7 was identical in 6 of 8. In addition, the -55 kDa protein and the -52 kDa protein were identified as the regulatory subunit of protein phosphatase 2A (PP2A) by protein microsequence analysis (Table 5). The Tl peptide was a perfect match to Drosophila PP2A alpha subunit in all 16 amino acids sequenced, while the T2 peptide was identical in 8 of 11 residues. The T3 peptide was identical in 9 of 10 residues to the porcine PP2A beta subunit. The same samples were subsequently stained with silver (Fig. 20A) and immunoblotted with antibodies directed against HSP-60 (Fig. 20B) and PP2A (Fig. 20C). The irrimunoblots confirmed the identification and presence of HSP-60 and PP2 A in the 87 Table 5 Alignment and Identification of Sequenced Fragments Band ID Protein MW Peptide Number Sequence Identification Band 1-52 kDa Tl GEYNVYSTFQSHEPEF PP2A-ALPHA T2 MFEEPEDPSNR PP2A-ALPHA Band 2-55 kDa T3 LSSGDWFTSR PP2A-BETA Band 3-60 kDa T4 IGGSSDVEVNEK HSP60 T5 XXXFXDN HSP60 u T6 GANPVEIR HSP60 ii T7 VQDAMNAT HSP60 Band 1 Alignment with PP2A-ALPHA PP2A 61ANPRRGEYNV YSTFQSHEPE FDYLKSLEIE EKINKIRWLQ QKNPVHFLLS11 Tl GEYNV YSTFQSHEPE F PP2A 181YSSSKGTIRL CDMRSAALCD RHSKQFEEPE NPTNRSFFSE IISSISDVKL T2 MFEEPE DPSNR Band 2 Alignment with PP2A-BETA PP2A 121PSDLEAHFVA LVKRLASGDW FTSRTSACGL FSVCYPRVSS AVKAELRQYF1 T3 LSSGDW FTSR Band 3 Alignment with HSP60 HSP60 121RSIAKEGFEK ISKGANPVEI RRGVMLAVDA VIAELKKQSK PVTTPEEIAQ170 T6 GANPVEIR HSP60 301KAPGFGDNRK NQLKDMAIAT GGAVFGEEGL TLNLEDVQPH DLGKVGEVIV T5 XXXFXDN HSP60 391NERLAKLSDG VAVLKVGGTS DVEVNEKKDR VTDALNATRA AVEEGIVLGG4 T4/T7 IGGSS DVEVNEK VQDAMNAT 55 -fi a, < 0-C C3 B '3 of) s-> i-C3 B o 00 53 co 1? "3 B 03 - o 3 « a on gj O < CN C+H o o .-a "C o cu C c HC CO O VO B o 60 03 O (X) o 03 IM 03 OH CJ OH . S 03 OH fl OH H-< too <+H CJ o 73 C C3 T3 3 03 PH CM 8 o 03 ^~ •15 -H K*1 O vo CO a 03 -rH 03 O CJ -3 CJ '«M> 03 4-* a cu 00 CJ IH CH CJ >H 00 cci a O T3 •rt cu «fi 4) cu cu > 5 s* B o ,3 oo 3 "3 c3 -a cu CJ CJ .2 U ^ <U 33 CJ es es 3 T3 CU oo CJ © © C s S SM es pq OH O »- < O- CH oo 'ii es Q fi CO - 15 ^° on o\ to co -H rt 03 CJ -3 H U o VC -a o -O '3 ol fH HH 00 *S r/N +-> C CJ O CJ o (H OH CH CO T3 5 03 CJ OH CJ 89 enriched preparation. In spite of repeated attempts, we were unable to obtain sequence data for a kinase in the preparation. We speculate that the sea star S6 kinase was masked by the presence of contaminating proteins in the final sample, especially PP2A. As the sequence data identified both the -52 kDa and the -55 kDa proteins as regulatory subunits of PP2A, it was possible that PP2A was much more abundant than the kinase in the enriched preparation, and thus prevented its identification by micro-sequencing. (See Discussion). 3.4.2 Co-immunoprecipitation of sea star S6 kinase, HSP-60 and PP2A It was found that the S6 kinase co-purified over a number of column chromatography steps with an HSP protein and PP2A. We therefore speculated that these proteins may form a multi-unit complex in vivo. It was also possible that the kinase activity, or the activity of PP2A or HSP-60 might be regulated in some way by the interaction. Co-immunoprecipitation experiments were undertaken to determine if the p70S6K was interacting with other proteins. The S6 kinase was first immunoprecipitated from mature sea star oocyte extract with the N/T antibody, and probed with the same antibody to confirm that the antibody could immunoprecipitate the kinase (Fig. 21 A). The antibody immunoprecipitated a protein at -65 kDa. No protein bands were detected at -52-55 kDa, possibly due to the presence of the immunoglobulin (Ig) band. It was also possible that the the 52 kDa form of the S6 kinase was simply a degradation product of the p70 isoform, which was not present in the mature extract, but was produced during the purification protocol. The same immunoprecipitates were then probed with anti-HSP-60 antibody. The S6 kinase and a protein of -80 kDa were co-immunoprecipitated and the -80 kDa protein was recognized by antibody directed against HSP-60 (Fig. 2IB). The S6 C/T antibody 90 vO CO IT) fT> O s H Z N CO © 03 CQ* F— Z to © TS = C3 NO CO ON ON NO tt NO co 91 and the subdomain III antibody failed to irnmunoprecipitate the S6 kinase in sea star (Data not shown). Protein phosphatase 2A (PP2A) was then immunoprecipitated from partially purified sea star extract with antibody directed against the regulatory alpha subunit (Fig. 21C). The immunoprecipitates were assayed in the presence of 40S ribosomes and were shown to contain phosphotransferase activity against the 40S ribosomal S6 protein under these conditions. There was no phosphotransferase activity seen in the control lane (antibody and protein A Sepharose beads in the absence of sea star extract). We were unable to obtain immunoreactivity of PP2A in p70S6K immunoprecipitates, or immunoreactivity of p70S6K in PP2A immunoprecipitates. It was previously demonstrated that a protein at ~55 kDa became phosphorylated when the enriched sea star preparation was incubated in the presence of ATP (Fig. 8, 9 and 13). This protein was distinguished from p52S6K by incubation in the presence of azido-ATP (Fig. 14), by immunoreactivity with the PP2A antibody (Fig. 19), and by micro-sequencing (Table 5). The -55 kDa PP2A protein band was subjected to phosphoamino acid analysis (Fig. 22). Incorporation of phosphate was seen exclusively on serine residues, demonstrating that PP2A was a substrate for and was phosphorylated by active p52S6K in vitro. These findings supported the possibility that PP2A, HSP-60 and p52S6K might be associated in a high molecular weight complex. It is important to note however, that the gel filtration data (Fig. 10) was inconsistent with the presence of a multi-protein complex, as the peak of phosphotransferase activity eluted from the column had an estimated molecular mass of-50-55 kDa. It is possible that unidentified molecule(s) required for maintenance of the complex had been purified away during the purification procedure, leaving only the highly active kinase (See Discussion). It is also possible that the PP2A and HSP-60 were simply contaminating proteins that had co-purified with the kinase. 92 Fig. 22. Phosphoamino acid analysis of p55 phosphorylated by enriched sea star p52s6K: The enriched S6 kinase preparation was incubated in the presence of ATP. The proteins were separated by SDS-PAGE, transferred to PVDF and the phosphorylated 55 kDa protein was subjected to phosphoamino acid analysis. 93 3.5 Functional Studies of Sea Star S6 Kinase 3.5.1 Immunoprecipitation of active S6 kinase from immature and mature sea star oocytes In Section 3.2,1 described the enrichment of an active S6 kinase from mature sea star oocytes and demonstrated that it was the sea star cognate of plQS6K. It was possible that the activation of plQS6K was generated by proteolysis during the purification procedure. Therefore, to compare activity from immature and mature sea star oocyte extracts, the S6 kinase was immunoprecipitated with an antibody directed against a carboxyl terminal region of the p70S6A (Santa Cruz), and the amino terminal (N/T) antibody. The immunoprecipitate formed with the C/T antibody was electrophoresed on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the same antibody (Fig. 23A). An immunoreactive protein was apparent at ~70 kDa in both immature and mature oocyte extracts, although there appeared to be slightly more immunoprecipitated kinase in the mature oocyte extract. Immunoprecipitations with both antibodies were then immunoblotted with the N/T antibody (Fig. 23B) and protein bands were detected at -70 kDa in both immunoprecipitates. It appeared that there was increased immunoprecipitated kinase present in mature oocyte extract as compared to immature. Immunoblots of crude mature and immature oocyte extracts showed that the same amount of protein was present in each sample, so it is unclear why more immunoprecipitatable S6 kinase was present in the mature samples as compared to the immature. No immunoreactive proteins were seen at -52 kDa, but if p52S6K was immunoprecipitated, the protein would be obscured by the presence of the immunoglobulin (Ig) protein band at -52-55 kDa. The immunoprecipitates were assayed with the S6 peptide substrate and the phosphotransferase activity was plotted as counts per min (Fig. 23C). The mature 94 A. anti-C/T IP I mm Mat B. 67 k — 51 k anti-N/T IP anti-C/T IP Imm Mat Imm Mat 67 kH 51 k—^ anti-C/T blot anti-N/T blot anti-C/T IP anti-N/T IP E a SJ *w/ O t> '& u 03 QJ in 03 J-,0) e 03 Q. O 1500 -1000 500 Mature Imm Mature Imm Fig. 23. Immunoprecipitation of active p70SVi/ir from immature and mature sea star oocytes A sample of 125 pi of crude immature or 90 min post 1-methyladenine-treated sea star cytosol was added to 125 pi high salt NEFT buffer containing 6% NP-40. The S6 kinase was immunoprecipitated with 5 pi of antibody as outlined in Materials and Methods. The proteins were electrophoresed on 12.5% gels, transferred to nitrocellulose and immunoblotted with anti-C/T (A) or anti-N/T (B) antibodies. The immunoprecipitates were assayed in duplicate using the S6 peptide substrate, and the results were pooled for determination of phosphotransferase activity (C). The data shown were from a single experiment. 95 oocyte extract contained about 2- to 3.5-fold of the phosphotransferase activity of the immature oocyte extract, consistent with the kinase being activated during sea star oocyte maturation. However, the increase in activity could be due to the fact that there was more immunoprecipitated kinase in the mature oocyte extract as compared to immature. The cytosolic extract used for the immunoprecipitation experiments was from more recent sea star preparations than the extract used for the previous experiments, and the S6 peptide kinase activity in these extracts had been inconsistent. It was therefore possible that the S6 kinase was already partially activated in the immature oocyte extract, or conversely, the S6 peptide kinase activity may have been reduced in the mature oocyte extracts. 96 3.5.2 Immunoblot analysis of heat shock-treated immature and mature sea star extracts Heat shock proteins interact with various cellular proteins in response to stresses such as osmotic shock and irradiation. For instance, PKB has been shown to interact with heat shock proteins in response to stress-stimuli (Konishi et al, 1997). As an abundant heat shock protein (HSP-60) was found to co-purify with the sea star S6 kinase, it was possible that HSP-60 and p70S6K may form a complex in vivo. To investigate this possibility, a time course of heat shock treatment was undertaken. Oocytes from individual P. ochraceus were resuspended in natural sea water preheated to 35°C. At various times, aliquots were removed and frozen at -70°C. The aliquots were fractionated on Resource Q, assayed for kinase phosphotransferase activity and probed with a variety of antibodies. The peak kinase activity fractions immuno-reacted with all three plQS6K antibodies (Fig. 24A). The subdomain III antibody recognized a protein at ~63 kDa that appeared to increase in expression with length of incubation time, as well as a ~65 kDa protein that decreased in amount with increasing time at 35°C. The C/T antibody also recognized the -65 kDa protein, as did the N/T antibody. We concluded that the -65 kDa protein corresponded to p7056^, which became degraded during heat shock. The N/T antibody also recognized a protein at -52 kDa, a size similar to that identified in the purified sea star kinase preparation. The same fractions were probed with anti-HSP-60 antibodies, and a protein at -65 kDa which was unaffected in expression levels during the heat shock period was seen (Fig. 24B). The same fractions were also probed with antibodies directed against the alpha subunit of PP2A (Fig. 24B). An immunoreactive protein could be seen at -65 kDa which was also unaffected in expression levels during the heat shock treatment. These results demonstrated that PP2A and HSP-60 may be present in the same fractions as p7056A:, and was consistent with the possibility that these proteins may interact in vivo. 97 ©N 00 V© V© 00 00 V© ri ca ©V 00 V© V© 00 00 se es s-+-M •-es .9. S E °-JH © XS S3 i e3 ed O >> -o CJ CO n3 CQ es -H T3 c es 41 la 3 es cu E cu T3 * •S 2 CJ rt cu CO rt 3 es cu -. <B i _M cu © rt -a CJ > O E ^ cu vo •-1 CO U IM L> ^ T3 CJ >> rt -a 9 -a CJ £ -8 CO g rt CO O w r-- o CM VO to CM a co 'rt ffi ox) ^ rt co T3 .9 cu rt o 3 cy co UM ™ -a CJ cu (50 cu * .a cu cu 3 cu in CJ D-X cj cu To a T3 O es cu •a © '35 >> "es a es rt -TJ a rt u o in rn rt a cu o o co CJ OH XJ O CJ CU CJ CJ cu It, T3 a o rt X> +-> 'a a a co rt J+M co a 3 T3 rt « rt 3 O •2 JE CU " cu <u CJ IM CJ £ o -3 co rt -fn rt -o cj X) H CJ CJ CJ rt PH O >» % « r> CO a x> y o rt cj X o a a E oi E co M3 U CJ ? co IM CO CO 8 § £ xT co es rt u cu X! CO f— rt IM <4M CO 3 rt i. -t-> O ,3 OH CO O CJ IM CJ OH CO S E rt rt Xi X3 OH H CO CO X) < CN CM PH t+-O 3 3 X) 3 co O rt "3 SO CJ ©v 00 v© v© 00 00 m V© 98 3.5.3 Expression of active recombinant S6 kinase from baculovirus Yields of enriched, active kinase obtained from mature sea star oocytes were very low, and the process was labour intensive. The S6 kinase is highly conserved among various species, therefore we speculated that expressed kinase from other species could be used to screen sea star extracts to identify potential upstream regulators of p7QS6K, or for a variety of other biological experiments such as micro-injection. We obtained clones for human p70S6K from Dr. James Woodgett (Ontario Cancer Inst.). The p70S6K was expressed and purified from baculovirus infected Sf9 cells as detailed in the Materials and Methods section. The GST-S6K baculovirus/Hi5 cells were stimulated with 25 or 50 nM PMA for 25 or 15 min to further activate the p70S6K phosphotransferase activity. The PMA-treated GST-S6K was then electrophoresed, transferred and immunoblotted with the N/T antibody (Fig. 25A). The antibody recognized an immunoreactive protein at ~85 kDa, the expected size of the expressed GST-linked kinase. Protein levels in all the samples were approximately equivalent (data not shown). To test the activity of the expressed kinase, the PMA-treated GST fusion protein was assayed in the presence of myelin basic protein (MBP) and the S6 peptide substrate (Fig. 25B). The kinase, at ~85 kDa, became phosphorylated during the incubation. In fact, incorporation of phosphate into the expressed kinase was enhanced in the presence of MBP (lanes 2 and 5 in Fig. 25B), and inhibited in the presence of the peptide substrate (lanes 3 and 6 in Fig. 25B). A possible explanation could be that in the presence of the preferred substrate (S6 peptide), there is less incorporation of phosphate into the kinase whereas in the presence of the less preferred substrate (MBP), more phosphate was incorporated into the kinase. In any event, the expressed kinase exhibited significant phosphotransferase activity against the substrates and will be useful for future experiments. The 25 min 25 nM PMA treatment produced the most highly active preparation of p70S6K. 99 tt UO i H in U TJ Cu CO Q. CU Da NO CO I I "i—r ON NO OO NO 00 00 ro NO <N CC o £ 55 CO ^ CD 4-T CD ,fD HH 03 X CD 8 43 cs TJ d CD 3 | NO +3 CO co e .5 cs > fl o co • 3 b E 2 -2 ^ d CJ c3 O JO 1- NO IM OO d ^ fl t>0 .2 o c/> >N W o ir, d of CD 4-» 03 X> fl CD fl W O < _>» -a °r CD ?? oo > 3 Q t3 ^ ^ Si 03 — s § ° CD i/-, fi eo IJ t+3 CD ^ 5 2 o 3 43 a °< CN >> 2 -fl c2 g B o fl I g VH .S -O O CD CD -H- »?H CN IH IH XJ °? H CD t-H "3 00 d 3 a g £ » _fl •3 £ o co H o « E o * CD CD fl CD CD i- 43 OH OH CD P 43 CD IH CD o d CD O - 43 CD co H^ ra ra 03 4D d CO CD T3 ra -a CD 43 <£ OH ^ OH S vo ts oo 0 CD 1 g E 3 I « •a & ra o "d S3 CD g c2 3 CD CQ o H—I -a CD CO C OH X CD -o C ra TJ CD CO fl ra IH CD OH X CD cu CiD C 100 3.5.4 Microinjection studies The human form of p70 was expressed in the baculovirus system. The kinase was cleaved from GST beads with thrombin, and prepared in PBS in the absence of proteases or phosphatase inhibitors. Although echinoderm and human are very divergent, the p7056/: is known to be very highly conserved amongst species. We therefore speculated that the expressed human form of the kinase may have an effect on the maturation process in sea star. To test this hypothesis, preparations of the active human kinase and PBS controls were microinjected into immature sea star oocytes to determine the effect of the active kinase on maturation as identified by germinal vesicle breakdown (GVBD). Control cells were treated with 1-methyladenine, and underwent normal maturation in every set of experiments (data not shown). Comparison of PBS-injected oocytes with S6 kinase-treated oocytes showed that microinjection of purified, active S6 kinase had no obvious effect on maturation of the oocytes (Fig. 26). The same proportion of oocytes appeared to undergo normal maturation in treated and untreated samples (Table 6). 101 Fig. 26. Microinjection of p!0S6K into immature sea star oocytes: Microinjection was carried out as detailed in the Material and Methods Section. Immature sea star oocytes were microinjected with active, expressed S6 kinase (A) or PBS (B). The pictures were taken 3 1/2 hours post injection. In panel A, the maturation was 2/26 while in panel B the maturation was 3/24 oocytes. 102 Table 6 Microinjection of purified human p70S6K into sea star oocytes Date Microinjected Number of oocytes matured 09-12-1996 p70S6K 2/30, 3/37, 5/38 Buffer B + 0.4 M NaCI 0/24, 1/34 06-06-1997 p70S6K 3/26, 1/38 PBS 0/26 07-31-1997 p70S6K 3/21,5/24, 5/18, 2/26 PBS 3/21,5/21 103 Chapter 4: Results and Discussion: Photodynamic Therapy and it's Effect on the Phosphorylation and Activation State of p70 4.1 Introduction and Model System In heat shock-treated sea star oocytes, the levels of p70S6K appeared to decline. In the next series of studies, we sought to investigate how p70s6K was affected in a mammalian cell system in which stress signaling responses were activated. It was previously reported that photodynamic therapy (PDT) with benzoporphyrin derivative mono-acid ring A (verteporfin) (BPD-MA) caused a marked, ROS-mediated activation of both stress-activated protein kinase (SAPK) and p38 HOG1 without affecting the activity of extracellularly regulated protein kinase 1 and 2 (ERK1 and ERK2) in the transformed keratinocyte cell line Pam212 (Tao et al, 1996). Although p70S6K does not appear to be involved in the apoptotic pathway, much evidence has placed it downstream of the growth factor-induced activation of PI 3-kinase and PKB, both of which have been shown to block the induction of apoptotis (Kennedy et ai, 1997; Kulik et al, 1997; Kaufmann-Zeh et al, 1997). We were therefore interested in analyzing the PI 3-kinase/PKB/p70S6* pathway in Pam212 cells treated with PDT, to evaluate the effect of PDT on this pathway. Keratinocytes are the major component of the epidermis that is affected by UV irradiation and PDT treatment. In response to these treatments, keratinocytes release cytokines such as TNFa, TGFp, IL-10, IL-8 and GM-CSF. We have observed many similarities between UV and PDT in both cell culture and animal models, therefore, increased understanding of the signaling pathways in PDT-treated keratinocytes may improve our understanding of the actions of PDT and ROS on p7QS6K and its regulation at the molecular level. We therefore undertook analysis of some of the signaling components in response to PDT in Pam212 cells. 104 4.2 Initial Observations: PDT Effect on p70 4.2.1 Effect of PDT on activation of v70MK Initially, we immunoprecipitated active p70S6K from crude Pam212 cell extracts treated with anisomycin, UV irradiation and PDT (Fig. 27). The immune complexes were assayed for phosphotransferase activity and electrophoretic mobility shift (see Material and Methods). PDT treatment caused a profound suppression of S6 peptide phosphotransferase activity in cells maintained in a medium supplemented with 5% FCS. The enzyme activity of the PDT-treated samples was about 20% that of untreated controls. The untreated control cells had a high basal S6 peptide activity that was stimulated 2- to 3-fold by anisomycin and UV irradiation. Immunoblot analysis revealed the presence of a high molecular weight, activated form of p70S6K in the samples derived from untreated cells, BPD-MA-treated, anisomycin-treated or UV-treated cells. This activated form of p70S6K was not detected in PDT-treated samples. These results indicated that PDT inhibited the phosphorylation and phosphotransferase activity of serum-activated p70S6K. 105 Fig. 27. Effect of PDT on activation of p705,i*: Pam212 cells cultured in DMEM supplemented with 5% FCS were treated for 5 min with 200 ng/ml BPD-MA only, 40 mg/ml anisomycin, 200 ng/ml BPD-MA plus 2 J/cm2 of light, or 4 min exposure of UV radiation. p70 S6 kinase was immunoprecipitated from crude cell lysates using anti-p7056Ar N/T antibody, and the immunoprecipitated kinase was incubated in the presence of the peptide substrate and assayed for phosphotransferase activity. The immunoprecipitates were electrophoresed, transferred and immunoblotted with the same antibody. Data from three independent experiments were pooled. Untreated controls exhibited an enzyme activity of about 10,000 counts per minute in a 10 minute incubation., -fc , statistically significant difference between samples derived from variously treated cells and from control cells. Bars, S.D. 106 4.2.2 Dose- and time-dependence of PDT effect on p70' We investigated the relationship between the dose of PDT and inhibition of the phosphorylation and phosphotransferase activity of p70 . Pam212 cells maintained in a medium supplemented with 5% FCS were treated with various doses of PDT (Fig. 28). The light dose was fixed at 2 J/cm2, and the BPD-MA dose varied from 5 to 250 ng/ml. Thirty min after treatment, cells were lysed, and p70S6K was immunoprecipitated from the crude cell lysates with anti-p705<5A: C/T antibody (Santa Cruz). The immunoprecipitates were assayed for phosphotransferase activity using the S6 peptide as a substrate, and were immunoblotted with the same antibody. The results show a correlation between the dose of BPD-MA and the extent of inhibition of both the phosphorylation and the phosphotransferase activity of p70S6K. We went on to evaluate the time-dependence of the kinase activity to treatment with PDT. Pam212 cells were maintained as above, and crude cell lysates were prepared at different times following PDT at a dose of 200 ng/ml BPD-MA and 2 J/cm2 of light (Fig. 29). p70S6K was immunoprecipitated, assayed and immunoblotted as above. The earliest inhibition of p70S6K was noted at 10 min following PDT treatment while maximum inhibition was observed 30 to 40 min following treatment. The duration of the inhibition persisted during the course of the experiment and there was no recovery of p70S6K activity up to 2 h after PDT treatment. Immunoblot analysis demonstrated that the change in kinase activity correlated with a shift in electrophoretic mobility. Thus, the inhibitory effect of PDT on the phosphorylation and phosphotransferase activity of p70S6K was both dose- and time-dependent. 107 BPD-MA concentration (ng/ml) Fig. 28. BPD dose response of PDT inhibition of p7056A:: Pam212 cells maintained in a medium supplemented with 5% FCS were pre-incubated with various doses of BPD-MA ranging from 5 ng/ml to 250 ng/ml for 12 h. Subsequently the cells were exposed to 2J/cm2 red light. The whole cell extracts were prepared 30 min after PDT treatment. p70S6K was then immunoprecipitated, assayed, and immunoblotted. Data from three independent experiments were pooled. Control activity was approximately 10,000 counts per minute in a 10 minute incubation. Bars, S.D. 108 ihosphotran of control) in © i 1 ..IT 100 If50 • GO R 0 -II i •.. I H 11 *S a .9 J3 j^&Jt*. ^g^^ ^^^^^ ^Mg^^ ^dgwk% n,n nrifWi M^TFFE •filfiiffi - P7056* 1- antibody 0 2.5 5 10 20 30 45 60 120 > Time (min) ^ Fig. 29. BPD time course of PDT inhibition of p7056* : Pam212 cells maintained in a medium supplemented with 5% FCS were treated with 200 ng/ml BPD plus 2 J/cm2 red light. After PDT, cultures were incubated at 37°C for additional periods of time ranging from 2.5 min to 2 h. p705<w was then immunoprecipitated, assayed, and immunoblotted. Data from 3 independent experiments were pooled. Untreated controls exhibited an enzyme activity of about 10,000 counts per minute in a 10 minute incubation. Bars, S.D. 109 4.3 Analysis of the PI 3-kinase /PKB/_p7(TA Pathway in PDT Treatment 4.3.1 Effect of insulin on PI 3-kinase. PKB and p7056*~ in serum-starved Pam212 cells It has been reported that mitogenic stimulation caused strong activation of PI 3-kinase, PKB and plQS6K (Alessi et al, 1996; Chung et al, 1994; Mukhopadhyay et al, 1992; Price etal, 1990). There is also circumstantial evidence that PI 3-kinase and PKB may be upstream mediators of p70S6K activation (Bourewijn et al, 1995; Chung et al, 1994). Since PDT had a profound inhibitory effect on p70S6/\ we were interested in the effect PDT may have on these upstream kinases. Initially, we investigated the effect of insulin stimulation on the three enzymes. Serum-starved Pam212 cells were treated with 100 nM of insulin for various periods of time (Fig. 30). PI 3-kinase, PKB and p70lS6/f were immunoprecipitated from crude cell lysates, and assayed for activity. PI 3-kinase activity was measured by immune complex assay using PI as a substrate. This was followed by separation with thin-layer chromatography and radiography. The activity of PKB and p70S6K was assayed in filter paper assays using HH2B or the S6 peptide as substrates. We observed a 2-fold increase in PI phosphotransferase activity 1 min following insulin stimulation. The peak activation was detected at 20 min post insulin treatment. A significant activation of HH2B phosphotransferase activity was noted at 5 min following insulin stimulation. A 5-fold peak activation was detected at 30 min post insulin treatment and the activation persisted during the course of the experiment. The time course of p70S6K activation was similar to that of PKB. These results demonstrated that Pam212 cells responded to insulin stimulation in a manner consistent to that previously reported for other cell types. Therefore, we could use the Pam212 cell system to study the effect of PDT on this pathway. 110 500 + i 400 300 200 100 Untreated 1 10 20 30 60 Time (min) Fig. 30. Insulin-induced activation of PI3-K, PKB and p70S6K. Pam212 cells were cultured in DMEM supplemented with 5% FCS until 75% subconfluent. The cells were then maintained in DMEM supplemented with 0.5% FCS for 36 h, then serum-starved for 12 h. The resting cells were stimulated with 100 nM insulin for various periods of time as indicated. Crude whole cell lysates were immunoprecipitated with antibodies directed against PI 3-K (N-SH2), PKB-PH, or p7056* N/T. The immune complexes were assayed using PI, HH2B or the S6 peptide substrate. The data shown were representative of two separate experiments. Untreated controls exhibited an enzyme activity of about 4,000, 500 and 2,000 counts per minute in a 10 minute incubation, for PI 3-kinase, PKB and p70S6K, respectively. Ill 4.3.2 Effect of PDT on insulin-induced activation of PI 3-kinase. PKB and p7(r° in serum- starved Pam212 cells The activity of PI 3-kinase was measured by immune complex assay using PI and PI 4.5-P2 as substrates (Fig. 31). There were 2-fold increases in PI and PI 4,5-P2 phosphotransferase activity detected at 1 min post insulin treatment. Pre-incubation with 100 nM wortmannin completely abolished the insulin induced PI 3-kinase activity, in agreement with previously published reports (Ui et al, 1995). As expected, pre incubation with 25 ng/ml of rapamycin did not affect the insulin-induced activation of PI 3-kinase. However, pre-treatment with PDT totally inhibited activation of PI 3-kinase. Immunoblot analysis indicated that the antibody immunoprecipitated approximately equivalent amounts of protein. PKB was assayed using HH2B as a substrate (Fig. 32). A 6-fold increase in HH2B phosphotransferase activity was detected at 10 min post insulin treatment. The insulin-induced HH2B phosphotransferase activity was partially inhibited by pre incubation with wortmannin. Rapamycin pre-treatment did not significantly affect the insulin-induced activation of PKB. Like wortmannin, PDT at least partially inhibited insulin-induced HH2B phosphotransferase activity. Immunoblot analysis of the immunoprecipitated PKB demonstrated that the amount of protein immunoprecipitated was approximately the same. p70S6K was assayed using the S6 peptide as a substrate (Fig. 33). An 8-fold increase in S6 phosphotransferase activity was detected at 10 min post insulin treatment. In addition, 8- to 10-fold increases in S6 phosphotransferase activity were detected at 10 min stimulation with 100 ng/ml of EGF. As expected, both wortmannin and rapamycin completely blocked the inductive effect of insulin and EGF. Pretreatment with PDT totally abolished insulin- or EGF-induced increase in S6 phosphotransferase activity. Anti-p705<5A: immunoblot analysis demonstrated that the pattern of electrophoretic 112 93 S-s e u ,<u !/> S3 T5 I- 41 *- -W O C3 o 5 © OH -5 IT) w u -J H tt © i * es B • * t f * Origin I- PI 3-K antibody ^c ^ ^ ^ * Fig. 31. PDT inhibits insulin-induced activation of PI 3-K: Serum-starved Pam212 cells were treated with 2 J/cm2 of red light. Thirty min after PDT or following a 15 min pre incubation with 100 nM wortmannin or 25 ng/ml rapamycin, cells were stimulated with 100 nM insulin for 1 min. The immunoprecipitates were assayed by TLC using PI as a substrate. Identical immunoprecipitates were electrophoresed, transferred and immunoblotted. Data from three independent experiments were pooled. Beads control represents protein A sepharose beads incubated with extract in the absence of antibody. Untreated controls exhibited an enzyme activity of about 4,000 counts per minute in a 10 minute incubation. statistically significant difference between samples derived from variously treated cells and that from untreated control cells, -fc , statistically significant difference between samples derived from variously treated cells and that from insulin-treated cells. Bars, S.D. 113 Fig. 32. PDT inhibits insulin-induced activation of PKB: Serum-starved Pam212 cells were treated with 2 J/cm2 of red light. Thirty min after PDT or following a 15 min pre-incubation with 100 nM wortmannin or 25 ng/ml rapamycin, cells were stimulated with 100 nM insulin for 10 min. The immunoprecipitates were assayed using HH2B as a substrate. Identical immunoprecipitates were electrophoresed, transferred and immunoblotted. Data from three independent experiments were pooled. Beads control represents protein A sepharose beads incubated with extract in the absence of antibody. Untreated controls exhibited an enzyme activity of about 500 counts per minute in a 10 minute incubation. statistically significant difference between samples derived from variously treated cells and that from untreated control cells. , statistically significant difference between samples derived from variously treated cells and that from insulin-treated cells. Bars, S.D. 114 Fig. 33. Effect of PDT on growth factor-induced activation of p70S6K : Serum-starved Pam212 cells were stimulated 10 min with 100 ng/ml EGF or 100 nM insulin. Cells were pre-incubated with 100 nM wortmannin, 25 ng/ml rapamycin for 15 min, or treated with PDT prior to growth factor stimulation. The immunoprecipitates were assayed, electrophoresed, transferred and immunoblotted. Data from three to five independent experiments were pooled. Untreated controls exhibited an enzyme activity of about 2,000 counts per minute in a 10 minute incubation. statistically significant difference between samples derived from variously treated cells and that from untreated control cells. , statistically significant difference between samples derived from variously treated cells and that from growth factor-treated cells. Bars, S.D. 115 mobility shift correlated with the results of the in vitro kinase assay. Both insulin and EGF stimulation resulted in an electrophoretic mobility shift that correlated with the activation of the kinase. From these results, we concluded that PDT caused a profound inhibition of insulin-induced activation of PI 3-kinase and PKB as well as the insulin- or EGF-induced activation of p70S6K. 116 4.3.3 Effect of UV irradiation on insulin-induced activation of PI 3-kinase, PKB and p70S6K in serum-starved Pam212 cells UV irradiation has been shown to induce stress-response and apoptosis in many cell types (Chen et al., 1996). Generation of ROS is thought to be the mechanism for the biological and biochemical effects of both UV and PDT (Zhang et al., 1997; Kochevar, 1995; Girotti et al, 1990). It was previously shown that the PDT-induced activation of SAPK and p38 HOG1 was at least partially mediated by ROS (Tao et al, 1996). In addition, both ERK1 and ERK2 were strongly activated by treatment with H2O2 and we had previously observed activation of PI 3-kinase and PKB by treatment with H2O2 (Data not shown). Therefore, there may be common upstream regulators modulated by ROS in the signaling pathways mediated by UV and PDT. We therefore wanted to compare the effect of either UV irradiation or PDT on the insulin-induced activation of PI 3-kinase, PKB and p7056* in serum-starved Pam212 cells. The cells were pretreated with PDT or 4 min UV irradiation. The dose and conditions of UV treatment were similar to those used to activate SAPK and p38 HOG1 (Tao et al, 1996). At various times after UV irradiation, cells were stimulated with 100 nM insulin for 10 min before cell extracts were prepared. Immunoprecipitates were analyzed with in vitro kinase assays as previously described. When the cells were given insulin 5 min after UV irradiation, there was a similar level of inhibition of PI phosphotransferase activity as was observed following PDT treatment (Fig. 34). The inhibition became less pronounced with the increase in the interval between UV irradiation and insulin stimulation. However, UV irradiation alone, PDT alone, or PDT plus UV irradiation did not significantly affect the basal level of PI phosphotransferase activity. UV irradiation did not inhibit the insulin-induced HH2B phosphotransferase activity of PKB (Fig. 35). Indeed, there was a potentiation of the insulin response 117 Insulin Fig. 34. Effect of UV irradiation on insulin-induced activation of PI 3-kinase: Serum-starved Pam212 cells were treated with 100 ng/ml BPD plus 2 J/cm2 of red light, 4 min exposure to UV light, or PDT prior to UV irradiation. At various times after treatment, cells were stimulated with 100 nM insulin for 1 min. The immunoprecipitates were assayed by TLC using PI as a substrate. Identical immunoprecipitates were electrophoresed, transferred and immunoblotted. Data from three independent experiments were pooled. Beads control represents protein A sepharose beads incubated with extract in the absence of antibody. Untreated controls exhibited an enzyme activity of about 4,000 counts per minute in a 10 minute incubation. statistically significant difference between samples derived from variously treated cells and that from untreated control cells. # , statistically significant difference between samples derived from variously treated cells and that from insulin-treated cells. Bars, S.D. 118 Fig. 35. Effect of UV irradiation on insulin-induced activation of PKB: Serum-starved Pam212 cells were treated with 100 ng/ml BPD plus 2 J/cm2 of red light, 4 min exposure to UV light, or PDT prior to UV irradiation. At various times after treatment, cells were stimulated with 100 nM insulin for 10 min. Immunoprecipitates with anti-PKB-PH antibody were subsequently assayed using HH2B as a substrate. Identical immunoprecipitates were electrophoresed, transferred and immunoblotted. Data from three independent experiments were pooled. Beads control represents protein A sepharose beads incubated with extract in the absence of antibody. Untreated controls exhibited an enzyme activity of about 500 counts per minute in a 10 minute incubation. statistically significant difference between samples derived from variously treated cells and that from untreated control cells. ^ , statistically significant difference between samples derived from variously treated cells and that from insulin-treated cells. Bars, S.D. 119 between 30 and 60 min following UV irradiation. Like that of PI 3-kinase, the phosphotransferase activity of PKB was not significantly affected by UV irradiation alone, PDT alone, or PDT plus UV irradiation without insulin stimulation. Except for a partial inhibition observed in cells treated with insulin 5 min after UV irradiation, the effect of UV on insulin-induced S6 phosphotransferase activity in the immunoprecipitates was not significant (Fig. 36). However, the phosphotransferase activity of the immunoprecipitates was increased 2- to 3-fold in the cells treated with UV irradiation alone. The UV-induced S6 phosphotransferase activity was completely abolished by pretreating the cells with PDT. Immunoblot analysis demonstrated the presence of the high molecular weight, activated form of p70S6K in the samples derived from insulin-treated cells. This high molecular weight species was not detected in the samples derived from the cells pretreated with PDT. The pattern of electrophoretic mobility correlated with the in vitro kinase assay results. These findings indicated that the effect of UV irradiation on insulin responses was different from that of PDT. Pretreatment with UV irradiation inhibited insulin-induced PI 3-kinase activation. However, insulin-induced PKB activiation was potentiated in cells stimulated with insulin 30 min after UV irradiation. UV irradiation did not significantly affect insulin-induced p7QS6K activation, but UV irradiation alone induced 2- to 3-fold C" IS CjC If activation of p70 . The UV-induced activation of p70 was blocked by pre-treating the cells with PDT. Therefore, although the effects of UV irradiation and PDT on insulin-induced activation of PI 3-kinase were similar, they were very different with respect to the insulin-induced activation of PKB and plQS6K. 120 Insulin Fig. 36. Effect of UV irradiation on insulin-activation of p70S6K: Serum-starved Pam212 cells were treated with 100 ng/ml BPD plus 2 J/cm2 of red light, 4 min exposure to UV light, or PDT prior to UV irradiation. At various times after treatment, cells were stimulated with 100 nM insulin for 10 min. The immunoprecipitates were assayed using the S6 peptide as a substrate. Identical immunoprecipitates were electrophoresed, transferred and immunoblotted. Data from three independent experiments were pooled. Beads control represents protein A sepharose beads incubated with extract in the absence of antibody. Untreated controls exhibited an enzyme activity of about 2,000 counts per minute in a 10 minute incubation. statistically significant difference between samples derived from variously treated cells and that from untreated control cells. * f statistically significant difference between samples derived from variously treated cells and that from insulin-treated cells. Bars, S.D. 121 4.3.4 Effect of PDT on anisomycin-induced activation of p70' We have demonstrated that PDT caused profound inhibition of growth factor-induced activation of PI3-kinase, PKB and p70S6K. It is unknown whether the inhibitory effect of PDT on p!0S6K phosphotransferase activity was a consequence of inhibition of the activity of PI 3-kinase, PKB, or other proximal targets upstream of p70S6K. That the inhibition of UV-induced activation of p70S6K by PDT was independent of PI 3-kinase and PKB favored the second possibility. To address the question of whether PDT inhibited upstream targets other than those in the PI 3-kinase /PKB pathway, we investigated the effect of PDT treatment on anisomycin-induced activation of p70S6K. Serum-starved Pam212 cells were treated with 40 pg/ml of anisomycin for various periods of time up to 90 min (Fig. 37A). PI 3-kinase, PKB and p70S6K were immunoprecipitated from the crude cell extracts and assayed in vitro as described previously. There was no significant increase in PI phosphotransferase activity in the PI 3-kinase immunoprecipitates for the duration of the experiment. Similarly, there was no significant change in the phosphotransferase activity of the immunoprecipitated PKB. However, 1 min after anisomycin treatment, there was a marked, 2.5-fold increase in the PiT Tf Q£ If S6 phosphotransferase activity of p70 . The anisomycin-induced activation of p70 peaked at 20 to 30 min post anisomycin treatment and lasted for the duration of the experiment. These results indicated that anisomycin-induced p7t)S6K activation was independent of PI 3-kinase and PKB. We then investigated the effect of PDT on the anisomycin-induced activation of p7QS6K (Fig. 37B). Thirty min treatment with 40 pg/ml anisomycin induced a 9-fold increase in S6 phosphotransferase activity of p70S6K. The activation was completely blocked when cells were pre-treated with PDT or 25 ng/ml rapamycin. The activation of p70S6K was also largely inhibited by pretreatment with 100 nM wortmannin. 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T3 3 03 CO M4 33 § & H3 CJ 3 cd X) 3 CJ fl I CJ HH 3 cj -a 3 CJ PH CJ 13 G CJ CJ 111 t> co *^ £ ^3 "3 2 £ 3 U -a -2 ,3 - 8 3 cu tJ •2 |"8 P 2 £ £ X) 3 CJ 3 O c+-H—• ca H3 HH -a 3 03 CJ CJ TS CJ H 03 CJ 3 cu CU X^ co 3 O D 3 a-S £ fe 8 ca 123 in the samples derived from insulin-treated cells. This high molecular weight species was not present in the samples derived from cells pretreated with wortmannin, rapamycin or PDT. These results support the conclusion that the inhibitory effect of PDT on anisomycin-induced p!0S6K activation was independent of the PI 3-kinase and PKB pathway. 124 Chapter 5: General Discussion, Future Directions and Conclusions 5.1 Introduction p7QS6K is a mitogenically activated protein-serine/threonine kinase whose only known physiological substrate is ribosomal protein S6 (Pullen and Thomas 1997). The kinase mediates increased protein translation of 5'TOP (terminal oligopyrimidine tract) mRNAs. This group represents a small family of transcripts essential for the onset of protein synthesis, such as ribosomal subunits and translation initiation factors. In spite of the fact that the number of individual genes containing 5'TOP sequences is small, these RNAs are predicted to make up 15-20% of total cellular RNA (Meyuhas et al, 1996), and it is likely that the kinase that plays a major role in the induction of these genes is essential for mitogenesis. Therefore, a clearer understanding of the role of plQS6K in signaling and during the maturation process will contribute to our understanding of these processes. This study was undertaken to attempt to purify and characterize the sea star homologue of plQiS6K, and to use the purified kinase to attempt to clarify the role the kinase plays in maturation in sea star oocytes. The signaling events leading to the activation of plQ>S6K are complex, and have been difficult to unravel. We had previously noted that photodynamic therapy (PDT) with benzoporphyrin derivative mono-acid ring A (verteporfm) (BPD-MA) led to activation of the SAPK and p38 HOG1 pathways in a transformed murine keratinocyte cell line, Pam212 (Tao et al, 1996). In this system, p!0S6K activity was profoundly inhibited in response to PDT. We therefore undertook an investigation of the role of p70S6K as well as PKB and PI 3-kinase in Pam212 cells treated with PDT. 125 5.2 p70 in Pisaster ochraceus 5.1.1 Identification of \>52S6K as a homologue of p70S6K and regulation of the kinase activity I have described, for the first time, the partial purification of a ribosomal S6 kinase from an echinoderm source. The kinase was identified by screening immature and mature sea star extracts that had been fractionated by MonoQ chromatography. An S6 peptide phosphotransferase activity that became activated during maturation was enriched through a series of chromatography steps. The seven column-step procedure yielded extremely active protein kinase phosphotransferase activity, although protein yields were very low. Immunoblot analysis of purified proteins from early stages of the purification produced immunoreactive bands at -70-75 kDa and -52 kDa with three different antibodies directed against the amino terminal, carboxyl terminal and internal sequences of p70S6K. During progressive chromatography steps, the antibodies immunoreacted with proteins at -70 kDa as well as at -52 kDa with the smaller form of the protein immunoreacting much more strongly with the amino terminal antibody. In the purified preparation, a protein at - 52 kDa was recognized by the amino terminal antibody only. I interpreted these findings to indicate that two distinct isoforms of p70S6K are present in sea star, and that the purification procedure detailed in this study substantially enriched the p52S6K isoform. As discussed earlier, previously characterized isoforms of p70S6K contain a pseudosubstrate, auto-inhibitory region in the carboxyl terminus that likely regulates activity. It is possible that this region was proteolytically cleaved during the purification procedure, leading to purification of a hyperactivated form of the kinase. This would be consistent with the observed result (see Section 3.3.5) that the partially purified kinase did not undergo a significant amount of autophosphorylation. Indeed, if the kinase was 126 already maximally activated, it would not be expected to undergo further autophosphorylation. Although the model of proteolytic activation during purification is one explanation for the observed results, it is also possible that a distinct 52 kDa isoform of plQS6K had been enriched. This will ultimately be established by molecular cloning of the 52 kDa isoform from sea star oocytes. This was not undertaken during the course of this study due to the difficulty in obtaining an mRNA library in sea star. The sea star p52S6K phosphorylated the S6 peptide substrate as well as the 40S ribosomal protein S6 and the phosphotransferase activity was stable for months at -70°C, allowing it to be analyzed over a period of time. p52S6K was further characterized with respect to ion and co-factor dependence and it was found that the kinase was inhibited by fluoride, but relatively insensitive to most other common treatments, similar to other published reports for p70S6K (Jeno etal., 1989; Ferrari et al, 1991), as discussed in Section 3.3.3. The sea star p52S6K efficiently utilized myelin basic protein and the 40S ribosomal protein S6 as substrates, and phosphoamino acid analysis of the S6 protein showed that the kinase phosphorylated S6 exclusively on serine. Two-dimensional phosphopeptide mapping of the 40S ribosomal proteins revealed phosphorylation of 8 distinct tryptic peptides, fewer than that of previously identified p70S6K isoforms (Bandi et al, 1993; Martin-Perez and Thomas, 1983; Martin-Perez et al, 1984). This result was likely due to insufficient incubation time of the kinase with its substrate as discussed in Section 3.3.6. I went on to examine the substrate specificity of echinoderm p52 using a series of synthetic peptides modeled after the sites in the S6 protein known to be phosphorylated by plQ>S6K (R232RLS*S*LRAS*TSKS*ESS*QK). The sites in plQS6K are phosphorylated in a sequential manner in vivo, in the order Ser-236>Ser-235>Ser-240>Ser-244>Ser-247 (Krieg et al, 1988; Martin-Perez and Thomas, 1983). Enzyme kinetics for Km were determined using the Michaelis-Menton rate equation and Vmax was estimated using 127 Lineweaver-Burke double-reciprocal plots of dose-response data. Kinetic analyses of the various peptides (see Table 4 and Section 3.3.8) provided some intriguing differences from previously reported consensus sequences for p!0S6K. Of particular interest was the finding that an arginine residue in the +2 position enhanced kinase activity, unlike previous reports (Flotow and Thomas 1992). This finding may at least partially explain why Ser-236 is the first residue to be phosphorylated by the p7056A" in vivo. The initial phosphorylation event may then allow phosphorylation of the other sites to proceed. Unfortunately, in spite of many attempts, we were unable to obtain micro-sequence data for the purified sea star kinase. One possible explanation for this was the fact that the enzyme was present in such small quantities. Two proteins that were present in the final purification, PP2A and HSP-60, are much more abundant in cells than protein kinases. It is possible, therefore, that these two proteins physically masked the presence of the kinase, preventing us from obtaining sufficient quantities for micro-sequencing to be successful. Nevertheless, the basic biochemical characterization of the kinase activity supports its identification as an echinoderm homologue of plQS6K. The kinase was recognized by antibodies to p705<5/\, the substrate specificity was similar to, though not identical to published reports and the phosphotransferase activity was active against 40S ribosomal subunits and was inactivated by dephosphorylation. It would be desirable in the future, to attempt to produce enough material to obtain micro-sequencing data. One potential strategy would be to utilize 2-dimensional gel electrophoresis to better separate the protein contaminants from the kinase. The recent availablility of more sensitive antibodies will also facilitate identification of the kinase band on immunoblots. 128 5.1.2 Multi-protein complex formation Interestingly, the two proteins that co-purified with p52 were identified by immunoblot analysis and by direct protein micro-sequencing, as a heat shock protein (HSP-60) and the regulatory subunit of PP2A (Table 5 and Fig. 19). Further, I have co-immunoprecipitated S6 kinase phosphotransferase activity with anti-PP2A specific antibodies, and co-immunoprecipitated S6 kinase with HSP-60 (Fig. 21). In addition, PP2A in the purified preparation was phosphorylated on serine residues, consistent with its phosphorylation by the p52S6K in the preparation (Fig. 22). These results raise the intriguing possibility that the sea star S6 kinase may form a multi-protein complex in vivo and the activity of the S6 kinase may be regulated by it's association with PP2A and/or HSP-60. In collaboration with Dr. Brian Wadzinsky (Vanderbilt University, Nashville), we have also observed that purified rat brain PP2A complexes with p70S6K (unpublished observations). Immunoblot analysis of heat shock-treated sea star oocytes demonstrated that the sea star S6 kinase, HSP-60, and PP2A were present in the same fractions and that the protein levels of S6 kinase seemed to decrease in response to this treatment (Fig. 24). Co-purification of the three proteins over seven different column chromatography steps, combined with the co-immunoprecipitation studies, as well as the results of the heat shock-treatment immunoprecipitations support the possibility that these proteins interact in vivo. However, gel filtration analysis of the enriched kinase (Fig. 10), contradicts this hypothesis since the phosphotransferase activity eluted at a molecular size of about 50-55 kDa. Although gel filtration is thought to be a gentle process that can maintain proteins and complexes in native form, it could be that the protein-protein interactions within the complex were weak, and the components may have become denatured during this step. It was also possible that an essential component for maintenance of the integrity of the 129 complex had been purified away during a previous purification step. Alternatively, it is also possible that the S6 kinase/PP2A complex interacted with the gel filtration resin, eluting with a lower apparent molecular weight. It was also possible that the larger isoform (p7QS6K) was required for interaction with PP2A, HSP-60 or both. This could explain why co-immunoprecipitation of S6 kinase with HSP-60 and immunoprecipitation of phosphotransferase activity against the 40S ribosome with anti-PP2A antibodies needed to be carried out using either crude, cytosolic sea star extract or partially purified (two or three steps) sea star S6 kinase. Several potential lines of investigation could be pursued from these results. Future experiments could evaluate which regions of p70S6K are important for binding to PP2A and HSP-60, and which subunits or regions of PP2A and HSP-60 are critical for binding to p70 . A series of p70 point and deletion mutants could be created, expressed, and tested for their ability to bind to PP2A and HSP-60 purified from other sources. In addition, we speculate that the formation of PP2A complexes with p70S6K may be reduced or altered in human tumours. Immunological techniques could be used to evaluate quantitiative changes of these proteins in human tumours as compared to matched, normal tissue. Functionally, the effect of p70S6K association with PP2A and HSP-60 on it's phosphotransferase activity could be evaluated. It would be expected that the association of PP2A with p70 would decrease the catalytic activity of p70 . This could be tested by assessing the activities of the various active, recombinant components through enzyme assays and gel electrophoresis. 5.2.3 Functional aspects of p70S6K signaling Decreased activity of p70 has been implicated as playing a role in GI arrest (Calvo et al, 1992; Morice et al, 1992). Presumably, this blockage is at least partially due to the decrease in protein translation in response to decreased protein S6 130 phosphorylation by p70S6K. During sea star oocyte maturation, dramatic increases in the rate of protein translation occur, and it is likely that the activation of p70S6K during maturation plays a critical role in the onset of increased translation. Active, human p70S6K was expressed using the baculovirus expression system and the active kinase was microinjected into immature sea star oocytes to measure the effect of this treatment on the maturation process. This had no discernible effect on the maturation process and injection of active p70S6K did not induce maturation in the absence of 1-methyladenine. As p70S6K activation is a relatively late event in the signaling pathway, many other cellular events would be expected to be required to support the processes leading to maturation. It seems likely that a number of alterations or pertubations in signaling pathways would be necessary to disrupt such an important process, and cells undoubtedly have other redundant routes of signal transmission. To identify an upstream activator of p70S6K, immature sea star extract could be fractionated on MonoQ and fractions assayed with expressed, inactive p70S6K. This would permit identification of fractions containing potential p70S6K kinases, which could then be purified and characterized. Further, the inactive, expressed kinase, or a dominant-negative S6 kinase could be microinjected into oocytes to assess whether such a construct could block or delay oocyte maturation. As the phosphorylation of the S6 protein is an integral and obligatory step in the initiation of translation of specific proteins required for mitosis, the purification of p52S6K described here will provide the means to further dissect the pathways leading to cell division and growth in a variety of systems. In addition, studies could be conducted to determine the role, if any, of HSP-60 and PP2A in the regulation of p70S6K or p52S6K. Specific kinase-phosphatase complexes may represent a novel means by which cellular signaling may be regulated. 131 5.3 PI 3-kinase/PKB/ p70 Signaling in PDT 5.3.1 Effect of PDT Although the molecular events by which PI 3-kinase activates PKB are rapidly being uncovered (Cohen et al, 1997), the signaling events leading to activation of FRAP, p!0S6K, and 4E-BP1 have proved much more difficult to unravel. Removal of the amino and carboxyl termini of p70 generates a rapamycin-resistant mutant that retains sensitivity to wortmannin (Dennis et al, 1996). This indicates that FRAP and PI 3-kinase signal to plQ>S6K via different routes. FRAP may be constitutively active, with downstream regulation of p!0S6K in response to mitogenic stimulation provided by signaling from PI 3-kinase. Recent data indicates that different kinases phosphorylate OA If Q/C If 4E-BPlandp70 . First, the rapamycin-sensitive sites in p70 are flanked by large, aromatic amino acids, while the rapamycin-sensitive sites in 4E-BP1 are proline-directed If C/C If sites (Pearson et al, 1995). Furthermore, overexpression of p70 blocks reporter p70 activation and prevents phosphorylation of the rapamycin-sensitive sites (Von Manteuffel et al, 1997). Significantly, overexpression of pl§S6K also blocks phosphorylation of the rapamycin-sensitive sites in 4E-BP1 (Von Manteuffel et al, 1997), indicating that the overexpressed p70 acts by sequestering an upstream activator, possibly FRAP. This hypothesis would be consistent with the notion that although FRAP lies upstream of p70S6K, and may be required for its activation, another kinase responsible for phosphorylation of the sites surrounded by bulky aromatic amino acids is also required for full p70S6K activity. Two recent publications have identified PDK1, known to activate PKB, as also activating p70S6K by phosphorylation of Thr-229 (Pullen et al, 1998; Alessi et al, 1998). The kinase that phosphorylates p70S6K at Thr-389 remains to be identified, although a recent report suggests that this site may be regulated by ILK (Delcommenne et al, 1998). To further investigate the role of p7QS6K, PKB and PI 3-kinase, we carried out 132 analysis of photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A (BPD-MA) treatment in a transformed murine keratinocyte cell line, Pam212. We have shown for the first time that PDT caused a dose- and time-dependent inhibition of serum-induced p70S6K activation in Pam212 cells. PDT also blocked insulin-induced activation of PI 3-kinase, PKB and p70 in serum-starved cells. In addition, PDT blocked anisomycin-induced activation of p70S6K in these cells. We analyzed the phosphotransferase activities of PI 3-kinase, PKB and p70 by immunoprecipitation followed by in vitro kinase assays using standard substrates. PI 3-kinase activity was assessed by demonstrating incorporation of phosphate into PI and PIP2 respectively, while PKB phosphotransferase activity was assessed by incorporation of phosphate into HH2B. The p70S6K phosphotransferase activity was measured both by incorporation of phosphate into the S6 peptide substrate, as well as by immunoblot analysis of the well established electrophoretic mobility shift due to increased phosphorylation during activation of the kinase. For the analysis of the effects of PDT on insulin, anisomycin and UV treatment of Pam212 keratinocytes we included control samples derived from the cells treated with well established pharmacological inhibitors of these kinases of interest. Rapamycin is a specific inhibitor of FRAP that blocks the activation of FRAP and p70S6K by all known mitogens (Brown et al, 1994; Sabatini et al, 1994). In Pam212 cells, rapamycin inhibited the activation of p7QS6K induced by all stimuli used, including serum, insulin, EGF, and anisomycin. As expected, rapamycin did not inhibit the activities of PI 3-kinase or PKB in these experiments. Wortmannin is an inhibitor of members of the PI 3-kinase family. This compound acts by binding covalently to the pi 10 subunit and inhibits PI 3-kinase at nanomolar concentrations (Yano et al, 1993). In Pam212 cells, wortmannin inhibited growth factor-induced activation of PI 3-kinase, PKB and p70 . Wortmannin also inhibited the anisomycin-induced activation of p7QS6K, though not to the same extent as it did the growth factor-induced activation. 133 Significantly, the results from immune complex assays indicated that the anisomycin-induced activation of p7QS6K was independent of PI 3-kinase and PKB, although the activation of p70S6K by anisomycin was wortmannin-sensitive. This can be explained by the fact that wortmannin may have other cellular targets, including FRAP (Brunn et al, 1996). Since FRAP may be an upstream component of growth factor-induced activation of p7QS6K, it is possible that the inhibitory effect of wortmannin on anisomycin-induced activation of p7t)S6K is a consequence of the inhibition of FRAP. We demonstrated high basal activity of p7QS6K, which was abolished by PDT, in Pam212 cells maintained in serum-containing medium. PDT treatment also blocked agonist-induced activation of p70S6K in serum-starved cells. As the signaling events leading to activation of p7QS6K are unclear, we investigated the role of two potential upstream signaling proteins, PI 3-kinase and PKB. We found that PDT also inhibited the insulin-induced activation of PI 3-kinase and PKB in these cells. It was unclear, however, whether the inhibitory effect of PDT was exerted directly on the individual kinases, or if it resulted from an upstream inhibition. To address this question, we investigated the effect of anisomycin on the three proteins. We found that anisomycin caused a strong activation of p7056^, in a PI 3-kinase and PKB independent manner. PDT completely blocked the anisomycin-induced activation of p70S6K. Thus, the PDT effect could be either direct, on p70S6K or on upstream kinases other than PI 3-kinase and PKB. We have also observed that UV irradiation and PMA activated p70S6K in a PI 3-kinase and PKB independent manner, and the activation was blocked by PDT. PMA is known to activate PKC, and thus PKC may be upstream of p70S6K activation in this system. It will be interesting to explore the relationship between PKC and p70S6K in anisomycin-and UV-induced p70S6K activation. Future investigations should characterize the effect of PDT on various isoforms of PKC and the role of PKC in anisomycin- and UV-induced activation of p70S6K in Pam212 cells. 134 The fact that PDT inhibited p70S6K by all agonists used indicated that PDT may act on proximal targets or may act directly on p70S6K by promoting dephosphorylation of serine/threonine residues required for activation of the kinase. Similarly, we could not conclude whether PDT effects on PKB and PI 3-kinase were direct or a consequence of inhibition above the level of PI 3-kinase. The data from the UV experiments provided some hints as to the answer to this question. We observed a significant inhibition of insulin-induced PI 3-kinase activation when the growth factor was given 5 min post UV treatment. The inhibition of PI 3-kinase decreased with increasing incubation time, yet this inhibition did not lead to a corresponding inhibition of PKB. Instead, there was a significant potentiation of the insulin-induced PKB activation, implying that the potentiation effect of UV irradiation on PKB was independent of PI 3-kinase activation. One explanation of these results is that UV irradiation could activate upstream kinases other than PI 3-kinase that act synergistically with insulin on PKB. A potential candidate for such a kinase is p38 HOG1. PKB has been shown to be activated by heat and osmotic shock in a PI 3-kinase independent manner (Konishi et al, 1996). UV, heat and osmotic shock are also known to activate p38 HOG1 (Kyriakis and Avruch, 1996). Therefore, in light of our results here, PKB may be activated via a p38 HOG1 dependent pathway. In this case, we should have seen a similar potentiation effect with PDT, since PDT also caused strong activation of p38 HOG1 (Tao et al, 1996), but we did not. Instead, insulin-induced activation of PKB was blocked when cells were given insulin 30 min later. Therefore, the effect of PDT on PKB may be mediated through two separate mechanisms, inhibition of PI 3-kinase, or a direct inhibition of PKB or its proximal activating kinase, PDK1. In Pam212 cells, UV irradiation and PDT inhibited insulin-induced activation of PI 3-kinase. The inhibition appeared to be more profound with PDT, but the mechanism of the inhibition is unknown. PDT targeted events may include ligand-binding-induced activation of the insulin receptor tyrosine kinase, phosphorylation of IRS-1 and 135 recruitment of signaling molecules, including PI 3-kinase to the membrane. Alternatively, growth factor-induced activation of Ras may be targeted by PDT. Further analysis of these upstream signaling events will facilitate our understanding of the mechanism of PDT-mediated inhibition of PI 3-kinase. In conclusion, our results demonstrated that PDT caused an inhibition of growth factor-induced activation of PI 3-kinase, PKB and p70S6K. PDT also inhibited agonist-induced activation of p70S6K in a PI 3-kinase, PKB independent manner. At present we have no experimental data indicating possible targets of PDT. We do not know if the inhibitory effects of PDT are direct or a consequence of upstream inhibition, although the evidence implies that the inhibition may be exerted independently on multiple kinases. One model in support of such a notion would be PDT activation of a serine/threonine protein phosphatase that was capable of dephosphorylating and inactivating all three kinases. Consistent with this hypothesis, the activation of PKB and p70S6K require multiple serine and threonine phosphorylations, although such a mechanism has not been proposed for PI 3-kinase activation. Future investigations in this system should include a thorough analysis of the various PKC isoforms and the PDT effect on the anisomycin-induced activation of p70S6K. One of the major difficulties that we have encountered in this series of experiments was that we had no assay for, or immunoblotting or immunoprecipitating antibodies against FRAP. Such antibodies would be of considerable use in unraveling the links between PKC, PKB, FRAP, 4E-BP1 and p70S6K. 136 5.3.2 Specificity of the PDT effect The PDT-induced inhibition of PI 3-kinase, PKB and p7(T" in Pam212 cells was not observed in cells treated with drug in the absence of light, or with light in the absence of drug. This indicated that the observations were the results of reactive oxygen species (ROS)-mediated photodynamic effects. It had been shown that H2O2 strongly activated ERK1 and ERK2 and we had previously observed that H2O2 also activated PI 3-kinase and PKB (Tao et al., 1996). We have shown here that the PDT effect is mediated differently than that of UV irradiation, which activated p7056A: and potentiated the effect of insulin on PKB. As the UV effect is also dependent on ROS, the effects of PDT reported here were not likely to be due to the ubiquitous effects of ROS, but specific properties of PDT with BPD-MA. Possible explanations for the unique properties of PDT with BPD-MA may be understood in terms of the characteristics of the photosensitizer. BPD-MA is a hydrophobic molecule that is localized in the cytoplasm in association with membranous structures (Margaron, 1998). The subcellular localization of many signaling molecules permits their interaction with substrates and other regulatory proteins. Similarly, the localization of BPD-MA, and a local increase in ROS may be responsible for the specificity of the photodynamic effect. To establish the role of subcellular localization of the photosensitizer in mediating the effects of PDT, direct evidence including functional interactions and physical co-localization of the signaling components needs to be obtained using biochemical and imaging methods. It will also be of interest to discover if the signaling components of this pathway undergo oxidation-mediated modifications during PDT. 137 5.3.3 PDT in apoptosis The inhibitory effect of PDT on PI 3-kinase, PKB and p70 was observed within a dose range of PDT between 50 to 100 ng/ml BPD-MA plus 2 J/cm2. At this dose range, significant cytotoxicity was observed in apoptosis assays and antioxidant treatment provided some protection against PDT-induced cell death (data not shown). Both PI 3-kinase and PKB have been implicated as playing a key role in prevention of apoptosis in response to survival signals (Kulik et al, 1997; Kennedy et al, 1997; Kaufmann-Zeh et al, 1997). The role that p7056^ plays in prevention or induction of apoptosis is unclear. Recent work that I performed in collaboration with Michael Scheid (Ph.D. candidate) at the Jack Bell Institute provided the first report that p7056* is not required for PI 3-kinase mediated cell survival in hemopoietic cells (Scheid et al, 1996). We showed that rapamycin was unable to induce apoptosis in cells stimulated with cytokines. Inhibition of PI 3-kinase with wortmannin or LY294002 was able to induce apoptosis in these cells, providing the first evidence of a bifurcation of the signaling pathways from PI 3-kinase. A second collaborative work with Dr. John Jackson at the University of British Columbia also dissociated the p70S6K from the PI 3-kinase pathway (Tudan et al, submitted). In this study, we showed that in neutrophils activated by calcium pyrophosphate dihydrate crystals, p70S6K was activated via a PI 3-kinase independent and PKC dependent pathway. Therefore, the available evidence indicates that p70S6K does not play a role in PDT-mediated induction of apoptosis. Nevertheless, the fact that PDT activated stress-activated pathways and inhibited PI 3-kinase, PKB and p70S6K is intriguing. It is possible that PDT triggers a pro-apoptotic signal leading to the activation of SAPKs and the inhibition of PI 3-kinase and PKB survival signals. The PDT effects on these kinases may arise as signaling effects that occur in parallel with apoptosis. To test these possibilities, the susceptibility to apoptosis of cells expressing mutant SAPKs which are 138 insensitive to PDT, or mutant PKB and p70 which are resistant to PDT-mediated inhibition should be tested. Pharmacological inhibitors of the SAPKs and MEKKs could also be employed in these studies. 5.4 Conclusions PI 3-kinase, PKB, FRAP and p70S6K appear to represent a branch of a larger signaling pathway that is involved in cell growth, survival, and the initiation of protein synthesis. Several components of the pathway appear to be required for prevention of apoptosis, while others appear to be involved in the translation of proteins required for growth or proliferation. The interactions between these proteins are complex, especially in the case of p70S6K. Some of the direct upstream activators are as yet uncharacterized, and it is clear that the regulation and activation of p70S6K is complicated and requires multiple signaling events and interactions with other regulatory proteins. The analysis of p70S6K has been facilitated recently by the availability of immunoprecipitating antibodies, specific inhibitors, and expression of recombinant, active kinase. The key novel findings of the research presented here include the first protocol for purification of an echinoderm homologue of p70S6K, and identification of distinct differences in substrate specificity from previously reported isoforms. In addition, I provide evidence supporting the possibility of multi-complex formation comprising p70S6K, PP2A and HSP-60. Finally, we show for the first time, that p7056* is profoundly inhibited in response to PDT, although we do not at present understand the mechanism of PDT-induced signaling. Our observations here will contribute to our understanding of the role of p70S6K in sea star oocyte maturation, and the mechanism of action of PDT in murine keratinocytes. 139 Aaronson, SA. (1991) Growth factors and cancer. Science 254, 1146-1153 Agarwal, M.L., Clay, M.E., Harvey, E.J., Autunez, A.R., and Oleinick, N.L. (1991) Photodynamic therapy induces rapid cell death by apoptosis in L51784 mouse lymphoma cells. Cancer. 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