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Identification and characterization of a maturation-inhibited protein kinase (MIPK) Morrison, Donna Lorraine 1998

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IDENTIFICATION A N D CHARACTERIZATION OF A MATURATION-INHIBITED PROTEIN KINASE (MIPK) by D O N N A LORRAINE MORRISON B.Sc. (Hons), Simon Fraser University, 1992 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF D O C T O R OF PHILOSOPHY i n T H E F A C U L T Y OF GRADUATE STUDIES (Department of Medicine) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA April 15, 1998 © Donna Lorraine Morrison, 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 l/Ybr^fMrve The University of British Columbia Vancouver, Canada •a te Afr , t23/qft DE-6 (2/88) ABSTRACT A novel homologue of p38 MAP kinase, called maturation-inhibited protein kinase (MIPK), has been cloned from the Pisaster ochraceus oocyte system. MIPK was first recognized as a major tyrosine phosphorylated protein in the immature oocyte, which underwent dephosphorylation in response to the natural hormone 1 -methyladenine. Using an antipeptide antibody based on the C-terminus of cyclin-dependent kinase 5 (Cdk5-CT) as a probe, MIPK was partially purified from seastar oocyte cytosol. Peptide sequence information was used to clone the full length MIPK cDNA. The predicted amino acid sequence of MIPK was found to be most closely related to the p38 MAP kinase. More specifically, MIPK was 65% identical to human p38, 62% identical to human p38p, 56% identical to human p38y, and 54% identical to p388. Analysis of the known p38 kinase family members from diverse species indicates a high degree of conservation (85-95% for p38) in primary structure. MIPK did not show the same level of identity with p38 as did the known p38 homologues, and was therefore defined as a novel member of the p38 MAP kinase family. MIPK was found to be activated in the oocyte system in response to high osmolarity medium and heat shock, confirming MIPK as a stress-activated protein kinase. Assessment of MIPK phosphotyrosine levels during embryonic development showed a dramatic activation of MIPK 12 h post-fertilization. This time course of activation correlated with the transition from synchronous cell divisions to differential cleavages, at a time when the overall rate of cell division decreased. MIPK appeared to be activated in cells arresting in the cell cycle, during meiotic maturation, under stress conditions, or during embryonic development. MIPK may therefore act as a cytostatic factor in the Pisaster ochraceus oocyte system. ii TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables ix List of Figures x List of Abbreviations xii Acknowledgements xv Dedication xvi CHAPTER 1: INTRODUCTION AND BACKGROUND 1.1 Protein phosphorylation 1 1.2 Protein kinases 3 1.3 MAPK signal transduction in yeast 5 1.3.1 The pheromone response pathway 6 1.3.2 Nutritional starvation responses 8 1.3.3 Cell wall integrity 10 1.3.4 Hyperosmotic medium response 11 1.4 The mammalian MAP kinase superfamily 12 1.4.1 Erkl/2 signaling in mammals 12 1.4.2 SAP kinases 16 1.4.3 p38 kinases 17 1.5 SAPK/p38 upstream activators 23 1.6 MAP kinase phosphatases 26 iii 1.7 Signal integration 28 1.8 Signal transduction during meiosis 31 1.8.1 Activation of G2-arrested oocytes 31 1.8.2 Signal transduction in P. ochraceus oocytes 32 1.9 Rationale and research objectives 37 C H A P T E R 2: MATERIALS A N D M E T H O D S 2.1 Materials 38 2.1.1 Chemical reagents and laboratory supplies 38 2.1.2 Photography supplies 42 2.1.3 Plasmids and bacterial strains 42 2.1.4 Antibody reagents 43 2.1.5 Sources of oligonucleotides 43 2.2 Manipulations of P. ochraceus oocytes 44 2.2.1 P. ochraceus oocyte maturation 44 2.2.2 P. ochraceus oocyte homogenization 44 2.2.3 Osmotic shock time course 45 2.2.4 Heat shock time course 45 2.2.5 Time course homogenization 46 2.2.6 P. ochraceus oocyte fertilization 46 2.3 Antibody production 47 2.4 Protein quantitation 48 2.5 ResourceQ fractionations 48 2.6 Superose 12 chromatography 49 iv 2.7 SDS-polyacrylamide gel electrophoresis 49 2.8 Staining of SDS-PAGE gels 50 2.8.1 Silver staining 50 2.8.2 Coomassie blue staining 50 2.9 Transferring to nitrocellulose membranes 50 2.9.1 Western blotting 50 2.9.2 Ponceau S staining of Western blots 52 2.9.3 Amido black staining 52 2.9.4 Stripping and reprobing of Western blots 52 2.10 Immunoprecipitation 53 2.11 Protein kinase assays 53 2.12 Purification of MIPK from P. ochraceus oocytes 54 2.13 Sample preparation for protein sequencing 56 2.14 Restriction digests 56 2.15 Ligations 56 2.16 Transformations 57 2.17 Cloning with T-tailed bluescript vector 57 2.18 Plasmid preparation 59 2.18.1 Small scale plasmid preparation 59 2.18.2 Large scale plasmid preparation 59 2.19 D N A sequencing 59 2.20 RNA isolation from P. ochraceus oocytes 60 2.21 RNA agarose gels 60 2.22 mRNA purification 61 2.23 Amplifying MIPK using polymerase chain reaction 61 2.23.1 Quantitation of oligonucleotides 61 2.23.2 Reverse transcription reaction 62 2.23.3 Specific PCR conditions for MIPK 62 2.23.4 Confirmation of identity of PCR clones 62 2.24 Rapid amplification of cDNA ends (RACE) 63 2.24.1 Adaptor ligated cDNA 63 2.24.2 RACE reactions 64 2.25 Construction of MIPK expression vectors 64 2.25.1 Amplifying full length Mipk cDNA clone using PCR 64 2.25.2 Construction of kinase-dead MIPK using PCR site-directed mutagenesis 65 2.26 Production and purification of GST-fusion proteins in bacteria 67 2.26.1 Expression and purification 67 2.26.2 Thrombin cleavage of fusion proteins 68 2.27 Measurement of MIPK upstream kinase activity 68 2.28 Microinjection of P. ochraceus oocytes 69 CHAPTER 3: RESULTS 3.1 Search for novel tyrosine phosphorylated proteins in P. ochraceus 73 3.1.1 Introduction 73 3.1.2 Analysis of P. ochraceus oocyte extracts for tyrosine 73 phosphorylated proteins 3.1.3 Initial characterization of p40mipk 76 3.1.4 Time course of dephosphorylation of p40m,pk during oocyte maturation 78 vi 3.2 Purification and characterization of p40 m , p k from immature 83 P. ochraceus oocytes 3.2.1 Introduction 83 3.2.2 Purification of MIPK from immature oocytes 83 3.2.3 Micropeptide sequencing of purified MIPK 87 3.3 Cloning and sequencing of P. ochraceus MIPK 91 3.3.1 Introduction 91 3.3.2 Degenerate PCR of MIPK from P. ochraceus 91 3.3.3 Cloning and sequencing of MIPK from immature seastar oocytes 94 3.3.4 Comparison of seastar MIPK with other proteins 100 3.4 Expression and characterization of MIPK 108 3.4.1 Introduction 108 3.4.2 Bacterial expression of MIPK as a GST-fusion protein 108 3.4.3 Antibody profiling of GST-MIPK 110 3.4.4 Western blotting comparisons of MIPK and p38 in P. ochraceus 114 3.4.5 Design of MIPK specific antibodies 114 3.5 MIPK activity in the P. ochraceus system 117 3.5.1 Introduction 117 3.5.2 Time course of activation by osmotic shock treatment 117 3.5.3 Time course of activation by heat shock treatment 118 3.5.4 Postfertilization activation of MIPK 121 3.5.5 Assessment of kinase activity of purified MIPK 124 3.5.6 Assessment of phosphotransferase activity of bacterially 128 expressed MIPK 3.5.7 Examination of in vivo effects of MIPK in the oocyte system 130 by microinjection vii 3.5.8 Identification of an upstream MIPK kinase 138 C H A P T E R 4: D ISCUSSION/FUTURE DIRECTIONS 4.1 Discussion 141 4.2 Future directions 147 4.3 Conclusions 152 B I B L I O G R A P H Y 154 APPENDICES Appendix I Description of antibodies 167 Appendix II Description of oligonucleotides 171 Appendix III Cloning and sequencing of a shorter MIPK transcript 176 viii LIST OF TABLES Table 1. Micropeptide sequencing results. 90 Table 2. Identity/similarity between p38 homologues and MIPK. 101 Table 3. Identity/similarity between p38 family members and MIPK. 102 Table 4. Names and accession numbers of kinases listed in the MIPK 106 homology tree. Table 5. Alignment of immunizing peptides with MIPK and p38 sequences. 113 Table 6. Protein kinase residues predicted to contact side chains 145 of peptide substrates. ix LIST O F FIGURES Figure 1. MAP kinase pathways in the budding yeast. 7 Figure 2. The MAP kinase superfamily. 13 Figure 3. 1-Methyladenine stimulation of P. ochraceus oocyte maturation. 33 Figure 4. Restriction map of P. ochraceus MIPK in the pGEX-4T3 vector. 66 Figure 5. Microinjection of P. ochraceus oocytes. 71 Figure 6. Western blot analyses of ResourceQ fractionated P. ochraceus 74 oocyte extracts. Figure 7. Western blot analysis of Cdk5-CT immunoprecipitations. 77 Figure 8. Western blot comparison of Cdk5-CT and Erkl-CT 79 immunoprecipitated proteins. Figure 9. Dephosphorylation of MIPK in homogenates of a P. ochraceus oocyte 81 maturation time course. Figure 10. Purification of MIPK from immature P. ochraceus oocytes. 84 Figure 11. Identification of purified MIPK protein band. 88 Figure 12. Degenerate PCR based on peptide sequencing results. 92 Figure 13. PCR amplification of full length MIPK from P. ochraceus. 95 Figure 14. Nucleotide and amino acid sequence of coding region of 97 P. ochraceus MIPK. Figure 15. Protein sequence alignment of P. ochraceus MIPK with p38 homologues. 99 Figure 16. Homology tree of MIPK with MAPK superfamily members. 104 Figure 17. Expression of GST-MIPK and GST-K62R in E. coli. 109 Figure 18. Immunoreactivity comparisons of GST-MIPK and GST-p38. 112 Figure 19. Identification of p38 isoforms in P. ochraceus oocytes by 115 Western blotting. x Figure 20. Phosphorylation of MIPK in homogenates of a P. ochraceus oocyte 119 osmotic shock time course. Figure 21. Phosphorylation of MIPK in homogenates of a P. ochraceus oocyte 120 heat shock time course. Figure 22. Phosphorylation of MIPK in homogenates of a P. ochraceus oocyte 122 fertilization time course. Figure 23. P. ochraceus embryo development time course. 123 Figure 24. Assessment of kinase activity of partially purified MIPK. 125 Figure 25. Autophosphorylation of partially purified MIPK. 127 Figure 26. Levels of tyrosine phosphorylation in bacterially expressed 129 GST-MIPK versus endogenous MIPK. Figure 27. Microinjection of kinase-dead MIPK and p38 inhibitor into 133 P. ochraceus oocytes. Figure 28. Antisense oligonucleotide microinjection into P. ochraceus oocytes, 134 10 h post-fertilization. Figure 29. Antisense oligonucleotide microinjection into P. ochraceus oocytes, 136 22 h post-fertilization. Figure 30. Identification of an upstream MIPK kinase in the P. ochraceus oocyte. 140 Figure 31. MIPK homologues in a mammalian system. 150 Figure 32. Identification of a shorter transcript of MIPK by PCR amplification. 177 Figure 33. Specific PCR amplification of a shorter transcript of MIPK. 178 Figure 34. Sequence alignment of MIPK with the shorter transcript of MIPK. 179 Figure 35. Expression of the shorter transcript of MIPK in E. coli. 181 xi L I S T O F A B B R E V I A T I O N S aa amino acid(s) bp base pair(s) cAMP cyclic adenosine 3',5'-monophosphate Cdk cyclin-dependent kinase cDNA complementary deoxyribonucleic acid CT C-terminus DNA deoxyribonucleic acid dNTP deoxyribonucleoside triphosphate Erk extracellular signal-regulated protein kinase GSH glutathione GST glutathione S-transferase GVBD germinal vesicle breakdown H O G high osmolarity glycerol IgG immunoglobulin G IP immunoprecipitate Jnk c-Jun N-terminal kinase kb kilobase kDa kiloDaltons M molar MAPK mitogen-activated protein kinase MBP myelin basic protein MIPK maturation-inhibited protein kinase xii Mkk/Mek MAPK/Erk kinase (ig microgram |ll microlitre \lM micromolar mg milligram ml millilitre mM millimolar Mpk M-phase-activated MBP kinase mRNA messenger ribonucleic acid ng nanogram NT N-terminus PCR polymerase chain reaction PPi inorganic phosphate RACE rapid amplification of cDNA ends RNA ribonucleic acid rpm revolutions per minute Sapk stress activated protein kinase TBS Tris buffered saline A deoxyadenosine T deoxythymidine C deoxycytidine G deoxyguanosine Single and three letter codes for amino acids: A Ala Alanine R Arg Arginine N Asn Asparagine D Asp Aspartic acid C Cys Cysteine Q Gin Glutamine E Glu Glutamic acid G Gly Glycine H His Histidine I He Isoleucine M Met Methionine F Phe Phenylalanine P Pro Proline S Ser Serine T Thr Threonine W Trp Tryptophan Y Tyr Tyrosine V Val Valine xiv A C K N O W L E D G E M E N T S I would first like to thank my research supervisor, Dr Steven Pelech, for giving me the opportunity to do my graduate studies in his laboratory, for introducing me to the seastar system, and for giving me the guided freedom to explore this project. I would also like to thank my thesis committee, Drs. Michael Gold, Vince Duronio, and Gerry Krystal for their advice and support. Special thanks to Dr. Jasbinder Sanghera, for the daily advice and support, and for giving me the training in protein chemistry needed to proceed through my graduate research. I would like to thank the many colleagues and collaborators I have worked with over the last six years. Thanks to Dr. Ruedi Aebersold for his assistance with the protein sequencing of MIPK, and to Drs. Allen Delany and Gabe Kalmar for helping with sequence alignments and homology tree construction. Additional thanks to Dr. Gabe Kalmar and to Dr. Arthur Yee for their patience while teaching me molecular biology techniques. Thanks to Dr. Diana Lefebvre for supplying me with the fertilized oocyte extracts, but more importantly for her friendship and support through the difficult times. I would like to acknowledge my sincere appreciation to Harry Paddon, for the microinjection experiments, for the seastar adventures, and for his special talents in fixing all the things we could break, and creating all things we could dream up. Thanks to Dr. Chrystal Palaty, the first Pelech graduate, for her perspective on life, science, and the importance of outdoor adventure. A very special thanks to Lorin Charlton, for the morning breakfast meetings, the afterwork "wings" sessions, and especially the workouts needed to keep us sane. I would like to gratefully acknowledge the friendship and support of Georgia Tai and Venska Wagey, and Drs. David Charest and Guy Mordret for the insight on work ethic and endurance. Thank you to all other graduate students and postdoctoral fellows with whom I had the privilege to work with during my studies, and to all of the undergraduate students who put in their time in the Pelech lab. A final thanks to my family and friends, for supporting me through the "stress" that is graduate studies. I would especially like to ackowledge my husband, Hamish Morrison, for surviving with me through it all. I would also like to acknowledge the financial support of the Medical Research Council of Canada. xv DEDICATION J would like to dedicate this thesis to my late mother, Cynthia Campbell, and to my father, Alexander Campbell. Their support and encouragement throughout my life has given me the inspiration to set my goals, the confidence to know I could obtain them, and strength to see them through to completion. xvi C H A P T E R 1 I N T R O D U C T I O N A N D B A C K G R O U N D 1.1 Protein phosphorylation. Cells are programmed to respond to a multitude of extracellular signals responsible for the regulation of homeostasis, growth, proliferation, differentiation, or programmed cell death (apoptosis). The ability of an extracellular signal to evoke appropriate cellular responses requires a network of intracellular signalling molecules connected within cascades, transmitting vital information to the nucleus of the cell. The control over transcription of both immediate and delayed early response genes ultimately commits a cell either to progress through the cell cycle, to arrest in GO phase, or to differentiate. Essential components in these signalling cascades include enzymes known as protein kinases and phosphatases. These proteins catalyze the addition (kinases) or removal (phosphatases) of phosphate groups on polypeptide chains. In doing this, kinases and phosphatases regulate the activity of many effectors within the cellular matrix. Protein phosphorylation was initially identified as a regulatory mechanism over forty years ago in the study of glycogen phosphorylase (Fischer and Krebs, 1955). Research has expanded with the realization that protein phosphorylation plays a pivotal role in the coordination of cellular activities. Protein kinases catalyze the transfer of a high energy phosphate from adenosine triphosphate (ATP), to the hydroxyl group of amino acids within a target protein. Specifically, the amino acids serine, threonine, and tyrosine are the major targets of phosphotransferase activities. The addition of a phosphate residue to these amino acids within a polypeptide chain can induce dramatic changes in a protein's enzymatic activity, localization, or binding properties. Protein 1 phosphatases are equally important for their ability to catalyze the reverse reaction. It is this reversible nature of protein phosphorylation that allows a cell to quickly respond to external stimuli, or rapidly attenuate signal intensities. The potential uses for this covalent modification are derived from the individual properties of the phosphorylated residues. Phosphotyrosine has an elongated structure which provides a convenient anchor for protein-protein interactions. Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains have been identified as conserved protein modules that bind directly to phosphotyrosine (reviewed in Pawson, 1995), leading to the formation of protein complexes. The main function of serine/threonine phosphorylation (and sometimes tyrosine phosphorylation) is to modify the conformation of proteins, resulting in changes in enzymatic activity. The effects of a phosphoserine or phosphothreonine residue can often be mimicked by an acidic residue such as glutamic acid, indicating that they act mainly through acquisition of negative charge. The initiation of a cellular response to an extracellular signal typically occurs at the level of the cell surface receptor. These receptors are usually membrane associated, and often contain intrinsic protein tyrosine kinase activity (eg. platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR)), or may be associated with cytoplasmic tyrosine kinases (eg. cytokine receptors). Activation of the tyrosine kinase activity through ligand binding results in multiple phosphorylation events, and leads to the assembly of protein-protein complexes. The receptor-specific recruitment of intracellular signalling molecules eventually leads to nuclear events, including transcriptional regulation. Although the same proteins may be activated in response to a variety of signals, it is the net influence of all the initiated pathways which ultimately defines the cell's fate. Enhanced understanding of the interactions between the signal transducers involved in the 2 regulation of cellular responses will eventually allow scientists to control the balances of cascades within the cell that are evoking the final cellular response. With this ability, attempts could be made to correct abnormalities in signalling mechanisms associated with many human diseases. 1.2 Protein kinases. The protein kinase family is a large group of enzymes, with the potential for many more family members yet to be identified. The total number of distinct kinase domains known in 1988 was only 100 (Hanks etal, 1988), but was nearing 400 by 1995 (Hardie and Hanks, 1995). Advances in molecular cloning techniques are largely responsible for the near exponential growth in kinase identification. In the fully sequenced genome of the budding yeast, Saccharomyces cerevisiae (S. cerevisiae), 113 protein kinases were identified, corresponding to approximately 2% of the total gene pool (reviewed in Hunter and Plowman, 1997). More than 60% of these kinases have either known or deduced functions, while the remainder await further characterization. Recent analysis of the GenBank and unfinished sequence databases for the nematode Caenorhabditis elegans (C. elegans), which account for about 60% of the worm genome, has so far revealed 270 unique kinases. By extrapolation, based on the fact that mammals contain four times as many genes as C. elegans, the human genome can be expected to contain over 1500 different protein kinases. The catalytic domain of a protein kinase is comprised of 250 to 300 amino acids, corresponding to about 30 kDa. The boundaries and characteristics of this domain were defined based on analysis of conserved sequences, as well as through assays of truncated enzymes. Crystal structure analyses have recently confirmed early predictions that protein kinases share similar three-dimensional structures. The conserved motifs found 3 in protein kinases have been used to define 11 subdomain regions, with residues from these subdomains being implicated in essential roles concerning enzyme structure and function (Hanks et al, 1988). The variable regions found within the catalytic domain, as well as the presence of additional domains or subunits, helps to define classes of protein kinases. The serine/threonine-specific and the tyrosine-specific classes have very similar primary structures,- yet, certain short amino acid sequences characterize each class, and can be used to predict the specificity of a kinase from the primary structure (Hanks, 1987). These predictions are not absolute, with proteins like the Weel kinase showing attributes of a serine/threonine protein kinase, yet being specific for tyrosine residues. New categories of protein kinases are currently being identified including the dual specificity, serine/threonine/tyrosine kinases, as well as the histidine kinases. Protein kinases can be regulated by several mechanisms, a feature making them ideally suited for controlling diverse cellular responses (reviewed in Johnson et al, 1996). Second messengers can bind to specific subunits or domains within a kinase, such as for cAMP- (PKA) and cGMP- (PKG) dependent protein kinases, respectively. Cyclin-dependent protein kinases (Cdks) earned their name based on their requirement for an additional subunit, a cyclin, whose expression is tightly regulated. Protein-protein interactions and consequent subcellular localization are key features modulating the kinase activity of proteins such as members of the Src family of tyrosine kinases. Some kinases contain autoinhibitory domains whose effect must be counteracted to allow for activation, such as for myosin light chain kinase (MLCK). Protein kinases are also common targets of other protein kinases and phosphatases, a concept which has lead to the recognition of kinase cascades. Activation of kinases by phosphorylation is usually achieved through phosphorylation of an activation loop in the center of the protein kinase domain. The activation loop spans the conserved sequences DFG and APE between 4 subdomains VII and VIII, corresponding to residues 184-208 in PKA (Taylor and Radzio-Andzelm, 1994). The activating phosphorylation sites found within this loop have been observed in various kinase families, yet the structure of this loop can be very different. One obvious variation is in the length of the polypeptide chain defining the loop, indicating that the 3-dimensional structure of this region is a key determinant of recognition and activation (Marshall, 1994). The specificity created by this loop allows for the formation of distinct signalling networks. A cell is therefore able to integrate the numerous mechanisms of regulation, and to evoke the necessary responses to extracellular stimuli. Some of the earliest and most successful studies of intracellular signalling focused on a family of mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated protein kinases (Erks). These highly conserved proteins are found in all eukaryotic organisms and appear to be part of a three kinase module involving a MAPK, which is activated by a MAPK/Erk kinase (Mek or Mkk), which is in turn activated by a Mek kinase (Mekk). MAPK-dependent pathways transduce signals in response to a wide variety of stimuli, ultimately leading to regulation of gene transcription in the nucleus of the cell (reviewed in Pelech and Charest, 1995). 1.3 MAP kinase signal transduction in veast. Five members of the MAP kinase family, Fus3, Kssl, Hogl, Mpkl/Slt2, and Smkl had been identified and functionally characterized prior to the completion of the genome project in budding yeast (reviewed in Waskiewicz and Cooper, 1995). The budding yeast has provided important genetic studies contributing to the understanding of MAP kinase-dependent pathways. Dissection of fundamental cellular processes in the yeast including mating, adaptation to nitrogen starvation, changes in external osmolarity, 5 polarized growth, and sporulation has revealed at least six independent MAPK-related cascades in yeast (Levin and Errede, 1995) (Figure 1). 1.3.1 The pheromone response pathway. The most highly characterized of the MAP kinase pathways in yeast transduces signals from cell surface receptors for the mating pheromones a-factor and a-factor, causing the haploid yeast to exit from the cell cycle prior to Start in Gl , and to conjugate with the opposite mating type irrespective of nutritional conditions. Sterile (Ste) mutants in the yeast have been exploited to delineate the downstream signalling events. The mating pheromone receptors are serpentine receptors associated with a heterotrimeric G protein. The activated mating pheromone receptors (Ste2 for a-factor, Ste3 for a-factor) induce the exchange of GDP for GTP on the OC-subunit of the G protein (Gpal) and its release from (3- (Ste4) and y- (Stel8) subunits. Release of the subunits results in the activation of the protein kinase Ste20 (Peter et al, 1996, Leberer et al, 1997). The cascade continues with the activation of Ste 11 (Mekk homologue), which phosphorylates and activates Ste7 (Mek homologue), which finally activates the MAP kinases Fus3 and Kssl. Also apparent in this cascade is the requirement for a protein known as Ste5, which seems to act as a scaffolding protein, holding the signalling proteins of the MAPK module in close proximity to their downstream targets. This feature may increase the rate of the transduction process, as well as control cross talk between similar signalling pathways. The MAPK homologues Fus3 and Kssl phosphorylate the transcription factor Ste 12, which induces transcription of mating-specific genes. Transcription of the Farl gene increases 3- to 5-fold in response to the mating pheromones. The cell cycle arrest 6 which occurs in response to the mating pheromones, results from the action of this Farl protein. Farl is a known substrate for Fus3 both in vitro and in vivo (Peter ef al, 1993,-Elion et al, 1993,- Tyers and Futcher, 1993), and it is thought that this phosphorylation activates Farl. Farl is then able to bind to and inhibit the cyclin-dependent kinases Cdc28-Clnl and Cdc28-Cln2 (Peter and Herskowitz, 1994), leading to cell cycle arrest. Farl therefore links the MAP kinase signalling pathway to the regulation of cell cycle control. MAP kinases play important roles in the response of the yeast cell to pheromones. This is especially apparent in consideration of the redundancy utilized in an organism with such a limited genome size. Either of the two MAPKs in this pathway (Fus3 or Kssl) are sufficient for the activation of Ste 12, as determined through observations of mutant strains (Elion etal, 1991). However, their functions are not fully redundant, as only Fus3 has the capacity to activate Farl (Peter et al, 1993,- Elion et al, 1993). Novel targets of Kssl will likely be discovered. Fus3 has been found to have several other substrates, some of which have aided in the understanding of MAPK signalling pathways. Fus3 is capable of phosphorylating Ste7 (Zhou et al, 1993) and the molecular scaffold Ste5 (Kranz etal, 1994). The consequences of phosphorylation of the N-terminus of Ste7 appears to be inactivation (Errede and Ge, 1996). The overall outcome of Fus3 activation and phosphorylation of both Ste5 and Ste7 is predicted to be the dissociation of the components of the signalling pathway from Ste5, impeding the signal transmission. 1.3.2 Nutritional starvation responses. Yeast cells are programmed to respond to the changing conditions of their environment, especially with respect to the nutritional status. Haploid strains (a- or CX-8 cell type) respond to nutritional starvation with what has been termed the invasive growth response (Roberts and Fink, 1994). This allows cells growing on agar to invade the agar and grow beneath it, while the colony remains anchored to the surface. Some of the proteins involved in this response pathway have been identified as the same proteins involved in pheromone response. The first three components of the pheromone response MAPK module (Ste20, Ste 11 and Ste7) are required for invasive growth, with the additional requirement for an as yet unidentified MAP kinase homologue, leading to the activation of the transcription factor Ste 12. The separation of these two very related signalling pathways could be achieved through the involvement a novel Ste5 protein. The a/a diploid yeast cell responds to nutritional starvation with a unique pseudohyphal differentiation (Gimeno et al, 1992). Cells change shape, exhibiting unipolar budding while remaining connected, resulting in chains which resemble the hyphae of filamentous fungi (Gimeno et al, 1992,- Kron et al, 1994). The signalling cascade triggered in these cells has the same requirements as seen for invasive growth of the haploid cell (Figure 1). In the diploid cell, it has also been observed that the synthesis of many pheromone response pathway proteins is turned off, including the subunits of the G protein, Ste5, and Fus3, as well as a potential decrease in synthesis of S t e l 2 (Fields and Herskowitz, 1987,- Liu et al, 1993). Analysis of mutant strains of yeast has shown evidence for the involvement of the small G protein Ras, feeding into the pathway at the level of Ste20. Downstream of Ste20, these pathways appear extremely similar, down to the level of transcription factor activation. What causes the differences in the outcomes of Ste 12 activation remains to be determined. The decrease in Ste 12 protein levels may be an important determinant, influencing the intensity of the signal. Alternatively, it may be the unique MAP kinase lying upstream of Ste 12 in this pathway which leads Ste 12 to induce the transcription of a different subset of genes. 9 Yeast sporulation is a meiotic process involving Ste20 and MAP kinase homologues, occurring in a/a cells in response to nutritional starvation. The Ste20 homologue Spsl (44% identical in the catalytic domain) (Friesen et al, 1994), is essential for spore formation. Also required is Smkl, a protein which is 40% identical to Fus3 (Krisak et al, 1994), and represents the MAPK component in the signalling cascade. Observation of mutant strains places the role of this pathway late in sporulation. These proteins appear to be partially under trancriptional control, representing a novel aspect of MAPK pathway regulation. 1.3.3 Cell wall integrity. Cell wall construction represents another MAPK dependent mechanism. This pathway differs from the other MAPK modules identified in the yeast system, with a requirement for a homologue of the mammalian a, (3, and y protein kinase C enzymes, PKCl (Watanabe et al, 1994). PKCl lies upstream of a MAPK cascade that is essential for cell wall integrity. This pathway includes a Mekk homologue, Bckl (bypass of C-kinase), two MAPKKs, Mkkl and Mkk2, and a MAPK, Mpkl (also known as Slt2; Torres et al, 1991). Mutations at any of these levels of signalling results in lysis of cells at high temperatures. The phenotype becomes much more severe with the PKCl mutant, with lysis occurring at all temperatures. This indicates that PKCl lies upstream of more than one signalling cascade, one of which being independent of the MAP kinase pathway (Lee and Levin, 1992). 10 1.3.4 Hyperosmotic medium response. A final MAPK cascade controls production of glycerol in response to high osmolarity. Hogl (high osmolarity glycerol-1) is the MAPK component of this signalling cascade, and is known to be tyrosine phosphorylated and activated by Pbs2 (polymyxin B sensitivity), a Ste7 homologue (Brewster et al, 1993). The yeast osmosensor Slnl contains both a receiver domain and a histidine kinase domain. Under conditions of low osmolarity, the histidine kinase domain is activated, resulting in phosphorylation of a histidine residue within the kinase domain. The phosphate group is transferred to an aspartate residue in the receiver domain in Slnl. This phosphate is then transferred to a histidine in a novel protein, Ypdl, and then to an aspartate in Sskl. This process is known as the multistep phosphorelay mechanism (Posas etal, 1996). In its phosphorylated state, Sskl is incapable of activating the Hogl pathway. Under high osmolarity conditions, Slnl in inactivated, releasing the activity of Sskl, resulting in the activation of Ssk2 and Ssk22 (MAPKKKs). This activation is thought to involve interaction with a non-catalytic inhibitory domain in the N-terminal region of Ssk2 and Ssk22 (Maeda et al, 1995). These kinases phosphorylate and activate Byrl, which in turn activates Hogl. This signalling cascade has many unique features, and shows again the diversity of signals which can result in the activation of a MAPK pathway. The elucidation of these mechanisms, in conjunction with the availability of the complete yeast genome sequence, will greatly increase the understanding of signalling pathways in mammalian systems. A second osmosensor, Shol, functions independently of Slnl and acts through a very different signalling mechanism (Maeda et al, 1995). Shol has four predicted transmembrane domains, and a C-terminal cytoplasmic region containing an SH3 domain. Shol interacts with, and activates, the Pbs2 MAPKK through Stel 1 MAPKKK (Posas and 11 Saito, 1997). These events lead to the activation of Hogl MAPK (Brewster et al, 1993), and to the production of glycerol, increasing the internal osmolarity of the cell. 1.4 The mammalian MAP kinase superfamilv. Many distinct MAPK subfamilies have been identified in mammalian systems (Figure 2), each of which has been characterized to a different level. These MAP kinases are grouped into subfamilies based on sequence similarity, sensitivity to activation by different upstream kinases, and mechanism of upstream regulation. MAP kinases are known to be proline-directed protein kinases, requiring the presence of a proline residue at the +1 position relative to the phosphorylatable serine or threonine. They are most closely related to the cyclin-dependent protein kinases (Cdks), which are also proline-directed kinases (Pelech and Sanghera, 1992). Family members include Erk3 (Boulton etal, 1991; Gonzalez etal, 1992; Zhu et al, 1994), a 97 kDa protein kinase whose role in signalling has not been well characterized. Very little is understood of the Erk5 signalling pathway. It is thought to lie downstream of Mek5, and becomes activated in response to some extracellular stresses. A 40 kDa human epidermal growth factor receptor-associated protein kinase (Hera) (Williams et al, 1993) represents another unique member of the MAP kinase family. The Erkl/2, Sapk, and p38 kinase families are areas of intense research, leading to discoveries of many interesting similarities and differences with respect to modes of regulation. 1.4.1 Erk 1/2 signalling in mammals. The originally identified and characterized mammalian MAP kinases were the 44 kDa Erkl and 42 kDa Erk2 protein kinases (Boulton et al, 1990,- Boulton et al, 1991), known to be activated by over 50 extracellular stimuli (reviewed in Pelech and Charest, 12 LO o bo c cu LO LO CL) cu E o on u 1/1 CO e £ .ro IS <-> o o o oo CM LU < on I I * ' Q- T LJMI •sr <_> m O J O J £ S i <-J LO (/> ra cu c to u - i 12 to J= o o £ LO cu \ O J Q_ 1— 1 \\ • L i -re Q £ 1 <_) O J cu \ • CQ L ) _ 1 CU a : Q. ro oo OJ c U < O J LO T—I CU > 2 cn U J LO ••— -Q "O C 3 _3 ro LO 0 _ ^ > 1 C LO I " § ro C bo rs C CU < -d m _> V ra c c V bo o E _c <u L» 3 -l-> u o c ro & c o u ra c Cu 2 H 3. 3 *d o B 13 1995). The Erkl/2 pathways can be activated by receptor tyrosine kinases, as evident in response to the platelet derived growth factor (PDGF) which is released by platelets at the site of a wound to stimulate proliferation of fibroblastic cells. Erkl/2 can also be activated in response to serpentine receptors and hemopoietic receptors and appears to be an important convergence point for incoming signals. All three receptor types use different protein subsets to activate the Erkl/2 kinases. For the PDGF receptor, binding of the dimeric PDGF molecule to the extracellular domain of the receptor activates the intracellular signalling cascade. Autophosphorylation of the PDGF receptor on tyrosine residues recruits a wide variety of signalling molecules to the cell membrane. These include numerous SH2 domain containing proteins such as the adaptor protein Grb2 (growth receptor binding-2). Grb2 binds to other proteins through two Src-homology 3 (SH3) modules which interact with proline-rich motifs. Grb2 specifically binds to the guanine nucleotide exchange protein (GNEP) Son-of-sevenless (Sos), recruiting it to the cell membrane. This important translocation brings Sos in proximity with the monomeric G protein, Ras. Sos can then stimulate the exchange of GDP for GTP and thereby activate Ras. Ras appears to be an important link to the cytoplasmic signalling proteins which transmit the signal to the nucleus (Figure 2). Ras leads to the activation of Rafl, through a complex mechanism which is yet to be fully understood. Rafl is the Mekk component upstream of Mekl/2, responsible for phosphorylating Ser-218 and Ser-222 in the activation loop of Mekl/2 (Pelech and Charest, 1995). Mekl/2 are then available to phosphorylate and activate Erkl/2 at the Thr-Glu-Tyr (TEY) motif in the activation loop, defining Mek as a dual specificity kinase. cAMP signalling through the Erkl/2 MAP kinase pathway utilizes a similar, yet distinct cascade of intermediary transducers. The small G protein, Rapl, takes the place of Ras, in this scheme. In the absence of Ras 14 activity, Rapl is able to stimulate the Erkl/2 pathway via the B-Raf protein kinase. In PC12 cells, this activity leads to neuronal differentiation (Vossler etal, 1997). Upon activation, both Erkl/2 and Mekl/2 are translocated to the nucleus, where Erkl/2 encounters its transcription factor substrates, including the ternary complex factor, Elk-1. This phosphorylation activates the transcription potential of Elk-1, leading to thr transcription of many mitogen-inducible genes, such as c-Fos (Cahill et al, 1996). Mekl/2, however, is rapidly exported from the nucleus as a result of a nuclear export signal sequence. Mek may be permitted to enter the nucleus briefly to allow phosphorylation of a small pool of constitutively nuclear, unphosphorylated Erkl/2, but must be quickly transported out to preserve the fidelity of the signal. The length of retention time of active Erk in the nucleus appears to be an important factor in the outcome of a signalling event (Lavoie et al, 1996). Half of the Erkl/2 molecules are bound to the cytoskeleton (Reszka et al, 1995), supporting the potential involvement of Erks in cytoskeletal reorganization, or the possible role of the cytoskeleton in controlling Erkl/2 localization. Isoforms of the Rsk (ribosomal S6 kinase) family are among the best candidates for physiological substrates of Erkl/2. These 85- to 92-kDa protein serine/threonine kinases, Rskl and Rsk2, are activated during meiotic maturation of oocytes and in the early response to growth factor treatment of somatic cells (Sturgill and Wu, 1991,- Blenis, 1991). However, phosphorylation by Erkl/2 is not sufficient for full activation of Rsk phosphotransferase activity, implicating other factors in Rsk activation. Rsk appears to be a broad specificity kinase, with potential targets including the glycogen-binding subunit of protein phosphatase-1, which activates its phosphatase activity. This results in the dephosphorylation and activation of glycogen synthase and enhanced glycogen 15 synthesis, and dephosphorylation and inhibition of phosphorylase kinase to reduce glycogenolysis (Dent et al, 1990). Erkl/2 are therefore important proteins used to transmit an extracellular signal to various internal components required to elicit an appropriate cellular response. This includes activation of transcription factors leading to mitogen-inducible gene transcription, as well as activation of other protein kinases, including Rsk, which go on to play their own roles in cell signalling. The Erkl/2 pathways are known to be activated by a multitude of extracellular signals, in many different cell types, indicating a central role in intracellular signal transduction. 1.4.2 SAP kinases. A second group of MAP kinase family members are the c-Jun amino-terminal kinases (Jnks) or stress-activated protein kinases (Sapks) (Figure 2). The first Sapk was identified as the major protein kinase activity responsible for the phosphorylation of the microtubule associated protein-2 (Map-2) in rats injected with cycloheximide (Kyriakis and Avruch, 1990). Characterization showed the requirement for both tyrosine and serine/threonine phosphorylation for activity, and the kinase was named p54mapk. Subsequent investigations determined that this kinase was found to phosphorylate the transcription factor c-Jun at its in vivo sites, Ser-63 and Ser-73 (Pulverer et al, 1991), leading to the name Jnk. Tryptic peptides from the purified kinase were used to create oligonucleotides for amplification and cloning of the p54 m a p k gene. The cDNA was then used to probe a human hepatoma cDNA library, from which eight classes of cDNAs were isolated, representing splice variants of three distinct genes (reviewed in Woodgett et al, 1996). These genes have been termed a, |3, and y, and encode highly homologous proteins (85-16 92% identity) of 54 kDa and 46 kDa. The 46 kDa variants arise from induction of a 5 base pair sequence into the C-terminal region, introducing a premature stop codon. The a gene also contains a differential splice site internally, resulting in a 15 amino acid substitution between subdomains IX and X of the kinase domain. Specific antibodies were used to assess the stimuli which could lead to activation of these kinases. Mitogens were found to be very poor inducers, while a variety of stresses potently activated the family. The profile was very distinct from Erkl/2, implying the use of separate signalling pathways. These new kinases were therefore named stress-activated protein kinases (Sapks). Sapks possess a Thr-Pro-Tyr (TPY) sequence in the activation loop between subdomains VII and VIII, equivalent to the TEY sequence of Erkl/2. This proline would likely induce a kink in the 3-dimensional structure of the polypeptide chain, creating a very different recognition site for an upstream kinase. It is therefore not surprising that Mekl/2 are not able to phosphorylate the Sapks. Sekl (Sanchez et al, 1994) (Sapk/Erk kinase) also known as Mkk4 (Derijard et al, 1995) or Jnkk (Lin ef al, 1995), and Mek7 (Tournier et al, 1997) are the only known upstream activators of the Sapks. Activities known as Sapkk4/5 have also been identified (Meier et al, 1996) but are yet to be sequenced and confirmed as novel activators. 1.4.3 p38 kinases. A third family of MAP kinases has been identified by many different groups, resulting in an assortment of naming strategies. Initial reports of p38 described it as an unknown, tyrosine phosphorylated protein in lipopolysaccharide (LPS) treated cells (Han et al, 1993; Sanghera et al, 1996). p38 was distinguished from known MAP kinase isoforms due to differences in mobility on an SDS-polyacrylamide gel, and due to a lack of crossreactivity with specific MAP kinases antibodies. The identity of the protein 17 remaining unknown, the protein was referred to simply as p38. In the year that followed, several more research groups discovered a 38 kDa protein of interest. Mpk2 was discovered in the Xenopus oocyte system and was used to generate specific antibodies. Investigations of the mitogen-activated protein kinase-activated protein kinase-2 (Mapkapk-2) lead to the identification of a 38 kDa upstream activator, known as reactivating kinase (RK) (Rouse et al, 1994). Mapkapk-2, a known in vitro substrate of Erkl/2, was found to be activated under conditions such as heat shock, osmotic stress, or treatment with sodium arsenite, but not by agonists known to activate Erkl/2. RK was found to be activated under these stress conditions, and further characterization revealed crossreactivity of RK with the antibody specific for Mpk2. It was therefore concluded that RK and Mpk2 represented members of a novel stress-activated signal transduction pathway, distinct from the known MAP kinases, and likely to be upstream of the Mapkapk-2 enzyme. Another source of this novel enzyme was in the form of a 40 kDa protein observed in response to interleukin-1 (IL-1) activation (Freshney et al., 1994). This group noted that this protein, p40, was activated in response to IL-1 via serine/threonine and tyrosine phosphorylation. Upon activation, p40 phosphorylated and activated a 50 kDa protein via serine/threonine phosphorylation, which in turn phosphorylated the heat shock protein, Hsp27. This group predicted that Mapkapk-2 was the 50 kDa protein in question, but succeeded only in partially purifying the new kinase. p40 was found to be clearly distinct from Erkl/2 and the newly emerging Sapk, but beyond that could not be characterized. Later in 1994, a study was published in collaboration with researchers at SmithKline Beecham Pharmaceuticals, looking at a protein kinase involved in the regulation of inflammatory cytokine biosynthesis (Lee etal, 1994). A series of pyridinal-imidazole drugs were found to inhibit the production of IL-1 and tumour necrosis factor 18 (TNF) from stimulated human monocytes. In search of the target for these compounds, a protein was identified which bound to the drug, and was therefore named CSAID (cytokine-suppressive antiinflammatory drug) binding protein (CSBP). Sequencing data revealed that CSBP had a predicted molecular mass of 41 kDa, and was closely related to the MAP kinase family. When the clones of the Xenopus Mpk2 and mouse p38 (Han et al., 1994, Derijard et al., 1995) were aligned, it was quickly realized that p38 represented a novel class of MAPK-like proteins, with a characteristic Thr-Gly-Tyr (TGY) sequence in the activation loop of the kinase domain. This information drew attention to the yeast Hogl protein, which also possesses a TGY motif. Mutant strains of S. cerevisiae demonstrated some functional overlap, with p38 and Sapk both able to complement Hogl deficient strains. With this range of discoveries, a new MAP kinase family was formed. The important role of p38 in inflammation prompted research groups to search for new family members as potential new targets. Several cloning studies from numerous research teams has resulted in the cloning of three novel groups of p38-like proteins. All subfamilies are characterized with the TGY motif, yet contain significant regions of variance from p38. p38f3 was identified through a search of the EST division of GenBank data base, looking for p38 related proteins (Jiang et al., 1996). p38p was found to be -74% identical to p38, with the largest regions of variance found in subdomains V and VI, where an 8 amino acid insert was located. Subsequent research from two independent groups has called this insert into question (Goedert et al, 1997,- Kumar et al, 1997), yet confirms that p38p represents a new class of p38 kinases. Northern blot analysis indicated that p38(3 mRNA was expressed at highest levels in heart and brain, but could also be found in all 19 other tissues tested. Its mRNA expression profile closely resembled that found for p38 in the same study (Jiang et al., 1996). The third member of the p38 family is known as Erk6 (Lechner et al, 1996), Sapk3 (Mertens et al, 1996), and p38y (Li et al, 1996). Erk6 was identified as a novel M A P kinase in skeletal muscle. It was found to have no effect on cell proliferation, yet induced differentiation to myotubes when overexpressed in C2C12 cells. Erk6 was believed to represent a tissue specific differentiation factor, and a distinct M A P K family member. Sapk3 and p38ywere both cloned through exploitation of their homology to p38, either using PCR or via searches of the EST data base. Sapk3 mRNA was found to be widely distributed, in contrast to the specificity seen with Erk6. However, p38ywas found to be expressed only in the skeletal muscle, leading to a questioning of the specificity of the Sapk3 probe, or stringency of the experimental procedure used. Adding to the confusion, Li et al. (1996) admitted that their cloning of p38y was from a human brain cDNA library, and suggested that p38y was expressed at low levels in tissues other than skeletal muscle. The latest p38-like protein to be discovered is known as Sapk4 (Kumar et al, 1997 ; Goedert etal, 1997) and p388 (Wang etal, 1997). p388 was identified by Wang et al and Kumar et al using the EST data base, while Goedert et al screened a human cDNA library with a portion of the EST data base p38p clone. p388 exhibits 6 1 % identity with p38, and is most highly expressed in testes, pancreas, prostate and small intestine (Goedert ef al, 1997). Wang et al. completed a very thorough assessment of the tissue distributions of all four p38 mRNAs, and found very unique patterns of expression for each isoform. Wang ef al. found p385 to be expressed at higher levels in the salivary gland, pituitary gland, 20 adrenal gland and placenta. This indicates that p388, like p38y, has a tissue specific role, in contrast to p38 and p38p* which seem to show a broader distribution. For clarity, the p38 family members will from this point on be referred to by their p38-like names, p38, p38p, p38y and p385. Of key importance throughout the study of novel kinases is not identification of a new sequence, but understanding of the role of the kinase in vivo. Although 4 different p38 family members have been cloned, their characterizations have been limited, especially with respect to p38y and p388. Thorough studies have been performed by Kumar et al. (1997) and Goedert et al. (1997) in attempts to find distinguishing features between the different proteins. The four kinases were found to be activated in response to stimuli such as IL-1, TNF, sorbitol, and UV light, while stimulation by phorbol esters and IGF-1 were unable to cause activation of any of the p38 isoforms. The four kinases therefore show remarkable similarity in their response to the stimuli tested. A second area tested was substrate preference. This information is key in understanding the potentially unique roles these kinases play in cell signalling. Commonly known substrates were assessed, such as MBP, Mapkapk-2, Mapkapk-3, and p53, as well as the transcription factors, Atf2, Elk-1, Sap-1, Sap-2, and c-Jun. Qualitative assessments showed that none of the kinases tested were capable of phosphorylating c-Jun, Sap-2 or p53. When activities were standardized to equivalent MBP phosphotransferase levels, some substrate preferences were noticed. p38 showed higher specificity for Elk-1, Mapkapk-2 and Mapkapk-3,- p38^ preferred Elk-1 and Mapkapk-2, with a lesser activity towards Atf2 and Mapkapk-3; p38y was able to phosphorylate Elk-1, Atf2, and Sap-1 with equal efficiency, as did p388. However, 21 relative preferences of the kinases for the different substrates could not be compared under the conditions used, as they assumed equal abilities for MBP phosphorylation, which was not tested. Kumar et a!. (1997) suggested a preference of p388 for Mapkapk-3, while Goedert et al. (1997) showed that p388 had minimal ability for phosphorylation. Further studies will be required to understand the roles for these kinases in the in vivo situation. An important tool providing valuable insight into the in vivo roles for p38 and p38p is the pyridinyl imidazole compound, known as either SB202190 or SB203580. These inhibitors were found to bind specifically to the ATP binding pocket of p38, with no effect of Sapk or Erk activities. Within the p38 family, the SB compounds were able to inhibit p38 and p38p*2 equally, while p38y and p388 were unaffected (Kumar et al, 1997). The SB compounds bind to Lys-53 and Asp-168, and act as competitive inhibitors of ATP. These amino acids must not confer the specificity of the drugs, as they are conserved within the p38 family. In reality, the entire ATP binding site has a quite conserved primary structure, adding to the intrigue regarding the SB compound's specificity. It is clearly a complex binding requirement, and will require further study before a prediction on sensitivity can be made for novel proteins which are identified. The SB compounds have been used to determine whether p38 or p38|3 are involved in intracellular signalling events in response to various stimuli. Valuable information has been compiled by exploiting this inhibitor in a variety of cell systems. p38 was found to be phosphorylated and activated in response to hyperosmolarity and bacterial endotoxin (Han et al, 1993, 1994), and is the direct upstream activator of the Mapkapk-2, responsible for phosphorylation of hsp27. p38 is also involved in the 22 regulation of IL-1 and T N F a synthesis in endotoxin-stimulated monocytes (Lee et al, 1994,- Lee and Young, 1996). These studies do not consider the role of p38y or p385, as the inhibitor is ineffective at blocking their activities. Isoform specific probes will be useful in assessing the role of the individual proteins in the intracellular signalling process. The antibodies currently available would likely not crossreact with new p38 family members due to a low level of conservation in the C-terminal region of the proteins. Only experiments looking at phosphotransferase activities in whole cell lysates would be likely to account for p38y or p388 activities in the cell's signalling networks, as they would not be under the constraints of antibody selectivity. In vivo, two upstream kinases responsible for the activation of the p38 kinases have been identified as Mkk3 and Mkk6 (reviewed in Paul et al, 1997). Other Sapkk activities have also been identified through column chromatography fractionation (Meier et al, 1996), however further characterization is required to confirm them as unique Mkk homologues. The large range of dual specificity kinases point to an increased complexity in the upstream regulation and activation of not only the p38 isoforms, but also the other MAP kinase family members. 1.5 Sapk/p38 upstream activators. The members of the signalling cascades linking the cell surface receptors to the MAP kinases are current areas of intense research. Defining the roles of known kinases as well as understanding the crosstalk between different cascades will aid in determining the signal mechanisms. As already stated, the major activators of the Sapk family appear to be Mkk4/Sekl and Mkk7, while the p38 family is activated by the Mkk3 and Mkk6 dual specificity kinases. Upstream of the Mkks, the signalling cascades are much less 23 understood. It is predicted from the sequential nature of the Sapk/p38 pathways that the relevant Mkk requires activation by phosphorylation by an upstream kinase equivalent to the Raf kinase of the Erkl/2 pathway (Figure 2 ) . Several potential players have been identified, yet the in vivo activators have only been predicted. The first Mek activator identified was termed Mekkl and was originally thought to lie upstream of Mekl. Co-transfection studies have suggested that Mekkl exhibits much higher activation potential for Mkk4 than for Mekl. Further evidence has shown that Mekkl participates in both growth factor and TNF-stimulated activation of Sapk (Minden et al, 1994). In these studies, Mekkl was found to only stimulate Mekkl/2 when highly overexpressed, confirming the specificity of Mekkl to the Sapk pathway. The number of Mekk isoforms identified has grown to include Mekk2, Mekk3 (Blank et al, 1996), and Mekk4 (Gerwins et al, \997; Fanger ef al, 1997). Expression of Mekk2/3 leads to the activation of the Sapk and Erkl/2 pathways, while p38 remains unaffected. Mekk2 was found to preferentially activate the Sapk pathway, whereas Mekk3 activated the Erkl/2 cascade. The Mekk-like protein lying upstream of the p38 pathways has remained elusive. One candidate is the transforming growth factor-P (TGF-P)-activated kinase 1 (Takl) (Yamaguchi et al, 1995), which is stimulated in response to TGF-P and bone morphogenetic protein. Two novel Takl binding proteins have also been identified (Tabs) which may stimulate Takl activity (Shibuya ef al, 1996). Tumour progression locus-2 (Tpl-2/Cot), originally identified as an oncogene product associated with the progression of T-cell lymphoma induced by Moloney murine leukemia virus, represents another potential Mekk (Salmeron ef al, 1996). At this time, the relative contribution of the known Mekk isoforms to the activation of individual Erk/Sapk/p38 isoforms is unclear. 2 4 The ability of a Mekk to initiate individual cascades at different intensities, may be a key method by which a signal's magnitude and response can be regulated. An important discovery which may aid in the understanding of the p38 signalling system was Mtk l (MAP three kinase 1). A human cDNA library was used to transform mutant strains of yeast, defective in Ssk2/Ssk22 or Shol genes. Tests for osmoresistant transformants revealed four positive cells out of 2 x 106. All four clones contained an identical insert, encoding a protein with a kinase domain similar to Ssk2 and Ssk22. Northern blot analysis demonstrated that Mtk l mRNA is expressed at high levels in heart, placenta, skeletal muscle, and pancreas, and at lower levels in other tissues. The kinase domain was found to be 98% identical to the mouse Mekk4 (Gerwins et al, \997, Fanger et al, 1997). Mtk l and Ssk2/Ssk22 were found to be highly homologous in their non-catalytic, N-terminal domains. Specifically, deletion of the N-terminus from Ssk2, Ssk22 or Mtk l constitutively activated their kinase domains, indicating that the N-terminal sequence contains a negative regulatory domain which interacts with and inhibits the kinase domain. Mtk l overexpression in cultured COS7 or HeLa cells resulted in the activation of p38 and Sapk, but not Erk2. Mtk l was found to phosphorylate Mkk3 and Mkk6 both in vitro and in vivo, however, dominant negative Mtkl(K/R) interfered only with the p38 pathway. Mtkl(K/R) specifically interfered with activation due to extracellular stresses, but was not capable of blocking activation of p38 by cytokines such as TNFot. TNFoc is thought to signal through the small G proteins Racl and Cdc42, suggesting, along with other supporting data, that Mtk l does not utilize the cytokine-Cdc42-Racl pathway. The high level of identity between Mtk l and Mekk4 indicates that they are homologs. However, Mekk4 from mouse was identified as a specific activator of the Sapk pathway (Gerwins et al, \997; Fanger et al, 1997)., and further comparisons will have to be 25 performed to assess whether the differences are due to the experimental systems being studied, or to differences in assay techniques (Takekawa etal, 1997). At the top of the specific MAP kinase module is often a homologue of the Ste20 protein of the budding yeast. Mammalian relatives of Ste20 are diverse and include the p21-activated protein kinases (Paks) and the mixed-lineage kinases (Mlks) (Manser et al, 1994). These kinases appear to be regulated by small G proteins of the Rho subfamily, Rac and Cdc42 (Macara et al, 1996). However, activation of Sapk in response to some stimuli, such as T N F a and anisomycin, appear to be Rac independent. This indicates that there are other mechanisms of transducing signals to downstream intermediates of the stress pathways which are independent of the activation of monomeric G-proteins. Ste20-related kinases identified recently include Ste20/oxidative stress response kinase (Sok)-l, Kinases responsive to stress (Krs 1 and 2), Thousand and one amino acid kinase (Tao)-l (Robinson and Cobb, 1997), germinal center kinase (Gck), and MAPK-upstream kinase (Muk), which was identified as a Mekk-like protein yet is most closely related to the Mlks (Pombo etal, 1996,- Taylor etal, \996; Hirai etal, 1996 ; Pombo etal, 1995). Despite the efforts of many research groups, the in vivo roles of these kinases are yet to be determined, and the signalling cascades of both Sapk and p38 remain to be clearly defined. 1.6 MAP kinase phosphatases. Equally important as the role of the upstream kinase in M A P kinase activation, is the role of phosphatases in turning off the MAP kinase cascade. Each MAP kinase pathway can be downregulated by a variety of phosphatases, which are also involved in maintaining a low basal kinase activity in unstimulated cells. A novel family of protein phosphatases specific for phosphorylated serine/threonine and tyrosine residues has 26 been identified which are specific regulators of the MAP kinases. Members of this family include MAP-kinase phosphatase-1 (Mkp-1), Pac-1, Hvh2, Pystl, Mkp-2, and Mkp-3 (reviewed in Byon et al, 1997). These phosphatases have unique substrate specificities, as shown using in vivo assessments (Chu et al, 1996). Pac-1 specifically recognizes Erkl/2 and p38, Mkp-2 specifically recognizes Erkl/2 and Sapk, while Mkp-1 recognizes all three subfamilies of MAP kinases. These dual-specificity phosphatases are immediate early gene products which are strictly localized to the nucleus, indicating a role in the regulation of nuclear MAP kinase isoforms. Other phosphatases must be involved in the regulation of cytoplasmic MAP kinases. Pystl is a phosphatase that is not inducible by stress or mitogens, and is found only in the cytoplasm. Pystl interacts only with the Erk family, with very little ability to dephosphorylate Sapk or p38 (Groom etal, 1996). In contrast to Pystl, Hvh2 is localized to the nucleus where it dephosphorylates active Erkl/2. Hvh2 is found to be expressed in a tissue specific manner, very different from that found for Mkp-1 or Pac-1, indicating a unique functional role for Hvh2 in controlling cell signalling. Another MAP kinase phosphatase which has been identified is protein phosphatase-2A (PP2A). Most of the evidence for PP2A downregulation of MAP kinases has been derived from in vitro studies and is largely circumstancial evidence. This includes colocalization of PP2A and Erkl/2 to microtubules (Fiore etal, 1993,- Sontag et al, 1995). Growth factor stimulation results in the tyrosine phosphorylation and inactivation of PP2A, potentially by a member of the Src family of tyrosine kinases. PP2A therefore appears to be appropriately regulated and positioned to maintain low basal MAPK activity and to downregulate MAPK following activation. The large diversity in MAP kinase-phosphatases seems to parallel the broad range of MAP kinase signalling modules identified. This variety allows for the arrangement of 27 very specific networks of kinases and phosphatases, ready to respond appropriately to the extracellular stimulus being applied. Although it is still unclear how these signalling modules can be so cleanly segregated, substrate specificity, time course of stimulus-dependent activation and induction, cell-specific expression, or subcellular compartmentalization could clearly work together to avoid inappropriate crosstalk between the pathways. 1.7 Signal integration. The complex network of signalling molecules present in a cell requires strict guides to ensure appropriate activations. The strong similarities between the cascades activated by dramatically different stimuli must utilize a very delicately balanced integration process coupled with strongly defined barriers to prevent unwanted crosstalk from occurring. This is apparent when considering data obtained from in vitro phosphotransferase assessments, as well as in vivo overexpression results. What is emerging is that the information obtained by these studies, while defining potential phosphorylations or activations, tend to overestimate the power and promiscuity of the signalling molecules being tested. This is becoming increasingly obvious when researchers attempt to delineate the activation pathways responding to stress-related stimuli. Critical information is being provided from the in vivo experiments, looking at the myriad of signalling events and trying to determine how a cell integrates the information and determines its appropriate outcome. Studies in PC 12 cells stressed the importance of not only the activation, but also the duration time of the activation as important determinants of cell fate (Marshall, 1995). Signalling events occurring in response to activation of receptor tyrosine kinases can lead to differentiation or to proliferation. Treatment of PC12 cells with NGF or FGF 28 leads to differentiation whereas EGF sends a proliferation signal. These two mechanisms were therefore thought to utilize different signalling networks. On first analysis, the two outcomes appeared to share many signal transducing events. Closer inspection revealed that NGF was capable of inducing a persistent elevation of Ras-GTP, whereas EGF resulted in a short-lived increase. This effect was magnified to the relative activations of the Erk pathway, which could still be observed hours after NGF treatment. These differences may reflect the mechanisms of receptor downregulation, but is thought to center more on subtle differences in the signalling cascade components. Specifically, association of Grb2-Sos complex either directly to the receptor (as seen with the EGFR), or via a third protein bridge, Shc-Grb2-Sos (as is seen with the NGF receptor, TrkA) appears to confer different signalling outcomes. The NGF signalling mechanism in PC12 cells is found to be more complex, involving the integration of a number of signalling cascades, when the survival and apoptotic processes are compared. Withdrawal of NGF from PC 12 cells leads to programmed cell death. The mechanism by which this occurs involves the sustained activation of the Sapk and p38 MAP kinase pathways (Xia et al, 1995). However, this alone is not sufficient to induce apoptosis, as evidenced by the IL-1 activation of Sapk and p38 which does not lead to cell death. The survival signal of NGF utilizes the Erk signalling module, and removal of NGF stimulation leads to inactivation of the Erk pathway, and apoptosis. When this inactivation is blocked by expression of constitutively active Mek l , apoptosis was prevented. This indicates that cell survival in PC12 cells involves activation of Erkl/2, with suppresion of Sapk and p38 pathways, while apoptosis requires the opposite conditions. These roles appear to be reversed when looking at B cells. Activation by CD40 on human B cells can activate the Sapk pathway and rescue cells from apoptosis, whereas surface IgM cross-linking leads to Erk 29 activation which can cause apoptosis (Sakata et al, 1995). It therefore appears that the opposing actions of the Erk and Sapk-p38 MAPK determine a cell's fate. It is clear that the many mechanisms of regulation of the MAP kinase pathways results in various forms of crosstalk, allowing the cell to integrate multiple signals. A second example of MAP kinase pathways engaging in opposing activities was observed in the fibroblast cell line C C L 3 9 , looking at cyclin Dl expression (Lavoie et al, 1996). In these cells, Erkl/2 activation was necessary and sufficient to induce cyclin Dl expression in response to growth signals, regulating entry into S-phase. In contrast, p38 signalling negatively influenced cyclin Dl synthesis. This effect could be reversed by treatment with SB203580, confirming the importance of the role of p38 in this process. The molecular mechanism of regulation by p38 of cyclin Dl expression is not clear. This situation does however place p38 in a role as a negative regulator of cell growth, and implicates p38 in the maintenance of cell quiescence in fibroblasts. Signal integration may occur at any level of the signalling cascade, including upstream of the MAP kinase modules, within the cascade, or at the level of downstream targets. Several protein kinases lie downstream of MAP kinases and may be regulated by more than one pathway, such as the Mapkapk-3 and Mnkl/2 (MAPK signal integrating kinase/MAP kinase-interacting kinase) (McLaughlin et al, 1996,- Sithanandam et al, 1996,-Fukunaga and Hunter, 1997,- Waskiewicz et al, 1997). Mnkl/2 are activated in response to stress and mitogenic signals, with the p38 kinase and Erkl/2 pathways implicated respectively. In vitro, Mnkl is capable of phosphorylating the eukaryotic initiation factor-4E (eIF-4E) at the physiologically relevant site, Ser-209. Mnkl/2 may therefore represent a convergence point for the Erk and p38 MAP kinase pathways, potentially leading to eIF-4E phosphorylation and transcriptional activation. 30 An obvious site of signal integration occurs at the level of gene transcription. Immediate early gene promoters contain elements that bind various combinations of transcription factors that act cooperatively to regulate transcription. Distinct M A P kinases may differentially regulate gene transcription in ways not evident from simple analysis of isolated transcription factors and promoter elements. Activation of a subset of transcription factors which work cooperatively to direct gene transcription, ultimately defines the response of the cell. 1.8 Signal transduction during meiosis. 1.8.1 Activation of G2-arrested oocytes. The parallels evident between yeast and mammalian cell systems confirm that information obtained in one cell type can be used towards research in other cell types. Current research on signalling cascades in human cells relies on the use of cell culture. These human-derived cells have inherent differences that allow them to grow in culture which must be accounted for in the interpretation of results. Human cell lines probably use the same signalling proteins as in normal human cells, with an unknown factor of the genetic alterations allowing immortal growth. It may therefore be equally valid to use non-human based systems to obtain signalling information to be applied to the human situation. The use of primary cell systems ensures the fidelity of the signalling molecules responding to extracellular signalling events. An abundant source of material for study can be obtained through use of the seastar oocyte. In most animal species, immature oocytes are arrested naturally in the first meiotic prophase (prophase I) or the late G2 phase (Masui and Clarke, 1979,- Murray and Hunt, 1 9 9 3 ) . Hormonal stimulation releases this block, promoting passage of the cell through meiosis I and stopping again at a second developmental arrest point in 3 1 preparation for fertilization (Sagata, 1996). The relationship between G2-arrested oocytes and GO-arrested somatic cells appears very distant, involving unrelated mitogenic signalling cascades, triggering different regulatory processes, and leading to different cell cycle stage specific responses. GO-arrested somatic cells are activated in response to growth factors or other mitogens. This activation leads to the initiation of complex signalling cascades, transcriptional activation, and eventually the activation of DNA synthesis. G2-arrested oocytes respond to hormones or species-specific mitogens in a manner reminicent of mitotic cell division, including disintegration of the nuclear envelope, chromosome condensation, and a profound reorganization of virtually all cellular structures as cells enter M phase of first meiosis. While the endpoints are obviously different, the signalling pathways and many of the earliest events occurring during cell cycle re-entry are remarkably similar. Activation of both somatic cells and oocytes rapidly leads to changes in phospholipid metabolism, transient elevation of free intracellular calcium, a rise in intracellular pH, alteration of the cytoskeleton, and dramatic switches in the patterns of gene transcription and mRNA translation (Ruderman, 1993). The information obtained from G2-arrested oocytes can therefore be used to predict signalling mechanisms used in the activation of quiescent cells. 1.8.2 Signal transduction in P. ochraceus oocytes. Echinoderms have long been a favorite tool of developmental biologists. Early development of Pisaster ochraceus has been described (Feder, 1956), with detailed studies looking at the characteristics of the P. ochraceus which make them advantageous experimental systems (Fraser etal, 1981). The seastar oocyte is relatively clear and has a large germinal vesicle and visible nucleolus in the immature phase (Figure 3, Panel A). These characteristics allow for detailed analysis of the cytoplasmic rearrangements 32 c E o E ro CM E o o £ £ u <L» > .y * J= ,2 V C V) *« • £ O ^ c c -5 o o ti « 3 ro +-» ro u, s 3 ro E w - a 5 « U u 6 g E 2 ^ a-^ " S ~u « a. c ro B S -a £ | K O * J _ u ro >\ ^ trt W O . & c o o o b"> > o > i i , c v - DO-S r e g c c c -ts 3 3 O ro fc c c E y u 6 0 «*i g .1 1 ^ £ £ C x i ° ~ a -C . zr ^ ° u o o O E c oa ^ ,E ro „ re c y ro "*J m ro i/T .y c 4= 3 t w .5 B E E "* ~ o (U > re ic C "5 j ; a i .a s >, J j i i g E j £ 3 C O 3 o C O c c o v ^ ? re w E o TS . . M C . oo re ro vi w i w u o J trt ro V a 3 3 §, w § .E ro g ; £ | - S E E E ro 33 occurring during the maturation process. The gradual dissolution of the nuclear membrane is clearly evident, with the oocyte appearing homogeneous when fully matured (Figure 3, Panel D). The ease of observation of the stages of germinal vesicle breakdown, combined with the relatively large size of the oocytes also make them ideal for microinjection experiments. Meiotic maturation of the seastar oocyte occurs in response to the natural hormone 1 -methyladenine (Kanatani etal, 1969), a nucleic acid analogue which binds to an external receptor on the oocyte surface. During oocyte maturation, prior to germinal vesicle breakdown (GVBD), there is a burst in net protein phosphorylation (Guerrier et al, 1977). Early experiments in microinjection of seastar oocytes showed that alkaline phosphatase and purified preparations of the catalytic subunits of protein phosphatases 1 and 2A prevent 1-methyladenine-induced maturation (Meijer et al, 1986), whereas the phosphatase inhibitor oc-naphthylphosphate was found to trigger maturation in the absence of 1-methyladenine (Pondaven and Meijer, 1986). The burst of phosphorylation observed implicates a concerted action of protein kinase cascades in signal amplification and ultimately the regulation of the processes required for cell division. Initial experiments showed evidence for at least five protein kinases undergoing activation with oocyte maturation (Pelech, etal, 1988). These kinases were distinguished on the basis of the time courses of their activation following 1-methyladenine stimulation, their substrate specificities, and their chromatographic properties. Information obtained in the study of Xenopus laevis oocytes revealed that more than 150 proteins undergo enhanced phosphorylation near the time of GVBD, including the 40 S ribosomal protein S6 (Hanocq-Quertier and Baltus, 1981,- Nielsen et al, 1982), nucleoplasmin (Cotton et al, 1986), and nuclear lamins (Miake-Lye and Kirschner, 1985). This information prompted the search for the kinases responsible for these phosphorylations. In separate 34 experiments in the mid-1970s, microinjection of cytoplasm from 1 -methyladenine treated oocytes into untreated immature starfish (Asterina pectinijera) oocytes led to the detection of an activity known only as maturation- or M-phase-prompting factor (MPF) (reviewed in Kishimoto, 1996). The two directions of research converged at the identification that MPF was the histone H i kinase under investigation by other researchers (Labbe et al, 1988,- Arion et al, 1988). More specifically, MPF was discovered to be a complex of Cdc2 (the homologue of the S. pombe Cdc2 gene product) and cyclin B. This has led to intense research on the regulation of Cdc2 kinase (or Cdkl) in the oocyte system (Doree and Galas, 1994). Discoveries have included identification and characterization of an Cdkl-upstream activating kinase, Cdk7 with its associated subunit cyclin H, and a third stabilizing partner Matl (menage a trois-1) (Devault ef al, 1995). Other research has centered on a panel of specific inhibitors of cyclin-dependent kinases (Meijer et al, 1997; De Azevedo etal, 1997; Abraham etal, 1995). Research has continued on the signalling cascades initiated upon 1 -methyladenine exposure. The receptor for the hormone is a serpentine receptor with an associated heterotrimeric G protein. The py-subunits o r t n e G-protein have been purified in an attempt to understand their role in the maturation process. Microinjection of the purified proteins is sufficient to induce oocyte maturation, including formation of the MPF complex. Studies have implicated that the Py-subunits play an essential role upstream of the activation of the Cdkl/cyclin B dimer (Chiba etal, 1993). 1-Methyladenine activation of the immature oocyte also involves the induction of protein synthesis. As stated earlier, the phosphorylation of the 40S ribosomal protein S6 occurs during the maturation process. The eIF-4F complex, eIF-2 and eIF-2B proteins have also been shown to play a role in increased protein synthesis in response to 1-35 methyladenine. The phosphorylation of eIF-4E, the mRNA cap binding protein, appears to correlate with the increase in protein expression. This phosphorylation is also coincident with the activation of Cdkl and MAP kinase, but these kinases are not able to phosphorylate eIF-4E at its in vivo phosphorylation site (Xu etal, 1993). One of the first MAP kinases was identified via the purification and characterization of an MBP kinase activity from the P. ochraceus oocyte (Sanghera et al., 1990), termed p 4 4 m p k . The exact role of MAP kinase in seastar oocyte maturation is unclear, and appears to differ from the activity of MAP kinase in the frog oocyte. In the Xenopus, M A P K appears to play a role in the formation of the active MPF complex, while in the starfish M A P K activation occurs after the initiation of GVBD, long after M P F activation (Abrieu et al, 1997). The routes of MAP kinase activation in the echinoderm oocytes is also quite distinct from the cascade found in the Xenopus. p 4 4 m p k possesses a TEY sequence in its activation loop, characteristic of the Erkl /2 family of MAP kinases. Mpk has been found to autophosphorylate on a serine residue, as does Erk l , which correlates with a modest activation of the MBP phosphotransferase activity (Sanghera et al, 1990). Mek and Raf are both detectable in seastar oocytes with a range of specific antibodies, yet there is very little activation measured in response to oocyte maturation (Pelech and Charest, 1995). An important regulator of M A P K activity in the frog oocyte is the cytostatic factor Mos ; however, existence of a Mos-like kinase has not been established in the seastar oocyte. The in vivo kinase responsible for the activation of Mpk has yet to be fully identified and characterized. Mpk activation may occur downstream of a Src-related kinase, as has been observed in vitro (Ettehadieh etal, 1992). 36 1.9 Rationale and research objectives. It is clear that the P. ochraceus oocyte system possesses a number of signal transducing enzymes that are homologous to those in mammalian systems. The information obtained from the study of oocyte maturation could therefore be used to search for mammalian counterparts of seastar enzymes, which are likely to be involved in the maintenance and release of cells from quiescence. With each seastar able to produce up to 50 ml of packed cells (1 x 107 eggs), the system is ideal for the purification of novel proteins involved in cell signalling. For this reason, the P. ochraceus oocyte system was chosen to search for unique protein kinases. The oocyte, as a translucent cell, is ideally suited for this research with the ease of assessment of the maturation process (Figure 4). During a time course of 1-methyladenine activation, the gradual disintegration of the germinal vesicle is clearly visible as a marker for the progression of maturation. This is especially important for microinjection experiments, where subtle changes in the maturation process can be monitored. The objective of this study was to analyze the process of oocyte maturation, in an attempt to understand the mechanisms controlling the progression of oocytes through the stages of meiosis. Specifically, analyses would be performed to identify proteins involved in the control of oocyte maturation, with tyrosine phosphorylation used as a marker of activity regulation. The ultimate goal of this study was to identify potentially novel proteins which regulate maturation, and to further characterize the roles of these proteins in the oocyte in response to a variety of different stimuli. Specific probes could eventually be developed to characterize these proteins in mammalian systems. Together this information could be used to assess the role of novel signalling molecules in the regulation of cell cycle progression. 37 CHAPTER 1 MATERIALS A N D M E T H O D S 1.1 Materials. 2.1.1 Chemical reagents and laboratory supplies. 3mm filter paper Acetic acid Acrylamide Adenosine 5'-triphosphate disodium salt (ATP) [y-32P]ATP Agar Agarose Agarose (Low melting point) Amido black 10B Ampicillin (D[-]-(X-Aminobenzylpenicillin) Bactotryptone Bactoyeast extract Benzamidine Benzamidine Sepharose-6B Bis-acrylamide Bovine serum albumin (BSA) Brij 35 5-Bromo-4-chloro-3-indoyl phosphate (BCIP) Bromophenol blue VWR Fisher ICN/Fisher Sigma Amersham/Mandel/ICN Difco-Fisher/VWR Gibco/BRL Gibco/BRL ICN Sigma Difco-Fisher/VWR Difco-Fisher/VWR ICN Pharmacia ICN/Fisher Sigma VWR Sigma ICN 38 OC-Casein, dephosphorylated Chloroform Coomassie Brilliant Blue G Coomassie Brilliant Blue R-250 N, N-Dimethyl formamide (DMF) Dithiothreitol (DTT) DNA 1 kb ladder dNTP kit Enhanced chemiluminescence kit (ECL) Ethanol Ethidium bromide Ethylene bis (oxyethylenenitrilo) tetraacetic acid (EGTA) Ethylene diamine tetraacetate disodium salt (EDTA) Formaldehyde Formamide Glutathione agarose (GSH-agarose) Glycerol (3-Glycerophosphate Glycine Heparin Sepharose Histones Hydrochloric acid (HQ) Hydroxylapatite (HTP) Isopropyl p-D-thiogalactopyranoside (IPTG) Sigma Fisher Aldrich Fisher Fisher BDH Gibco/BRL Pharmacia Amersham Fisher Molecular Probes Inc. Fisher/ICN Fisher/ICN Gibco BRL Gibco BRL Sigma Anachemia ICN ICN/Fisher Sigma Sigma Fisher BioRad Promega 39 Lysozyme Sigma Magnesium chloride (MgCl2*6H20) Fisher Manganous chloride (MnCl 2 »4H 2 0) Fisher (3-Mercaptoethanol Fisher Fisher Methanol 1 -Methyladenine Sigma Sigma DL-threo-p-Methylaspartic acid Sigma Mineral oil Sigma Mixed bed resin Gibco BRL MMLV Reverse transcriptase Pharmacia MonoQ Sigma/ICN MOPS 3-[N-Morpholino]ethanesulfonic acid Kinetek/Sigma Myelin basic protein Fisher Nitric acid Sigma Nitro blue tetrazolium (NBT) Gelman Science Nitrocellulose BDH Nonidet P-40 (NP-40) VWR P81 filter paper Perkin Elmer PE-RNA PCR kit ICN Phenol Sigma Phenyl methylsulphonyl fluoride (PMSF) Pharmacia Phenyl-Sepharose Gibco Phosphate buffered saline (PBS) Fisher Phosphoric acid (H 3P0 4) PKI - cAMP-dependent protein kinase peptide inhibitor Sigma Polylysine agarose Sigma Ponceau S concentrate Sigma Phosvitin Sigma Potassium dichromate (K. 2Cr 20 7) BDH/VWR Potassium dihydrogen orthophosphate (KH 2P0 4) BDH/VWR di-Potassium hydrogen orthophosphate 3-hydrate (K 2HP0 4) BDH/VWR Prestained SDS-PAGE standards BioRad/Kinetek Protamine chloride Sigma Protein A Sepharose CL4B Pharmacia Q-Sepharose Pharmacia Qiagen kits Qiagen Inc. Random primers (5'-pd(N)6) (100 pmol/pll) Pharmacia ResourceQ Pharmacia Restriction enzymes NEB/Promega/BM RNasin Promega/Fisher Silver nitrate (AgNOa) Fisher Skim milk Safeway Sodium acetate (NaOAc) BDH/VWR/Fisher Sodium azide Fisher Sodium carbonate (Na 2C0 3) BDH/VWR Sodium chloride (NaCI) Fisher Sodium dodecylsulphate (SDS) Fisher Sodium fluoride (NaF) BDH/VWR/Fisher 41 Sodium hydroxide Sodium orthovanadate (Na 3V0 4) Soybean trypsin inhibitor (SBTI) Superscript II RNase H RT T4 DNA ligase Taq polymerase Terminal transferase Thrombin Tris (hydroxylmethyl) methylamine (Tris) Tris hydroxylmethyl aminomethane hydrochloride (Tris-HCI) Fisher Tween-20 (polyoxyethylene-20-sorbitan monolaurate) Fisher Vent DNA Polymerase NEB X-gal Gibco BRL Xylene cyanol Sigma Fisher Fisher Sigma Gibco BRL Gibco BRL Perkin Elmer Boeringer Mannheim Sigma ICN/Fisher 2.1.2 Photography supplies. Developer and fixer Reflection autoradiography film ISO 3000 Polaroid film 667 ISO 100 Polaroid film Medtec Mandel Medtec Medtec 2.1.3 Plasmids and bacterial strains. Bluescript II KS M l 3 ( + ) plasmid pGEX-4T3 vector Stratagene Pharmacia 42 D H 5 a bacteria UT5600 protease-bacteria BRL NEB 2.1.4 Antibody reagents. 4G10 antiphosphotyrosine antibody UBI Blotting grade affinity purified goat anti-rabbit IgG (H+L) BioRad alkaline phosphatase conjugate Blotting grade affinity purified goat anti-mouse IgG (H+L) BioRad alkaline phosphatase conjugate Goat anti-rabbit IgG - horse radish peroxidase conjugate Calbiochem Goat anti-mouse IgG - horse radish peroxidase conjugate BioRad Unique antibodies created in this laboratory are described in Appendix I. 2.1.5 Sources of oligonucleotides. A detailed description of all oligonucleotides can be found in Appendix II. U B C / S F U Oligonucleotide Synthesis Labs 43 2.2 Manipulations of P. ochraceus oocytes. 2.2.1 P. ochraceus oocyte maturation. The common purple seastars, P. ochraceus, were collected from beaches in the Greater Vancouver area. Ovaries were surgically removed from the arms of the P. ochraceus and kept on ice in natural sea water. Ovaries were gently teased apart with forceps and razor blades to release the oocytes, and were strained through a large mesh filter to remove connective tissue and residue. Oocytes were washed three times with cold sea water, and pelleted by centrifugation at 400 x g for 5 min. Four hundred ml of packed oocytes (-2.1 x 10 s eggs/ml,- Fraser et al., 1981) were resuspended in 4 1 of natural sea water containing 4 JLlM 1-methyladenine at 14°C for 90-120 min. Maturation was achieved by the onset of germinal vesicle breakdown (GVBD), as evidenced by the disappearance of the nucleus within the oocyte when viewed under high magnification. Mature oocytes were harvested when GVBD occured in over 8 0 % of the oocytes, or 2 h after initiation of maturation. For the maturation time course, measured volumes of oocyte suspension were removed at discrete time points after the addition of 1-methyladenine. 2.2.2 P. ochraceus oocyte homogenization. Oocytes were pelleted by centrifugation at 400 x $ for 5 min. To every 200 ml of packed cells, 400 ml of homogenization buffer (50 mM (3-glycerophosphate, 20 mM MOPS, 5 mM EGTA, 2 mM EDTA, 1 mM N a 3V0 4, 0.25 ujvt DTT, 5.0 \LM P-methylaspartic acid, 1.0 mM PMSF, 1.0 mM benzamidine, pH 7.2) was added. The oocytes were homogenized in a Waring blender in 2 x 15 sec bursts, and centrifuged at 9000 rpm for 15 min in a Beckman J2-HS centrifuge to remove particulate matter and 44 organelles. The post-mitochondrial supernatant was centrifuged in a Sorval Combi Ultracentrifuge (Dupont, Canada) at 250,000 x g for 30 min and the supernatants immediately aliquoted and frozen at -70°C until required. For time course samples the oocytes were homogenized by 2 x 30 sec bursts with a sonicator, and centrifuged at 250,000 x g for 30 min. Supernatants were stored in 1 ml aliquots at -70 °C until required. 2.2.3 Osmotic shock time course. Oocytes from individual P. ochraceus were resuspended in natural seawater containing 1.0 M, 1.5 M, 2.0 M, or 3.0 M NaCl based on the initial concentration of NaCl in sea water of 0.5 M. A first volume of oocyte suspension was removed immediately, pelleted, and the sea water removed. The oocytes were quick frozen in a dry ice/ethanol bath. The remaining suspensions were incubated at 14°C, with aliquots removed at discrete time points after the addition of the high salt sea water. Frozen, packed cells were stored at -70°C. 2.2.4 Heat shock time course. Oocytes from individual P. ochraceus were resuspended in a measured volume of natural sea water preheated to 25°C, 35°C, or 45°C. The zero time point of oocyte suspension was removed, pelleted, the sea water removed, and the oocytes quick frozen in a dry ice/ethanol bath. Suspensions were incubated at the given temperatures, with aliquots removed at discrete time points after the addition of the preheated sea water. Frozen, packed cells were stored at -70°C. 45 2.2.5 Time course homogenization. Due to the treatment of the cells, cell volumes changed significantly during both the osmotic and heat shock time courses. For example, the osmotic shock samples cells swelled from 1 ml starting volume to a final volume of 5 ml. To keep the protein concentrations relatively constant, the oocytes were homogenized in a standard total volume. To each aliquot of packed cells, 900 \l\ of 5x homogenization buffer was added. Samples were diluted to a final volume of 6 ml and homogenized by two 30 sec bursts with the sonicator. The extracts were then centrifuged at 250,000 x c) for 30 min, the supernatants divided into 1 ml aliquots and stored at -70°C until required. 2.2.6 P. ochraceus oocyte fertilization. Fertilized oocyte extracts were a generous donation of Dr. D. Lefebvre (University of Toronto) and were prepared as follows. Sea stars were induced to spawn by injection into the body cavity of a minimum of 1 ml per arm of 0.14 mM 1-methyladenine in Millipore filtered seawater (Fraser et al, 1981). Shedding of mature oocytes typically commenced between 60-90 min following primary injection. During spawning, sea stars were inverted over 400 ml beakers containing filtered seawater. After approximately 60 min of shedding, the oocytes were allowed to settle and were washed with three changes of filtered seawater. The oocytes were resuspended at a concentration of 1 % (v/v) in filtered seawater. The container was placed in a refrigerator, equilibrated to 12±0.5°C and the seawater was gently aerated and oscillated at 40 rpm. The oocyte suspension was allowed to equilibrate at this temperature for at least 1 h prior to addition of sperm. Sperm was collected from male sea stars and diluted 1:200 (v/v) with filtered seawater. Sperm viability and motility were verified by phase microscopy. Equilibrated mature oocytes were fertilized by addition of 1:100 (v/v) of 46 the sperm dilution (effective dilution 1:20000 (v/v)). Fertilization of oocytes was confirmed in an aliquot of oocytes at approximately 1 h following sperm addition by observing the elevation of fertilization membranes. Embryo cultures with less than 70% fertilization of oocytes were discarded. At specific time points during development, the embryos were pelleted at 4°C in a Beckman J2-HS centrifuge (1500 rpm for 5 min). A 33% (v/v) suspension was prepared in chilled homogenization buffer and the embryos disrupted with 2 x 30 sec bursts at 19,000 rpm of a Polytron (PT3000, Brinkman, USA). Homogenates were immediately centrifuged in a Sorval Combi Ultracentrifuge at 10,000 x g for 10 min. The supernatant was then decanted and centrifuged at 250,000 x g for 30 min. Supernatants were quickly aliquotted and stored at -70°C. 2.3 Antibody production. The affinity-purified antibodies Cdk5-CT, Erk l -CT, Cdc2-CT, PSTAIRE, Cdc2-IX, and CdCK-2B were prepared as described (Sanghera et al, 1992) and many are commercially available from Upstate Biotechnology Inc. (Lake Placid). The immunizing peptides were produced on an A.B.I, automated peptide synthesizer. The peptide was cleaved from the synthesis resin, and purified by reverse-phase-high performance liquid chromatography (RP-HPLC). Purity was assessed by analytical RP-HPLC and the sequence confirmed by amino acid analysis. This peptide was coupled to K L H prior to immunization into rabbits. New Zealand White rabbits were subcutaneously injected with KLH-coupled immunizing peptide in PBS with Freund's incomplete adjuvant every 4 weeks. The sera from these animals was applied onto an agarose column to which the immunogen peptide was thio-linked. Antibody was eluted from the column with 0.1 M 4 7 glycine, pH 2.5. Subsequently, the antibody solution was neutralized to pH 7.0 with saturated Tris. Antibody affinities were assessed by ELISA. 2.4 Protein quantitation. Proteins were quantitated by the method of Bradford (1976). A series of protein standards were prepared ranging from 0-30 \ig BSA with 2.5 ml of Bradford reagent (100 mg Coomassie Blue G, 50 ml ethanol, 100 ml H 3 P0 4 , 850 ml dH 20). The protein to be quantitated was diluted with dH 2 0 to within 5-20 fig/10 jLLl. The appropriate dilution was added to 2.5 ml Bradford reagent and mixed by gentle vortexing. After 5-10 min incubation, the absorbances of the solutions were measured at 595 nm and the concentrations of the samples calculated through linear regression plotting of the standards. For samples containing Brij, Bradford assays could not be performed. Instead, the absorbance at 280 nm was measured for each fraction, and the protein concentration calculated as follows: [protein] (mg/ml) = A280 x 1.55 2.5 ResourceO fractionation. Cytosolic extracts from P. ochraceus were fractionated by fast protein liquid chromatography (FPLC) on 1 ml ResourceQ columns (Pharmacia). Columns were equilibrated before and after use with 2 ml of 2.0 M NaCI, and washed by running the program gradient, including the post-gradient equilibration steps. All buffers were filtered (0.22 \L) before use, and samples filtered (0.45 (l) before application to the column. 48 Samples were prepared as described, and the protein concentrations quantitated if necessary by Bradford assay. Up to 5 mg of protein was diluted to 2.1 ml with buffer A (10 mM MOPS, pH 7.2, 25 mM p-glycerophosphate, 2 mM EDTA, 5 mM EGTA, 2 mM N a 3 V 0 4 ) . Two ml of sample was applied to the column and a standard elution program including a 10 ml 0-0.8 M NaCl linear gradient was performed. The eluate was collected in either 0.25 ml or 0.50 ml fractions. These samples could then be further analyzed by protein kinase assay, SDS-PAGE, immunoprecipitation, or Superose 12 chromatography. 2.6 Superose 12 chromatography. Apparent molecular weight was determined by elution profile on a Superose 12 gel filtration column (Pharmacia). The 20 ml column had a maximum sample application volume of 200 Jill, so samples were initially concentrated on a ResourceQ column. The column was eluted with 20 ml of 0.2 M NaCl in KII buffer. One mg of each molecular weight standard protein was used to calibrate the column and create a standard curve from which the molecular weight of the unknown protein could be estimated. 2.7 SDS-polyacrvlamide gel electrophoresis. Proteins were separated using sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). Proteins were boiled for 5 min with 1 volume of 4x sample loading buffer (125 mM Tris-HCI (pH 6.8), 4 % SDS (w/v), 2 0 % glycerol (v/v), 0.3 M P-mercaptoethanol, 0 . 0 1 % bromophenol blue (w/v)). Proteins were subjected to electrophoresis on 1.5 mm thick polyacrylamide gels with 4 % stacking gels and 1 0 % separating gels. The gels were electrophoresed in running buffer (25 mM Tris, 49 192 mM glycine, 3.5 mM SDS) at 10 mA overnight (for large gels), or 180 V for 90 min (for minigels), until the dye front reached the bottom of the gel. 2.8 Staining of SDS-PAGE gels. 2.8.1 Silver staining. In preparation for silver staining (Merril et al. 1981), gels were first soaked in fixative 1 ( 4 0 % methanol/10% acetic acid (v/v/v)) for 30 min and fixative 2 ( 1 0 % ethanol/5% acetic acid (v/v/v)) for 2 x 1 5 min. The gels were incubated for 5 min in oxidizer (3.4 mM KjCrjG^, 3.2 mM nitric acid) followed by three washes with d H 2 0 . The gels were then stained with 0.204% A g N O B (w/v) for 20 min. Gels were washed briefly in d H 2 0 and developed with 0.28 M N a 2 C 0 3 in 0.166% formaldehyde solution (v/v). Development was stopped by soaking the gel in 5 % acetic acid (v/v) and the gel then stored in d H 2 0 . 2.8.2 Coomassie blue staining. Gels were immersed in Coomassie stain ( 0 . 5 % Coomassie Brilliant Blue R/45% methanol/10% acetic acid (w/v/v/v)) for 15-30 min, then soaked in 4 0 % methanol/10% acetic acid (v/v/v) until the gel was sufficiently destained to allow appearance of protein bands and clearing of the background gel. 2.9 Transferring to nitrocellulose membranes. 2.9.1 Western blotting. The gel was first equilibrated for 5 to 15 min in transfer buffer (20 mM Tris, 120 mM glycine, 2 0 % methanol (v/v), pH 8.6) to remove SDS. Nitrocellulose membrane was hydrated in transfer buffer for at least 1 min before transfer. The gel and membrane 50 were assembled into a transfer sandwich between 6 pieces of 3 M M filter paper. Proteins were electrophoretically transferred in a Hoeffer transfer cell at 4°C for 3 h at 300 mA. Transferred proteins were first visualized using Ponceau S dye by incubating the membrane in the stain for 1 min, then washing the excess stain away with water. Membranes were blocked in 5 % skim milk (w/v) in TBS (50 mM Tris base, 150 mM NaCl, pH 7.5) for 2 h. Membranes were rinsed briefly with TTBS ( 0 . 0 5 % Tween-20 (v/v) in TBS) to remove excess blocking solution. The primary antibody was diluted to the optimum concentration (usually 1/500-1/1000), in TTBS with 0.1% azide (w/v) and incubated with the membrane overnight at room temperature with agitation. The blots were washed extensively before incubation with the appropriate secondary antibody, diluted to the optimum concentration in TTBS for 2 h at room temperature. Alkaline phosphatase conjugated secondary antibodies were diluted 1/2000 while horseradish peroxidase conjugated goat anti-rabbit antibodies were diluted 1/20,000 and discarded after each use. Excess secondary antibody was removed by thoroughly washing with TTBS, and Tween-20 rinsed away with TBS. Alkaline phosphatase Western blots were developed in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) colour development solution (mixture of 3 % NBT in 1 ml 7 0 % dimethylformamide (DMF) and 1.5% BCIP in 1 ml 1 0 0 % DMF before addition of 100 ml of AP buffer (0.1 M Tris, pH 9.5, 0.1 M NaCl, 5 mM MgG2)). Development was stopped by soaking in dH 2 0. ECL Western blots were incubated for 1 min with ECL solution and dried briefly. The Western blot was then exposed to film to visualize the immunoreactive proteins. During protein purification steps, membranes were blocked for 15 min, primary antibody incubated with the membrane for a maximum of 1 h, and alkaline phosphatase 51 secondary antibody incubation time reduced to 30-45 min. The blots were then developed as described. For antiphosphotyrosine blots, the membranes were blocked overnight at room temperature using low-salt TBS (20 mM Tris, pH 7.5, and 50 mM NaCI) containing 3 % BSA (w/v). Primary antibody was incubated for 4 h, and alkaline phosphatase conjugated secondary antibody incubated for 2 h. All washes were performed with low-salt TBS containing 0 . 0 5 % Nonidet P-40 (NP-40). Blots were developed as described above. 2.9.2 Ponceau S staining of Western blots. Western blots were developed with alkaline phosphatase as described. After rinsing in water, Western blots were placed in Ponceau S solution for 30 sec to stain, and washed thoroughly in water. This method allows staining of the protein bands over the background of the blocking solution. The bands developed in the Western blot show clearly through the Ponceau S stain. 2.9.3 Amido black staining. Nitrocellulose membranes were immersed in 0.1% amido black/45% methanol/10% acetic acid (w/v/v/v) for 15-30 min at room temperature with shaking. Destaining was carried out in a 4 5 % methanol/10% acetic acid (v/v/v) solution until bands were visible and background was reduced. 2.9.4 Stripping andreprobing of Western blots. Membranes were stripped by incubation in 100 mM P-mercaptoethanol, 2 % SDS (w/v), 62.5 mM Tris-HCI (pH 6.7) at 50°C for 30 min with occasional agitation. The 52 membranes were then washed with TTBS until the P-mercaptoethanol could no longer be detected. The membranes were reblocked in 5 % skim milk in TBS for 1 h and Western blotted as described. 2.10 Immunoprecipitation. Protein A-Sepharose CL.4B was swollen for 15 min in 3 % NETF ( 3 % NP-40 (v/v) in 100 mM NaCl, 5 mM EDTA, 50 mM Tris-HCI (pH 7.4), 50 mM NaF). The beads were washed twice in 3 % NETF and resuspended in an equal volume of 3 % NETF. Cytosolic extracts were brought to 1 % SDS (w/v) with the addition of 2 0 % SDS. The extracts were then diluted with an equal volume of 6 % NETF ( 6 % NP-40 in NETF buffer). Extracts were precleared with 10 |il of Protein A-Sepharose slurry, incubated at 4°C for 15 min with rotation. The beads were removed by centrifugation for 1 min at 15,000 rpm. Ten |lg of antibody were incubated with the supernatants for 1 h at 4°C with rotation. To the mixture were added 20 JLLl of Protein A-Sepharose slurry and the antibody was allowed to complex for 45 min at 4°C with rotation. The beads were washed 2 x with 6 % NETF and once with NETF. Immunoprecipitates were boiled for 5 min in 4x SDS sample buffer and subjected to SDS-PAGE and Western blotting as described. 2.11 Protein kinase assays Kinase assays were performed on column fractions, immunoprecipitations as well as expressed proteins. Phosphotransferase reactions were carried out in ADB (20 mM MOPS, pH 7.2, 25 mM p-glycerophosphate, 20 mM MgCl 2, 5 mM EGTA, 2 mM EDTA, 1 mM DTT, 5 |lM P-methylaspartic acid and 1 mM sodium vanadate) in a total volume of 53 30 (ll containing, 0.5 \lM PKI, 50 |lM [y-32P]ATP (-2000 cpm/pmole), 5 pig of protein substrate or 2 \ig of peptide substrate. Reactions were incubated at 30°C for 10 min and stopped by spotting 20 |il onto P81 filter paper squares (Whatman). Filter papers were washed with 1 % H 3 P0 4 (v/v) to remove free ATP, placed into scintillation vials containing 200 jxl scintillant and counted in a scintillation counter. For protein substrates which did not bind to P81 paper, reactions were stopped with 4x sample buffer, boiled 5 min and analyzed by SDS-PAGE. Proteins were transferred to nitrocellulose and Ponceau S stained to visualize the substrate bands. Substrates were excised from the membrane and counted as described for the filter papers. Alternatively, gels were Coomassie stained to visualize the substrate bands, allowing the bands to be excised and the radioactivity counted. 2.12 Purification of MIPK from P. ochraceus oocytes. Approximately 16 g of cytosolic extract were thawed, diluted to 1:10 with KII buffer (5 mM MOPS, pH 7.2, 5 mM EGTA, 5 mM NaF, 1 mM Na 2 V0 3 , and 0.25 mM DTT) and applied to twenty-four 10 g hydroxylapatite columns. MIPK was eluted with 50-100 ml per column of 40 mM potassium phosphate buffer, pH 7.2. A total of 1 1 of eluate was diluted to 6 1 with KII and applied to six 25-ml Q-Sepharose columns. Proteins were eluted with a 280 ml, 0-0.8 M NaCI gradient in KII and through immunoblotting, MIPK was found to elute between 0.23-0.33 M NaCI. The pooled peak fractions, 240 ml total, were brought up to a final concentration of 1 M NaCI, with 300 ml 4 M NaCI in KII and 660 ml KII. This was applied to three 25-ml phenyl-Sepharose columns and eluted with a 300 ml linear gradient, 1-0.67 M NaCl/0-0.5% Brij 35 in KII. MIPK was found to elute between 0.73 M NaCl/0.4% Brij 35 and 0.67 M NaCl/0.5% Brij 35. The pooled peak fractions (150 ml) were diluted to 1.2 1, and adsorbed to three 25-ml polylysine-agarose 54 columns. The columns were developed with a 280 ml linear gradient of 0-0.8 M NaCl, with MIPK eluting between 0.24-0.32 M NaCl. The final step in the purification involved fractionation of the pooled peak fractions on a 1-ml MonoQ column. The polylysine fractions were diluted 1:1 with KII prior to application onto the MonoQ column, and eluted with a 10 ml linear gradient of 0-0.8 M NaCl. MIPK eluted with 0.42-0.44 M NaCl. The 2 peak fractions were pooled and prepared to be sent for sequencing, as described below. An alternative method tested for the purification of MIPK is outlined in Figure 10 and was used to validate that MIPK corresponded to the major tyrosine phosphorylated protein observed in the immature oocyte cytosol. In this purification protocol, 3.4 g of immature cytosol protein was applied to 8 - 10 g hydroxylapatite columns. These columns were eluted with a 350 ml 0-0.14 M phosphate linear gradient. MIPK eluted 0.034-0.05 M phosphate. These peak fractions were diluted and loaded onto two 25-ml Q-Sepharose columns. The columns were eluted with a 280 ml linear gradient, 0-0.8 M NaCl, and the MIPK found to elute 0.25-.30 M NaCl. The pooled peak fractions were diluted and applied to a 25 ml heparin-Sepharose column. MIPK did not bind to the heparin-Sepharose, and the flow through was applied to a polylysine-agarose column and eluted as described. The peak fractions, which eluted at 0.29-0.33 M NaCl, were then brought up to a final concentration of 1 M NaCl and applied to a 5-ml phenyl-Sepharose column and eluted with a 75 ml linear gradient, 1 M NaCl, 0 % Brij to 0 M NaCl, 1.5% Brij. MIPK eluted at 0.57 M NaCl, 0.65% Brij to 0.53 M NaCl, 0.7% Brij. At this point MIPK responded uncharacteristically and would no longer bind to ResourceQ resin. The results were used to assess the efficiency of the chromatography steps but could not be used for sequencing. 55 2.13 Sample preparation for protein sequencing. Peak ResourceQ fractions containing the 40 kDa protein were determined by Western blotting with Cdk5-CT. The 2 peak fractions (500 |0,1) were loaded 250 |il at a time into one lane of an 11% SDS-PAGE gel. The gel was transferred onto a nitrocellulose membrane with a second nitrocellulose membrane as backup. Both membranes were stained with amido black revealing an abundance of protein on both membranes. The edge of the lane was cut off to allow Western blotting with Cdk5-CT. The membranes were realigned and the appropriate band identified. The band of interest was excised from both membranes and placed in d H 2 0 to prevent drying out. This band was then sent to Dr. Ruedi Aebersold at the University of Washington, Seattle, for sequencing. 2.14 Restriction digests. From 0.5 to 5.0 p ig of D N A were combined with 2 |ll of appropriate lOx reaction buffer as supplied with the enzyme, and 1-2 [l\ of enzyme in a final volume of 20 \i\ with s d H 2 0 . Reactions were incubated at 37°C for 1-2 h or overnight, and analyzed on an agarose gel. 2.15 Ligations For the majority of ligations, the vector, the insert, or both were present in low melting point (LMP) agarose and the following ligation protocol was used. This general recipe was adapted from Kalvakolanu and Livingston (1991). The amount of agarose in the mixture was never more than 25 (ll per 50 |ll reaction. To 18.5 |Xl of s d H 2 0 , 5 (0.1 5x ligation buffer (Sambrook et al, 1989) were added, and 0.5 \i\ 100 mM ATP (pH 7), and the mixture heated to 65-70°C. Vector agarose was also heated to melting for about 2-5 56 min at 65-70°C. Approximately 250 ng of vector was added to the preheated buffer mixture and the mixture cooled to room temperature on ice. To each tube was then added 1 U T4 ligase (1 U=amount of enzyme which will catalyze the exchange of 1 nmol 3 2PPi into [a/P-32P]ATP in 20 min at 37°C). The insert agarose was then melted at 65-70°C for 2-5 min and 20 \l\ added to the ligation mixture and mixed gently. The ligation was incubated at 16°C overnight. 2.16 Transformations. Products of ligation reactions were heated to 65°C to melt. Ten u\l of the ligation mixture were added to 50 |_Ll prewarmed sdH 20 and placed on ice. Two hundred \i\ of competent DH50C E. coli cells were added and incubated on ice for 30 min, 42°C for 90 sec, and on ice for 2 min. One ml of LB media (10 g bactotryptone, 5 g bactoyeast extract, 10 g NaCI, pH 7, Sambrook et al, 1989, A.l) was added and incubated at 37°C, 225 rpm for 1 h. The cells were plated on LB/ampicillin/X-gal (for pBluescript vectors) agar. For pure vector, a quick transformation was performed. Fifty |ll of competent DH50C or UT5600 E. coli cells were combined with a few ng of DNA (lfll) and incubated at 37°C for 1 min and chilled on ice for 1 min. Two hundred |il of LB media were added and the mixture plated on LB/ampicillin agar. 2.17 Cloning with T-tailed bluescript vector. This method of cloning is specific for PCR reactions involving Taq polymerase and is based on the procedure by Holton and Graham (1991). Five Jig of pBluescript KS+ vector were digested with 20 U of EcoRV for about 2 h in a 20 \i\ volume with BRL 57 React 2 buffer. While the vector was being digested, the PCR reaction was carried out to amplify the fragment to be cloned, including a final 10 min 72°C step to finish all the ends of the PCR products. This procedure worked best if the vector and PCR products were used immediately after preparation or stored at -20°C after preparation. Storage of the PCR products at 4°C overnight seriously affects the efficiency of cloning. After EcoRV digestion, the vector was incubated at 65°C for 10 min to denature the enzyme. The vector was precipitated in 1/10 volume (2 |il) 3M NaOAc, pH 5.2 and 2 volumes (45 100% ethanol for 20 min at -20°C. The mixture was centrifuged for 5 min at 15000 rpm, the supernatant aspirated off, the pellet washed with 70% ethanol and centrifuged again. The supernatant was again aspirated off and the pellet dried. The Boehringer terminal deoxytransferase kit was used to T-tail the vector in a 50 (ll total volume. Five |lg or vector in 28 |ll sdH 20, 10 JJLl 5x TdT buffer, 3 (J.1 25 mM CoCl 2, 5 (111 100 JlM ddTTP (Pharmacia) and 4 (ll of TdTransferase (Boehringer 25 U/u] with 1 U equal to the amount of enzyme that incorporates 1 nmol dAMP into acid-insoluble products within 60 min at 37°C using d(pT)6 as primer) were incubated at 37°C for 1 h. The above reaction conditions are 25 mM Tris-HCI, pH 6.6, 200 mM potassium cacodylate, 250 |Xg/ml BSA, 1.5 mM CoCl 2, 10 (lM ddTTP and 100 U of terminal transferase. The T-tailed vector was gel purified in 0.9% low melting point agarose, the band excised and weighed to estimate DNA concentration. To clone the unpurified PCR products, ligations were set up in a total volume of 20 (ll, using 200 ng of vector per ligation and a vectoninsert ratio of 1:1 to 1:3. The vector agarose was melted at 65°C for 5 min and pipeted into a microfuge tube containing the correct amount of dH 2 0. Up to 10 |ll of PCR product were added along with 2 ul lOx ligation buffer (Sambrook et al, 1989), 0.5 mM ATP, pH 7, 1 U T4 ligase (BRL) and the mixture was incubated at 14-15°C overnight. The ligation mix was warmed 58 at 65°C for about 1 min and 5 |ll were added to a microfuge tube containing 20 \i\ prewarmed sdH 20. Transformation into DH50C competent E.coli cells was performed as described above. 2.18 Plasmid preparation. 2.18.1 Small scale plasmid preparation. Small quantities of plasmid were prepared using the Qiagen, QIAprep plasmid preparation system. The QIAprep Plasmid Kit procedure is a modified version of the alkaline lysis method of Birnboim and Doly (1979) based on the adsorption of DNA onto silica in the presence of high salt (Vogelstein and Gillespie, 1979). From 2 to 4 ml overnight cultures of E. coli in LB medium were used in each preparation, depending on the copy number of the vector. DNA prepared for sequencing was eluted using sdH 20, while all other DNA preparations were eluted using TE (10 mM Tris-HCI, 1 mM EDTA, pH 8.0) buffer. 2.18.2 Large scale plasmid preparation. Large quantity DNA preparations were performed using the Qiagen Plasmid Midi Kit. One hundred ml overnight cultures of E. coli in LB medium were used in each preparation for low copy number plasmids such as pGEX-4T3. Pellets were resuspended in 100 |ll of TE buffer for storage. 2.19 DNA sequencing. Clones were sequenced by automated fluorescent DNA sequencing at U C D N A (University of Calgary) and the DNA Sequencing Laboratory (University of British Columbia). Reactions were performed using the standard dideoxy chain termination 59 method (Sanger et al, 1980) with custom primers (described in Appendix II) using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA polymerase (Pharmacia). Reactions were performed using a DNA thermal cycler model 480 (Perkin Elmer) and products purified by ethanol precipitation protocol I. 2.20 RNA isolation from P. ochraceus oocytes. RNA isolation was performed using the RNeasy isolation kit from Qiagen. Briefly, immature oocytes were extracted from the arm of one starfish for RNA extraction. Three hundred |ll of packed cells was diluted in 3.5 ml of lysis buffer with p-mercaptoethanol and lysed using a Polytron homogenizer (Brinkman) 2 x 1 0 sec. The lysate was centrifuged for 3 min at maximum, and 350 \i\ of supernatant was used for each purification column. The sample was purified with a phenol/chloroform extraction, and centrifuged for 10 min at 10,000 x g. The supernatant was extracted with 1 volume of chloroform, mixed and centrifuged again for 10 min at 10,000 x g. One volume of 7 0 % ethanol was added to the sample before application to an RNeasy spin column. Columns were washed with the appropriate buffers before elution with 50 \l\ of sdH20. The quantity of RNA was assessed on a formaldehyde gel. 2.21 RNA agarose gels. One percent (w/v) agarose was dissolved in 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0. After cooling to 55°C, 5 % deionized formaldehyde was added and the gel poured. Two volumes of RNA loading buffer (600 (xl lOx MOPS buffer, 2.1 ml deionized formaldehyde, 6.0 ml deionized formamide, 4.0 ml glycerol, bromophenol blue) were added to each RNA sample, and the samples heated at 60°C for 15 min. One |ll of 1 60 mg/ml ethidium bromide was added to each sample before loading on the gel. The gel was electrophoresed at 75 V until the bromophenol blue was 75% through the gel. 2.22 mRNA purification. mRNA preparations were made using the Qiagen Oligotex mRNA kit. Approximately 250 ul of total seastar RNA (-440 |lg) was diluted to 500 jULl with sdH 20 and 500 (ll binding buffer was added. Thirty JJll of 37°C preheated Oligotex resin was added and the sample incubated for 3 min at 65°C. The sample was incubated at room temperature for an additional 10 min prior to centrifugation for 2 min at maximum speed. The supernatant was aspirated off and the resin resuspended in 400 (ll of wash buffer. This slurry was applied to a spin column and washed a second time. The mRNA was eluted with 2 x 30 |ll of 70°C elution buffer. The final concentration of mRNA was estimated at 72-360 ng/lil. 2.23 Amplifying MIPK using polymerase chain reaction. 2.23.1 Quantitation of oligonucleotides. Oligonucleotides were synthesized in the 40 nmole scale as described in Appendix II. Primer pellets were dissolved in 100 (ll of sdH 20 and vortexed to resuspend. Five |ll of primer was diluted to 1000 |ll in sdH 20 and an absorbance reading measured at 260 nm in a spectrophotometer. The molarity of the solution was calculted according to the following equation: cone (M) = A260 x 33 mg/L x dilution factor # base pairs x 330 g/mole x 1000 mg/g 61 2.23.2 Reverse transcription reaction. The Perkin-EImer GeneAmp RNA PCR kit was used in the synthesis of P. ochraceus cDNA. Ten ng of mRNA was added to a reaction mixture containing 5 mM M g C l 2 , 2 (ll lOx PCR Buffer II (500 mM KC1, 100 mM Tris-HCI, pH 8.3), 1 mM dNTPs, 1 U RNasin (1 U is the amount of RNasin required to inhibit by 5 0 % the activity of 5 ng of ribonuclease A), 2.5 U,M random hexamer primers, and 50 U Moloney murine leukemia virus (MMLV) reverse transcriptase (1 U of M M L V RT incorporates 1 nmol d l IP into acid-precipitable material in 10 min at 37°C using poly(A)» oligo(dT)12 1 8 as template-primer) in a 20 Jll reaction. The mixture was incubated for 10 min at room temperature, 60 min at 42°C, 99°C for 5 min, and held at 4°C until ready to proceed. 2.23.3 Specific PCR conditions for MIPK. The 20 (ll reverse transcriptase reaction mixture was combined with 3 U.1 lOx PCR Buffer II, 24.5 (J.1 sdH 2 0 , 1.5 (ll of each primer, and 0.5 |ll Taq polymerase. The PCR reaction conditions were an initial 1 min at 99°C, 45 cycles of 99°C for 30 sec, 35°C for 60 sec, 72°C for 90 sec, followed by a 10 min incubation at 72°C to fill all ends. The PCR reaction was frozen immediately if not used right away for ligation reactions. 2.23.4 Confirmation of identity of PCR clones. This protocol was adapted from Sathe et al. (1991) and Barnes (1994). Positive colonies from transformation plates were picked with sterile toothpicks and swirled into 20 [ll of s d H 2 0 in microamp PCR tubes. The toothpicks were then stabbed into numbered spots on a LB/ampicillin plate to be grown overnight as stock cultures. To the PCR tube were added 13 111 sdH 2 0, 5 \i\ ImM dNTPs (final 100 | lM), 5 ul lOx Vent Pol. buffer (final 2 mM MgSQ 4 ) , 5 \i\ 1 mg/ml BSA (final 100 plg/ml), 1 |ll l M Tris base (final 62 20 m M Tris p H 9.1), 1 | l l each 20 | l M M l 3 Forward and Reverse primers, and 0.5 jLll TaqrVent mix (2.5U:0.025U). The P C R reaction conditions were 5 min at 94°C, 30 cycles of 94°C for 1 min, 1 min annealing at 55°C, 2 min elongation at 72°C, followed by a 10 min final extension at 72°C. Five hundred |lg of RNAse were added per ml of 6x gel loading buffer (0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF, 30% (v/v) glycerol in water) and 10 ul was added to each reaction tube. Twenty (ll of the reaction was electrophoresed on an agarose gel to assess the insert size. 2.24 Rapid amplification of c D N A ends ( R A C E ) . 2.24.1 Adaptor ligated c D N A . 5' and 3' R A C E reactions were performed using the C L O N T E C H Marathon c D N A Amplification Kit. This kit involves the production of an adaptor ligated c D N A library to be used for P C R amplification. Five (ll (~1 |lg) seastar m R N A was used for first strand synthesis. m R N A was combined with 1 |LXl 10 m M (1 m M final) c D N A synthesis primer and heated to 70°C for 2 min followed by 2 min on ice. T w o (ll 5x 1st strand buffer, 10 | l M d N T P s , 100 U M M L V - R T , 100 U Superscript II (1 U incorporates 1 nmole of d 1 1P into acid-precipitable material in 10 min at 37°C using poly(A)«oligo(dT) 2 5 as template-primer) were added, and the reaction incubated at 42°C for 1 h. The tube was placed on ice to terminate the reaction. For second-strand synthesis, the 10 | l l first-strand reaction was combined with 16 | l l 5x second-strand buffer, 0.5 m M dNTPs, 4 \i\ 20x second-strand enzyme cocktail, and s d H 2 0 to a final volume of 80 | l l . The reaction was incubated at 16°C for 1.5 h. Ten U T4 D N A Polymerase (1 U incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 min at 37°C) were added, and the reaction continued for 45 min at 16°C. The reaction was terminated with EDTA/glycogen mixture. The D N A was 63 purified by extraction and precipitated with 9 5 % ethanol. The pellet was resuspended in 10 al sdH20. Adaptor ligation was performed on the entire 10 ul of ds cDNA. Two uM Marathon cDNA adaptor, 4 (ll 5x DNA ligation buffer, 2 U T 4 ligase (Clonetech), 2 U T 4 ligase (Boehringer), and 250 | iM ATP were incubated with the cDNA at 16°C overnight. The reaction was terminated by heating at 70°C for 5 min. The adaptor ligated (Ad)-cDNA was diluted with sdH20 (1/250, v/v) for use in RACE reactions. 2.24.2 RACE reactions. The 5' and 3' RACE reactions were performed using the Clonetech Advantage KlenTaq polymerase mix. To 5 [4,1 of diluted Ad-cDNA were added 5 [ll lOx KlenTaq buffer, 20 [XM dNTPs, 0.2 [XM gene specific primer ( S4C or S5D), 0.2 |lM adaptor primer 1 (API). A touchdown method of amplification was used, with the conditions as follows: 94°C for 1 min; 5 cycles of 94°C for 30 sec, 72°C for 3 min; 5 cycles of 94°C for 30 sec, 70°C for 3 min; 25 cycles of 94°C for 30 sec, 68°C for 3 min; final elongation at 72°C for 10 min. The reaction products were purified on a LMP agarose gel and the appropriate product used for ligation using a T-tailed vector. 2.25 Construction of MIPK expression vectors. 2.25.1 Amplifying full length MIPK cDNA clone using PCR. Specific primers were designed containing the start and stop codons of MIPK as described in Appendix II. Approximately 0.5 [Xl of adaptor-ligated cDNA was used to amplify the full length cDNA. To the reaction mixture were added 2 (ll 25 mM dNTPs, 10 [xl lOx Vent buffer (NEB), 1 (Xl 100 mM MgSO* 5 [Xl 20 uM ATGE primer and STOPS primer, and 1 U Vent polymerase (1 U is the amount of enzyme that will incorporate 10 64 nmoles of dNTP into acid-insoluble material at 75°C in 30 min in l x buffer). The mixture was preheated at 94°C for 3 min followed by 30 cycles of 1 min at 94°C, 1 min at 65°C, and 2 min at 72°C. The product ends were filled using an additional incubation of 10 min at 72°C. The PCR product was purified using the Qiaquick PCR purification protocol. The fragment and the pGEX-4T vector were digested with EcoRI and Sail to prepare for ligation. The PCR product was again purified using the Qiaquick procedure while the vector was gel purified on a 0 .9% low melting point-agarose gel. The vector band was excised from the gel and used directly in the ligation (see protocol under T-tailed vectors). Ligations were transformed into DH50C cells as described above and colonies screened by direct PCR. Positive clones were tested for expression levels and an appropriate clone selected and sequenced. The plasmid map of the resulting vector is shown in Figure 4. 2.25.2 Construction of kinase-dead MIPK using PCR site-directed mutagenesis. This protocol is based on that of Ali and Steinkasserer (1995) and required four primers, as described in Appendix II: mutagenesis primer 1 (KR), primer 2 (STOPS), primer 3 (S5G), and primer 4 (ATGE). First round PCR was carried out using the primer pairs 1 and 2, and pairs 3 and 4. The PCR conditions for a 50 |J,1 reaction were 10 mM KC1, 10 mM (NH4)2S04, 20 mM Tris-HCI, pH 8.8, 3 mM MgS04, 500 uM each dNTPs, 0 . 1 % Triton X-100, 1 flM primers, approximately 100 ng of linearized template, and 1 U of Vent Polymerase (NEB). Thermal cycling parameters were 2 min at 94°C, followed by 25 step cycles of 1 min at 94°C, 1 min at 52°C, 1 min at 72°C, then a final extension of 10 min at 72°C. Yield was assessed by running 2 |ll on an agarose gel. The PCR products were ligated by combining 2 |0.1 of each PCR reaction, 4 |ll of 5x BRL 65 Figure 4. Restriction map of P. ochraceus MIPK in the pGEX-4T3 vector. The full length open-reading frame of MIPK was amplified by PCR using primers having EcoRI and Sail tails. The fragment was then inserted into the EcoRI and Sail sites of pGEX-4T3. The map of kinase-dead MIPK is the same as shown above. 66 ligation buffer, 1 (0,1 10 mM ATP, and 400 U T4 ligase (NEB). Ligation was performed at 14°C overnight. The second round of PCR used the same reaction conditions as the first round with primers 2 and 4 only, and 1 |ll of the ligation product as a template. A 5 (ll aliquot was electrophoresed on an agarose gel to assess yield. The PCR product was then cleaned, digested, and ligated into pGEX-4T3 as described for full length MIPK cloning. 2.26 Production and purification of GST-fusion proteins in bacteria. 2.26.1 Expression and purification. Five ml of 2xYT media (16 g bactotryptone, 10 g bactoyeast extract, 5 g NaCI, p H 7-Sambrook et al, 1989, A.3) containing ampicillin was inoculated with freshly transformed D H 5 a or UT5600 E.coli cells and grown overnight at 37°C, 225 rpm. Approximately 2.5 ml of the overnight cultures was diluted into 50 ml 2xYT media containing ampicillin, and the culture grown at 37°C, 225 rpm until an A600 of 0.800 was reached. The culture was cooled to room temperature before induction of protein synthesis by the addition of 100 |J,M IPTG. Protein expression was continued at room temperature, 225 rpm for 4 h or overnight. Cells were pelleted and resuspended in 1-5 ml of lysis buffer (1 ml STE-500 (500 mM NaCI in TE buffer), 1 mg lysozyme, 1 (0,1 SBTI, 1 (xl PMSF). The suspension was incubated on ice 15 min, sonicated 3x15 sec, and centrifuged in an Eppendorf tube at 15,000 rpm for 5 min. One hundred (ll GSH-agarose slurry were added to the supernatant and allowed to incubate for 1-2 h with rotation at 4°C. The beads were washed with STE-500 buffer and PBS before being resuspended in an equal volume of PBS. These beads were then either stored at -70°C, or used for thrombin cleavage. Yield was assessed by SDS-PAGE and Coomassie staining. 67 2.26.2 Thrombin cleavage of fusion proteins. One U of thrombin was added to 100 \i\ of GST-fusion protein agarose slurry in PBS and allowed to cleave at room temperature for 1 h. The reaction was stopped with 10 ]LLl benzamidine-Sepharose slurry (in PBS) incubated for 1 h at 4°C with rotation. The beads were pelleted and the supernatant aliquoted to be stored at -70°C until required. Yield was assessed by SDS-PAGE and Coomassie staining. 2.27 Measurement of MIPK upstream kinase activity. Approximately 5 mg of protein from the control and 30 sec, 1 M NaCl time course samples were diluted to 2.1 ml and fractionated on a 1 ml ResourceQ column, as described. The eluates were collected in 500 ill fractions, and assayed for phosphotransferase activity towards GST-MIPK bound to GSH-agarose beads. To 1.25 (ll of GST-MIPK beads were added 3.75 ul buffer A, 5 ul 100 mM MgCl 2 /MnC l 2 , 100 (ll ResourceQ fraction, and 10 |ll of 250 \lM [y-3 2P]ATP (-2000 cpm/pmole). The reaction was incubated at 30°C for 60 min and placed on ice to stop the reaction. The beads were pelleted by centrifugation, and the 100 [l\ of supernatant removed to decrease the background. The beads were diluted in 50 (ll 4x sample loading buffer, boiled, and electrophoresed on an 1 1 % SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose membrane and Western-blotted with 4G10, antiphosphotyrosine antibody to assess increases in phosphorylation levels. The membranes were also analysed by autoradiography. On similar gels, 125 |xl of each ResourceQ fraction was electrophoresed, and Western blotted with an Ste7-VIII antibody, to detect M E K isoforms. In a separate experiment, 25 (ll GST-MIPK agarose beads was incubated with 25 (ll PBS, 200 fll buffer A, and 200 jxl of 5 mg/ml crude extract from Immature, 60 min 1-68 methyladenine treated, 120 min 1-methyladenine treated, and 12 h post-fertilization time points. The mixture was incubated with rotation for 2 h at 4°C. The beads were washed 2x with 1 %NETF ( 1 % NP-40 in NETF buffer), and once with KII buffer. The beads were subjected to a phosphotransferase assay with the addition of 25 JLXl KII buffer, 5 (ll 100 mM MgCl 2/MnCl 2, and 10 \i\ 250 U.M [y-32P]ATP (-2000 cpm/pmole). The reaction incubated for 1 h at 30°C, and was stopped with the addition of 50 |ll 4x sample loading buffer. The mixtures were analysed by Western blotting with anti-Ste7-VIII antibody as well as autoradiography. 2.28 Microinjection of P. ochraceus oocytes. P. ochraceus were kept in the lab in a 100 gallon aquarium, held at a constant temperature of 10°C. The specimens were fed mussels collected from local beaches at low tide. For each day of microinjection, fresh oocytes were removed from the gonad tissue by inserting a large guage needle into an arm of the seastar and gently aspirating a small volume, potentially containing a few thousand oocytes. The oocytes were placed in 10 ml of filtered, natural sea water (NSW) and kept on ice. Oocytes were sorted visually with a dissecting microscope (WILD-M3B) at 16 x power. At this magnification, oocytes (150 (lm in diameter) were inspected and healthy immature oocytes are picked up individually by hand using a pulled capillary pipet, and transferred to the microinjection slide. Usually about 20 oocytes were placed in 100 (ll of NSW on each slide. The NSW was localized within a ring of rubber cement, 1 cm in diameter, made with a PAP pen (The Binding Site, Institute of Research and Development, Birmingham, UK). The glass slides are kept at a temperature of 12-13°C on a 3/8 " thick aluminum plate on a thermoelectric cold plate (Thermoelectrics Unlimited 69 Inc.). Evaporation was minimized by using a blue plastic cap from a 15 ml centrifuge tube (Falcon) to cover the seawater ring. Microinjection of the oocytes was performed using a Leitz Labovert FS inverted microscope with differential interference contrast optics. The microscope was flanked by right and left Leitz micromanipulators which hold the injection needles and holding pipets respectively. During the microinjection, the slide was kept at 1 0 - 1 2 ° C using a refrigerated microscope stage based on a Peltier element, receiving variable direct current 12 V, 0-2 Amp. The microinjection needles were made on a needle puller set at 3 3 5 ° C , Gas 75 velocity 10, pull 7, from 1.0 mm glass capillaries with filament (World Precision Instruments, Inc.). The holding pipets were made from the same pulled needles which have been broken, heated, and manipulated with a microforge to produce a blunt tip with an inside diameter of 10-20 Um. The microinjection needles were backfilled using pulled capillary pipets into which approximately 1-2 |ll 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 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 (Figure 5). The volume of the oocyte was approximately 2500 picolitres, and therefore injections of 50 -200 picolitres of samples were performed. The volume was judged by eye. The instrument settings required to produce an injection of a certain volume varied with each needle, and during the course of injections, as the needle frequently became partially plugged with cell debris. Microinjected oocytes were monitored as described in the data analysis. 7 0 Figure 5. Microiniection of P. ochraceus oocytes. The holding pipet is shown in the top left corner of the panel, with one oocyte attached. The needle is entering the panel from the bottom center and is injecting the oocyte held. The surrounding oocytes are all immature, just after injection. For MIPK.-K.62R and p38 inhibitor studies, oocytes were injected and monitored for -24 h for spontaneous maturation events. In subsequent studies, oocytes were injected and allowed to mature in the presence of 40 |J,M 1-methyladenine. Meanwhile, 2.5 |ll of sperm was diluted in 1 ml of natural seawater in preparation for fertilization. After 4 h, oocytes were incubated with 2.5 |ll of diluted sperm per 100 (ll seawater. Embryos were observed over 24 h post-fertilization. For oligonucleotide injections, samples were diluted to 100 ftM in sterile water, and filtered through 0.45 |Im filters. The oocytes could then be injected with the various oligonucleotide samples. Oocytes were allowed to incubate 22-24 h to allow protein turnover to occur. Spontaneous maturations were quantitated prior to maturation of the oocytes with 40 | lM 1-methyladenine. The oocytes were matured for 4 h and fertilized as described above. Embryonic development was followed for 24 h post-fertilization. 72 CHAPTER 3 RESULTS 3.1 Search for novel tyrosine phosphorylated proteins in P. ochraceus. 3.1.1 Introduction. Tyrosine phosphorylation is a commonly exploited mode of protein regulation within the cell, especially with respect to protein kinase regulation. Tyrosine phosphorylation comprises less than one percent of total phosphorylation of cellular proteins yet is thought to be a key component in early levels of signal transduction from the cell membrane to the nucleus. It is therefore important to identify proteins undergoing changes in tyrosine phosphorylation during a signalling cascade in order to understand the events required for signal progression to occur. To this end, the P. ochraceus oocyte system has been used to search for cytosolic proteins that show changes in tyrosine phosphorylation upon stimulation with the natural hormone 1-methyladenine. Results 3 .1 .2 Analysis of P. ochraceus oocyte extracts for tyrosine phosphorylated proteins. To identify proteins undergoing changes in tyrosine phosphorylation with oocyte maturation, cytosolic extracts from immature and 1-methyladenine matured oocytes (Figure 4) were fractionated on a ResourceQ anion exchange column. Fractions were collected, further separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and subjected to Western blotting with an anti-phosphotyrosine antibody, 4 G l O (Figure 6, Panels A,E). Several major bands of tyrosine phosphorylation were identified using 7 3 kDa IMMATURE MATURE 46H 46" 26-46H 26-97-67H 4 6 H 26-1 A mm m» E -> B F C G » -> D -> — H •o — 4G10 P-TyrAb Erkl-CT MAPK Ab PSTAIRE Cdkl/2 Ab Cdk5-CT 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction number 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction number Figure 6. Western blot analyses of Resource O fractionated P. ochraceus oocyte extracts. Cytosolic extracts from either immature (A-D) or mature (E-H) oocytes were fractionated over a ResourceQ ion exchange column, and proteins further separated by SDS-PAGE. Western blots were probed with antibodies as indicated to the right of the blots in bold type. p44mPk is identified by the filled arrow (•*•), p 3 4 c c ^ 1 by the sharp arrow (•*-), and p40mipk D v t r i e 0 p e n arrow (-> ). Resource Q fraction numbers are shown below the blots, migration of Mr standards shown to the left. 74 this procedure, some of which underwent significant changes in response to 1-methyladenine stimulation. To characterize these 4G10 immunoreactive proteins, antibodies were used to assess elution profiles of various known tyrosine phosphorylated proteins. The most prominant protein showing an increase in phosphotyrosine signal was 44 kDa in size. Western blotting with Erkl-CT (Figure 6, Panels B,F) indicated that this protein belonged to the MAP kinase family. Further characterization allowed the identification of this protein as the previously characterized p 4 4 m p k (Sanghera etal, 1990). This enzyme is the major MAP kinase that is activated during meiosis and is known to be activated by tyrosine phosphorylation (Sanghera et al, 1991). p44mpk is most closely related to the mammalian MAP kinases, Erkl/2, containing the characteristic Thr-Glu-Tyr (TEY) dual phosphorylation site in the activation loop, between subdomains VII and VIII in the kinase domain (unpublished data). A major band shown to decrease in tyrosine phosphorylation was identified as a member of the Cdk family through crossreactivity with a polyclonal PSTAIRE antibody (Figure 6, Panels C, G). This antibody is known to immunoreact with Cdkl and Cdk2 in the sea star oocyte system. Western blotting with a specific Cdkl antibody (data not shown) confirmed that the tyrosine phosphorylation occurred on Cdkl in the immature oocyte cytosol, and that it was activated in response to 1-methyladenine. Cdkl was first identified as a histone Hi kinase in the oocyte system which underwent activation with oocyte maturation (Pelech et al, 1987, Meijer et al, 1987), which led to the sea star oocyte being the source of the first purified Cdkl (Labbe etal, 1988/ Arion etal, 1988). Although several bands did not crossreact with any of the antibodies tested, the major protein undergoing a decrease in tyrosine phosphorylation with oocyte maturation had an apparent molecular mass of 40 kDa, and was found to react with a polyclonal 75 Cdk5-CT antibody (Figure 6, Panels D,H). Cyclin-dependent kinase 5 (C d k 5 , also called PSSALRE and nclk for neuronal cdc2-like kinase) expression is highest in adult mouse brain. In humans, Cdk5 is also significantly expressed in heart, placenta, kidney, lung, liver, pancreas, placenta and skeletal muscle. Cdk5 from brain is associated with a novel member of the cyclin family. This cyclin appears to be expressed exclusively in brain and provides for marked activation of the Cdk5 catalytic subunit. With a molecular mass of 40 kDa, this protein did not exhibit the expected 31 kDa molecular mass of a Cdk5-like protein (Meyerson et al, 1992) and therefore further characterization was required to determine the identity of this protein. Cdk5-CT was found to recognize C d k 5 when tested in other systems (data not shown). Close analysis of the immunizing peptide used to generate the antibody indicated the potential for crossreactivity within the Cdk and MAP kinase families. The molecular weight indicated a closer relationship to the MAP kinase family and the protein was tentatively named maturation-inhibited protein kinase (MIPK, p 4 0 m i p k ) . 3.1.3 Initial characterization of p40mipk. To confirm that p40m,pk was the protein identified through 4 G 1 0 Western blotting, immunoprecipitation experiments were performed. Under denaturing conditions ( 1 % SDS) p40m,pk could be immunoprecipitated with the Cdk5-CT antibody, coincident with a depletion of phosphotyrosine signal in the ResourceQ fraction (data not shown). p40m'pk was also subjected to Western blotting with a panel of antibodies. MIPK was first immunoprecipitated with Cdk5-CT, followed by Western blotting with antibodies from the MAP kinase and cyclin-dependent kinase superfamilies of protein kinases (Figure 7 and data not shown). Only the C d k 5 - C T antibody crossreacted with the 40 kDa protein. Two other Cdk5 monoclonal and polyclonal antibodies were tested, and both failed to 76 9 10 11 12 / / / / / / </ </// p44mPk p40mipk Figure 7. Western blot analysis of Cdk5-CT immunoprecipitations. Mature oocyte cytosolic extracts were subjected to immunoprecipitation by Cdk5-CT. Immunoprecipitated proteins were Western blotted with various polyclonal and monoclonal antibodies. Lane 1, Cdk5-CT; lane 2, Cdc2-NT; lane 3, PSTAIRE,- lane 4, Cdc2-CT; lane 5, Cdc2-IX; lane 6, CdCK-2B; lane 7, Cdk6; lane 8, Erkl-CT ; lane 9, Cdk2; lane 10, Hogl-NT,- lane 11, Cdk5-CT-Santa Cruz,- lane 12, Cdk5-monoclonal-Santa Cruz. The open arrow (^—) indicates p40m'Pk, filled arrow (-^—) indicates p44mpk Migration of Mr standards (kDa) is shown on the left. 77 crossreact with MIPK. p40 m , p k also failed to react with Cdc2-NT, another antibody known to immunoreact with Cdk5 (Meyerson etal, 1992). This, along with the molecular weight, strongly indicated that p40 m i p k was not the seastar cognate of Cdk5. Interestingly, a 44 kDa protein was identified in the immunoprecipitation which Western blotted with Erkl-CT, but did not immunoblot with Cdk5-CT (Figure 7, lane 8). The crossreactivity with Erkl-CT indicated that this protein was p44 m p k . This was Confirmed through Western blotting with a polyclonal anti-p44mpk antibody (data not shown). To further investigate the relationship between p 4 4 m p k and p40 m i p k , Cdk5-CT and Erkl-CT were compared in reference to their ability to immunoprecipitate p 4 4 m p k and p 4 0 m i p k (Figure 8). Results showed that p44 m p k could be immunocomplexed with both antibodies, while p40 m i p k was only present in the Cdk5-CT immunoprecipitations. It was also apparent that the amount of p 4 4 m p k present in the Cdk5-CT immunoprecipitations was significantly less than that immunocomplexed with Erkl-CT. The lack of MIPK in the Erkl-CT immunoprecipitations indicated that the presence of p 4 4 m p k in the Cdk5-CT immunoprecipitations was due to crossreactivity of the antibody with p44 m p k . Another explanation was that a small percentage of cellular p44 m p k was involved in a complex with p40 m , p k . This complex would have to be stable to denaturation with 1% SDS, and binding must mask the epitope in p 4 4 m p k recognized by the Erkl-CT antibody. Although this seemed unlikely, it could not be ruled out as a possibility. 3.1.4 Time course of dephosphorvlation of p40mipk during oocyte maturation. The crossreactivity of Cdk5-CT with p44 m p k again supported p 4 0 m i p k as a member of the MAP kinase superfamily. This family is known to be activated via tyrosine and threonine phosphorylation in a position equivalent to the Thr-Glu-Tyr site in the activation loop of Erkl. It is therefore possible to predict MAPK activity based on 78 1 2 3 4 IP Cdk5-CT IP Erkl-CT IB Cdk5-CT IB Erkl-CT p44MP K p4Qmipk + + Figure 8. Western blot comparison of C d k 5 - C T and Erkl-CT immunoprecipitated proteins. Mature oocyte extract was denatured with 1 % SDS and subjected to immunoprecipitation with either C d k 5 - C T (lanes 1,3) or Erkl-CT (lanes 2,4) as indicated below the blot by IP "+". Western blots were probed with both antibodies as shown below the blots by IB "+". The solid arrow —) indicates p 4 4 m P k , the open arrow (^—) indicates p 4 0 m , P k . Migration of Mr standards (kDa) are shown on the left. 79 phosphotyrosine levels. The time course of dephosphorylation and potential inhibition of MIPK was assessed during 1-methyladenine induced maturation. At discrete timepoints following 1-methyladenine addition, aliquots of oocytes were removed and processed to yield cytosolic extracts. Immunoprecipitation with Cdk5-CT was used to isolate p40m,pk. Eighty percent of the immunoprecipitation was analyzed for phosphotyrosine levels (Figure 9, Panel A), while the remainder was used to assess p40 m , p k protein levels through Western blotting with Cdk5-CT (data not shown). Densitometric analysis was used to quantitate the phosphotyrosine signals, which could then be plotted as shown in Figure 9 (Panel B). Although there was some variability in the first 15 minutes post 1-methyladenine stimulation, a consistent decrease in phosphotyrosine levels was evident through the maturation process. A minimum signal was observed at the 50 minute time point, coincident with the appearance of tyrosine phosphorylation of p44mpk. This activation of p44mpk continued through the maturation process to full activity at the end of maturation, 90-120 minutes post-1-methyladenine addition. The inactivation of p40mipk, as assessed by tyrosine phosphorylation state, was complete in a period prior to the activation of known maturation activated protein kinases including p44mpk, p34cdkl, and ribosomal S6 kinase (Pelech et al, 1988). This suggests that MIPK dephosphorylation may be required for the activation of maturation-promoting enzymes to occur. This may implicate MIPK as a type of cytostatic factor, potentially involved in holding the oocyte arrested in prophase. This hypothesis was tested once the gene encoding MIPK had been cloned (Section 3.5.7) In summary, p40mipk has been identified as a potentially novel protein kinase which is highly tyrosine phosphorylated in the immature P. ochraceus oocyte. Although initially discovered through its immunoreactivity with an antibody modeled after the C-terminus of human Cdk5, p40m,pk was not recognized by any other Cdk5-specific antibodies. The 80 kDa 46 H 3 H p44mP k p40mipk 5 10 15 20 25 30 40 50 60 90 120 Time (min) 0 5 10 15 20 25 30 40 50 60 90 120 Time (min) Figure 9. Dephosphorylation of mipk in homogenates of a P. ochraceus oocyte maturation time course. Panel A. P. ochraceus oocyte timecourse homogenates were immunoprecipitated with Cdk5-CT and Western blotted with either Cdk5-CT (not shown) or antiphosphotyrosine (4G10). Detection of p44mPk is indicated by the solid arrow ( — ) , p40m'pk is indicated by the open arrow (^—). The numbers below the blot show the time course of maturation in minutes from 0 (immature oocytes) to 2 h post-1-methyladenine addition. On average GVBD had occurred in 80% of the oocytes by 90 min. Migration of Mr standards (kDa) are shown on the left. Panel B. Alkaline phosphatase signals from the Western blots were quantitated by densitometric analysis and phosphotyrosine levels corrected for protein levels. Values were standardized to 1 Unit = 0 min levels for MIPK < • ),- 1 Unit = 120 min for Mpk ( • ). Data shows the mean±S.E.M. of 3 independent time courses. 81 molecular weight supported p40mipk as potentially a member of the MAP kinase superfamily of protein kinases. The 1-methyladenine-induced maturation of the seastar oocyte resulted in dephosphorylation and potentially inactivation of this enzyme. Based on this information the kinase has been named maturation-inhibited protein kinase, MIPK. 82 3.2 Purification and characterization of p40"" p k from immature P. ochraceus oocytes. 3.2.1 Introduction. p 4 0 m i p k was identified as a protein that cross reacted with an antibody developed for the protein kinase, C d k 5 . This protein was a major tyrosine phosphorylated protein in the immature oocyte cytosol and became tyrosine dephosphorylated upon oocyte maturation. This was all that could be determined with the tools currently available. To discover more about this potentially new kinase, it was necessary to purify the protein from the seastar system. On the assumption that MIPK was active in its tyrosine phosphorylated state, attempts were made to purify MIPK from the immature oocyte. Results 3.2.2 Purification of MIPK from immature oocytes. Immature ooctyes were homogenized as described to obtain a cytosolic extract. The extract from 100 ml of packed cells (~2 x 10 7 oocytes) was used in the purification of MIPK for sequencing. The extract was fractionated through a series of column chromatography steps including those based on ion-exchange and hydrophobic columns. The elution profile of MIPK was monitored by Western blotting with Cdk5-CT (Figure 10, Panels F-J). 4G10, antiphosphotyrosine antibody was used to confirm that in following MIPK elution, the major tyrosine phosphorylated protein in the immature oocyte cytosol was also being tracked (Figure 10, Panels A-E). Cytosolic extract was adsorbed onto hydroxylapatite (HTP) resin and eluted with either a linear 0-0.14 M phosphate gradient (Figure 10, Panels A,F,K), or 40 mM phosphate in a step elution. The major band of tyrosine phosphorylation was found to 83 bo -d c « | o j a c. o O* ra <u V c H (3 a c fc. <U - d tU IU CL O J * " o 2 E .§ E fc. 3 <u * bo O re ~ as o 1/5 • oo 3 O C _ o ?r in O ra 4 £^ fr. re H 1/1 -s> o a-5 -5 « u re -5 o .2J f ° c „ . S B 3 r° <u E x < u C _ - o in ra p e t ; IU ra c IU E is 1/1 o J2 -c i; 3 a cr ro P w re* a ~ IU o -= T» e fc/ +2 ™ o wl <u w _ ra _ C * d ,13 ra r-tu u c ^ <u c O +2 - C v 4J ^ E o, fc.1 CL. fc! 3' O. - d c ra a O J O _ — fc c X o S « ra ui -rt iu 4r c IU .s c ^ E a ° 1% u o •5 J3 Q ° h U T iu C Q , ra ra ° £ £ p I act ra ra > , * d <= t i n . y ra iu — " > ' u ^ 1 1 '5 o ra •<-> 6 0 - d u fc. ra X T ID ^Ji a u u c " c ^ J2 .5 O ra W J_) w IU bo"2 ra ra _ c U IU ra £ bO, « in in & c h [O x p -j= (J H -fc-J O i n -E U fc. "S <u .JL. ^ * J +3 C ra . . U ra ra ra c E _3 o c c c E SS 3 O p c 5. " 'S 'S " +j ro ro c/5 0 ^ '5 a a uIU (U ^ 84 85 correspond to the 34 kDa protein Cdkl. The 40 kDa tyrosine phosphorylated protein coeluted with the MIPK protein. The most concentrated MIPK fractions were pooled and, after dilution, applied to Q-Sepharose resin. The total protein eluted from the Q-Sepharose column over a span of 30 fractions. By pooling the MIPK peak fractions, the majority of the contaminating proteins could be excluded (Figure 10, Panels B,G,L). The resin was also able to resolve MIPK from all other tyrosine phosphorylated proteins in the mixture. The pooled fractions were diluted and loaded onto a heparin-Sepharose column (Figure 10, Panels C,H,M). Although MIPK did not bind to this resin, contaminating proteins were retained by the column. The wash through material was directly loaded onto a polylysine agarose column (Figure 10, Panels D,I,N). MIPK eluted in a very sharp peak from this column, allowing for the exclusion of a large portion of contaminating proteins. The elution of MIPK also exactly matched the elution profile of the 40 kDa tyrosine phosphorylated protein. The peak fractions were pooled and brought up to 1 M NaCl for loading onto a phenyl-Sepharose column. The column was developed with a linear 1 M NaCl/0% Brij 35 to 0.67 M NaCl/0.5% Brij 35 gradient. Using this protocol, the total protein in the sample was reduced from 1 g of starting material, down to 200 Jig of protein. Unfortunately, after this step, MIPK would not bind to any subsequent resins and could therefore not be further purified. This experiment did however show the efficiency of the purification columns chosen, and confirmed that MIPK coeluted with the tyrosine phosphorylated 40 kDa protein over five consecutive column steps. A second, similar purification scheme was used to obtain a concentrated sample of MIPK to send for sequencing. The initial steps of hydroxylapatite and Q-Sepharose chromatography were consistent with the above procedure. The peak fractions from the Q-Sepharose column were applied to the phenyl-Sepharose, followed directly by the 86 polylysine-agarose column. At this point it was possible to concentrate MIPK on a ResourceQ column, collecting 250 (ll fractions. The fractions were subjected to SDS-PAGE and silver staining to resolve the protein bands present in the sample (Figure 11, Panel A). Although clearly not a pure preparation of MIPK, the protein bands appeared to be quite well resolved. To definitively identify the MIPK protein band, a duplicate gel was Western blotted with Cdk5 - C T . For ease of alignment of the two gels, the Western blot was Ponceau S-stained to give a banding pattern similar to that on the silver stain (Figure 11, Panel B). From this data it was clear that MIPK was being resolved from other proteins present in the sample. The next task was to prepare a sample to send for protein sequencing. Using the purification protocol outlined above, MIPK was concentrated into two major fractions from the ResourceQ column. These fractions were carefully loaded into one lane of an SDS-PAGE gel. The proteins were transferred to nitrocellulose membrane and visualized by amido black staining. To ensure identification of the correct protein band, the edge of the lane was cut off of the membrane and subjected to Western blotting with Cdk5-CT (data not shown). The lane was then reassembled and the MIPK band on the amido black stain identified. This band was excised from the membrane and sent for micropeptide sequencing. 3.2.3 Micropeptide sequencing of P. ochraceus MIPK. The discrete protein band representing MIPK was subjected to trypsin digestion followed by peptide separation by reverse phase-high performance liquid chromatography (RP-HPLC). Approximately 9 0 % of the sample was collected following RP-HPLC, and approximately 1 0 % of the sample was diverted into the mass spectrometer. The masses of the peptides were determined and, for a few peptides, 87 22 23 24 25 26 27 28 29 30 Fraction number 31 22 23 24 25 26 27 28 29 30 Fraction number Figure 11. Identification of purified MIPK protein band. After sequential column chromatography steps, MIPK was concentrated on a MonoQ ion-exchange column. Peak fractions from the column were further separated by SDS-PAGE and silver-stained (Panel A). To identify the specific protein band, an identical gel was Western blotted with anti-Cdk5-CT. Following the Western blot procedure, the membrane was Ponceau S-stained (Panel B) and the profile compared with the silver-stained gel. The arrow indicates the common band in both panels representing MIPK migration. Molecular weight markers are indicated on the left, and fraction numbers are below the panels. 88 collision-induced fragmentation data was obtained, which aided in the interpretation of the Edman sequencing data. Sequence information was obtained for a total of six unique peptides. Data base searches were performed to aid in the identification of MIPK, with the results as summarized in Table 1. Results indicated that MIPK was related to the MAP kinase superfamily of protein kinases, with closest homology to the p38 family of stress activated kinases. In summary, MIPK was shown to coelute over 5 chromatography steps, with a major tyrosine phosphorylated protein in the immature oocyte cytosol. The protein was partially purified to a state where it could be resolved by SDS-PAGE from all contaminating proteins in the preparation. This partially purified MIPK was identified by a combination of staining and Western blotting techniques, and the protein band sent for micropeptide sequencing. This resulted in the peptide sequencing of six discreet peptides indicating homology between MIPK and the p38 family of protein kinases with at least four of these peptides. These peptide sequences could be used for the development of strategies for the cloning of seastar MIPK. 89 Table 1: Micropeptide Sequencing Results Peptide ID. Peptide Sequence Peptide Homology Percent Match Si TLFPGYIEIGEL Stage V sporulation protein 59% S2 LSDEHVQFLIYQILR p38 - mouse, Xenopus 92%, 92% S3 MLLLDVDK p38 - Xenopus, mouse 81.6%, 76.3% S4 TTWEVPVQYQK p38 - mouse, Xenopus 67%, 65% S5 ITAEEALSHPYVAK Spkl - S. pombe Erk3 - rat, Erkl - human, rat 8 7 % 81%, 73%, 73% S6 ELTFQLIQAV RNA replication protein 70% 9 0 3.3 Cloning and sequencing of P. ochraceus M I P K 3.3.1 Introduction. The most powerful tool for the characterization of MIPK in the P. ochraceus oocyte system would be the cDNA clone and predicted amino acid sequence of the protein. Peptide sequencing results indicated that MIPK was a member of the MAP kinase superfamily of protein kinases. The closest homology appeared to be with the p38 family of stress activated protein kinases. From this information it was possible to design a strategy for the cloning of MIPK utilizing the polymerase chain reaction (PCR). PCR is a powerful method of rapidly amplifying a specific DNA sequence from a sample of total D N A or cDNA (reviewed in White et al, 1989). From the information obtained by protein sequencing, degenerate primers could be designed to allow direct amplification of MIPK from seastar cDNA. Results 3.3.2 Degenerate PCR of MIPK from P. ochraceus. The first step in designing a PCR strategy for MIPK cloning was the alignment of the sequenced peptides with the intact p38 sequence (Figure 12, Panel A). This allowed the creation of PCR primers in the correct orientation for amplification. Four of the peptides were the best candidates for primer design based on the confidence of peptide sequencing and peptide alignment with p38: S i , S2, S4 and S5. Degenerate primer sequences were designed as described in Appendix II. The most promising PCR results came from the primer pairs of S4A-S2C, and S2B-S5A (Figure 12, Panel B). Using very low stringency conditions to compensate for the degeneracy of the primers, PCR 91 ^ 00 O. ro S£ 00 Q_ CO ^ 00 a. ro S£ 00 D_ CO w a. * oo o_ ro H i Du ^ 00 Q_ ro oo a. ro ID C v 3 c o * 1 a 0 u £> a « ^ v cu c =3 iu tu +-» Pi w U N CL, '5i «j "3 fo -l-> L- rj ti +- c it ro 3 En UU OJ s-u c ro Cu VI c ^ u c (U 3 O oo u -d on a c ro E , 3 Ol X co -o IU in ro U CL. <U o VO m U , CN OJ £ A ro i CL, < on E u ro c bo i- -ro w U C V 3 er v i/i C 'C o L-a a •»-> r> - rs C -O r s < 2 O s-JD C O '-3 ro b-ro a <u u C L c i-o u > u <U J3 C Q O +-» C -d v c o u V i-V •S u H <2 c £ ro o t ro U -d u c be § .Sf S 3 -3 u C (DC ._ .„ ^ > -a ^  ro <u "3 JS bo C -a u u c 3 u 92 products of the expected size could be generated. These products were preferentially amplified over background, as was clearly established by loading lesser amounts of the reaction on an agarose gel (data not shown). It was therefore decided that these products could be cloned into pBluescript vector without the need of a purification step. The PCR products were cloned by exploiting the properties of the Taq polymerase used in the PCR reaction. Taq polymerase has been observed to add a single non-template-directed deoxyadenosine (A) residue to the 3' end of duplex PCR products (Clarke, 1988). Use of terminal transferase to add a single deoxythymidine (T) residue to a blunt ended vector created a sticky end which was specific for the Taq polymerase generated PCR product. This allowed for direct and efficient cloning of the PCR product without the need for enzymatic modification (Holton etal, 1991). Duplicate PCR reactions were performed for the two working primer pairs, and six clones from each PCR reaction were tested for correct sized inserts by direct PCR. In total, four of twelve clones showed appropriate insert size from the S4A-S2C reactions, while five of twelve clones indicated appropriate insert size from the S2B-S5A reactions. These clones were further analyzed by restriction mapping. Three of the four clones from the S4A-S2C reactions gave identical restriction maps and two were sent for sequencing. Three of the five clones from the S2B-S5A reactions gave appropriate sized inserts upon restriction digest, although they did not appear to be identical in size. As a result, all three of the clones were sent for sequencing. DNA sequence information confirmed that the PCR products amplified would encode a protein that was related to the p38 family of protein kinases. The S2B-S5A fragment contained the Si peptide sequence, indicating that the fragment matched the information obtained from the MIPK protein and likely was a part of the MIPK gene. The S4A-S2C fragment did not contain any peptides sequenced from the purified kinase and 93 therefore its identity as part of the MIPK gene could not be confirmed at this stage. Due to the placement of the working primers within the cDNA, the two fragments had no overlapping regions. It was necessary to clone the 5' and 3' regions of both fragments to ensure that one gene was responsible for giving rise to the two sets of PCR products. 3.3.3 Cloning and sequencing of MIPK from immature seastar oocytes. A series of sequence specific primers were designed based on the two DNA fragments generated by degenerate PCR. As it was still uncertain that the two fragments generated by the degenerate PCR came from the same gene, both fragments were used to design primers for both the 3' and 5' RACE reactions. These primers were tested for their ability to amplify internal sequences and all were found to work under these conditions (see Appendix III, Figure 32, Panel B). The primers chosen to amplify MIPK would give rise to the largest products in the RACE reactions, with the largest areas of overlap. An adaptor-ligated cDNA library was created for use in the RACE reactions. Using an adaptor specific primer in conjunction with a gene specific primer, high stringency conditions were used to amplify the MIPK gene (Figure 13, Panel A). Although a distinct band could not be identified, a wide band could be resolved that contained appropriate sized product in both RACE reactions. These wide bands were excised from a low-melting point agarose gel and ligated into pBluescript vector. The resulting clones were characterized by internal priming and amplification to show that both RACE reactions gave MIPK specific sequences. Both reactions resulted in overlapping sequences, indicating that both primers had amplified products of the same gene. The sequences obtained were used to design primers for the start and stop codon regions of the gene. The full length clone of MIPK was amplified, resulting in one major 94 Figure 13. Cloning of full length MIPK from P. ochraceus. Panel A: PCR amplification of the 3' (lane 2) and 5' (lane 3) ends of the MIPK cDNA using RACE. The region indicated by the bracket was excised from the gel, purified, and cloned into Bluescript vector. Sequence data was then used to design primers for the PCR amplification of the full length cDNA (panel B, lane 2). The cDNA was cloned into the pGEX-4T vector for expression of the GST-fusion protein, GST-MIPK. Migration of the 1 kb ladder is shown in lane 1 of both panels. 95 product (Figure 13, Panel B). A lower band was also identified and further characterized (see Appendix III). The open reading frame of MIPK was 1089 bp, which predicted a 363 amino acid protein (Figure 14). The protein sequence contained all of the conserved domains (I-XI) characteristic of a serine/threonine protein kinase. The Thr-189 andTyr-191 residues in the activation loop between subdomains VII and VIII are in an equivalent position to the TEY, TPY or T G Y sequences in known MAP kinases and stress-activated kinases. The sequence shares a TGY sequence with the p38 kinase family. As in p38, kinase subdomain VII is separated by only 6 amino acids from the activation region in subdomain VIII, whereas the gap is 8 residues in the Jnk family and over 12 amino acids in many other known MAP kinases, such as Erkl and Erk2. The predicted amino acid sequence and overall structure identified this protein as a member of the p38 family. All of the peptides sequenced in the purified MIPK protein (Table 1) were identified in the predicted amino acid sequence. All of the peptides matched perfectly, with the exception of peptide Si. The first 5 residues matched amino acids 230-234 of MIPK, and the differences are likely due to inaccuracies in the peptide sequencing procedure. This confirmed that the gene cloned encoded the protein purified from the immature oocyte. A search of various nucleotide and protein databases revealed that the deduced amino acid sequence of MIPK was a novel sequence, most closely related to p38 from Xenopus. Protein sequence alignments of MIPK with p38 from various species indicated a high homology in the kinase domains (Figure 15). There were significant differences in MIPK which were not apparent when comparing the p38 homologues. The N-terminus of MIPK contains an extra 8 amino acids which are not found in any of the p38 kinases. There is a stretch of 10 amino acids between subdomains IV and V which is 80% different than p38. The largest region of sequence variation between MIPK and p38 96 Figure 14. Nucleotide and amino acid sequence of coding region of P. ochraceus MIPK. The amino acid sequence of the coding region is shown above the nucleotides, numbered in bold type. The nucleotides sequenced are numbered starting at "1" for the first residue of the start codon. Oligonucleotides used to amplify Mipk from P. ochraceus cDNA were based on regions underlined, arrows indicate sense (>) and anti-sense (<) primers. Roman numerals indicate protein kinase subdomains. Residues that are identical between all protein kinases are shown in bold type. 97 144 216 G C C G G A C A C A T C C G T A C A T T C A G C C T G G G A T T A T A A G A A A A C T N A T T T A G T C A A A G T A A A T T A G A A T T A G T C A T T C G A T T T T G A T T T G G T A G A G C A C T A A A A A A T A C T C T T A G T C T T A G A G T T A G T G T T A C C A T A C C A A T T A A C T T A A T T A A A T C M N N P V T G S G E T L S D D G Y H R Y E L N K 24 A T G A A C A A C C C A G T A A C A G G A T C A G G A G A A A C G T T A T C T G A T G A C G G G T A T C A T C G A T A T G A A C T G A A T A A A 72 I T T W E V P V Q Y Q K L S A V G A G A Y G S V C 48 A C T A C A T G G G A G G T G C C G G T T C A G T A C C A A A A A C T C T C C G C A G T G G G A G C T G G T G C A T A T G G A T C C G T G T G C n 4c> S S L N T K T G I K I A I K K L S R P F Q S A I 72 T C A T C C T T A A A C A C A A A A A C T G G C A T A A A G A T T G C T A T C A A G A A G C T T T C T C G A C C A T T T C A G T C T G C G A T T m iv H A K R T Y R E L R L L Q H M D H E N I I S L L 96 C A T G C C A A G A G A A C G T A C C G A G A G C T T C G A C T T C T A C A G C A T A T G G A T C A T G A A A A C A T C A T C A G T C T A C T A 288 V D V F C R G D T L S S F R D V Y M V T H L M G A 120 G A T G T G T T T T G T A G A G G A G A T A C C T T A T C A A G C T T T C G G G A C G T A T A C A T G G T G A C A C A T T T G A T G G G T G C A 360 D L N S I T K T Q K L S D E H V Q F L V Y Q I L 144 G A T C T G A A T A G T A T T A C A A A A A C A C A G A A A C T C T C T G A T G A A C A T G T G C A G T T C C T T G T G T A T C A A A T A C T T 432 VI R G L K Y I H S V G V I H R D L K P S N L A V N 168 C G T G G G C T C A A G T A C A T T C A T T C A G T T G G T G T A A T C C A T C G T G A T C T G A A G C C C A G T A A C T T G G C T G T G A A T 5 04 vn E D C E L R I L D F G L A R Q A D D E M T G Y V 192 G A A G A C T G C G A A T T G A G G A T A C T A G A T T T T G G T C T T G C T C G T C A A G C T G A T G A T G A G A T G A C A G G T T A C G T A 57 6 vm A T R W Y R A P E I M L N W M H Y T N T V D M W 216 G C T A C A C G A T G G T A T A G A G C A C C A G A A A T C A T G C T G A A T T G G A T G C A T T A C A C C A A T A C T G T G G A T A T G T G G 648 IX S V G C I M A E L L T G K T L F P G S D H I D Q 240 T C T G T T G G A T G T A T A A T G G C A G A A C T T C T C A C A G G T A A A A C G C T A T T T C C T G G A T C G G A T C A C A T T G A T C A G 720 X L S R I M D L T G T P D D E I L A K I Q S E D A 264 T T G A G T C G C A T C A T G G A T C T A A G T G G T A C A C C T G A T G A T G A A A T C C T T G C C A A A A T C C A G A G T G A A G A T G C A 792 R N F V K S Q P K T K K K D F R G Y F A G A N E 288 C G G A A C T T T G T T A A A T C T C A A C C T A A A A C T A A G A A A A A A G A T T T T C G T G G A T A T T T T G C T G G A G C A A A C G A A 864 X I I A V D L L E K M L L L D V D K R I T A E E A L 312 A T T G C T G T T G A C C T T C T G G A G A A A A T G C T T C T G T T G G A T G T A G A C A A G C G T A T C A C T G C T G A A G A G G C A C T G 93 6 <5D S H P Y V A K Y H D E S D E P I G K Q F D D S F 336 A G T C A T C C T T A T G T T G C C A A A T A T C A T G A T G A A A G T G A T G A G C C T A T T G G T A A G C A G T T T G A T G A T T C C T T T 1008 E Q Q D L T V Q Q W K E L T F Q L I Q A V R H Q 360 G A A C A G C A A G A C T T G A C T G T G C A G C A G T G G A A A G A G C T T A C T T T T C A G C T G A T T C A A G C A G T A A G A C A T C A A 1080 S R R * 3 6 3 A G C A G A A G G T A A A T A G C T A C A A C A T T G A A A T C A A G G C T T G G G G A C G A N G G C T G G T C A A C A A T G T T A A G T A A T 1089 A C N A A T A T N K K I N N A A A A A A A A A A A A A A A A A A A A A A A A 98 HUMAN p3 8 MSQE RP TFYRQELNKTIWEVPERYQNLSPVGSGAYGSVCAAFDTKTGLRVA RAT p3 8 M V H MOUSE p3 8 M : CARP p38 M--K E-- --H V V T—S-Y-E K--XENOPUS p3 8 M-SN QSYV L- D T SS R-A—I-SEASTAR MIPK MNNPVTGSGETLSDDGYH-Y T VQ--K--A--A SSLN IKI-HUMAN p3 8 VKKLSRPFQSIIHAKRTYRELRLLKHMKHENVIGLLDVFTPARSLEEFNDVYLVTHLMGA RAT p38 MOUSE p3 8 CARP p3 8 T XENOPUS p3 8 S — K-F SEASTAR MIPK I A Q—D I-S CRGDT-SS-R M HUMAN p38 DLNNIVKCQKLTDDHVQFLIYQILRGLKYIHSADIIHRDLKPSNLAVNEDCELKILDFGL RAT p38 MOUSE p38 CARP p3 8 XENOPUS p3 8 G SEASTAR MIPK S-T-T S-E V VGV R HUMAN p3 8 ARHTDDEMTGWATRWYRAPEIMLNWMHYNQTVDIWSVGCIMAELLTGRTLFPGTDHINQ RAT p3 8 ; D-MOUSE p3 8 D-CARP p38 M XENOPUS p38 E D-SEASTAR MIPK —OA TN M K S D-HUMAN p3 8 LQQIMRLTGTPPAYLINRMPSHEARNYIQSLTQMPKMNFANVFIGANPLAVDLLEKMLVL RAT p3 8 -KL-L--V G-E-LKKIS-ES A MOUSE p38 -KL-L--V G-E-LKKIS-ES A CARP p38 S--S T--N--P R--SE Q XENOPUS p3 8 -KL-L—V EPE-LQKIS-EA PY ED--L Q SEASTAR MIPK -SR—D DDEILAKIQ-ED FVK-QPKTK-KD-RGY-A EI IN-HUMAN p3 8 DSDKRITAAQALAHAYFAQYHDPDDEPVADPYDQSFESRDLLIDEWKSLTYDEVISFVPP RAT p38 E F MOUSE p3 8 CARP p38 -T --E P E-E-F E-D-E-—RQ—E E — XENOPUS p38 -T E S I-E E-D-E R E—TC SEASTAR MIPK -V EE--S-P-V-K ES IGKQF-D QQ--TVQQ--E—FQLIQAVRHQ HUMAN p3 8 PLDQEEMES RAT p38 MOUSE p38 CARP p3 8 VF-GD XENOPUS p38 S SEASTAR MIPK SRR Figure 15. Protein sequence alignment of P. ochraceus MIPK with p38 from human, rat mouse, carp, andXenopus. Dashes indicate residues identical between all species, and gaps have been inserted to optimize the alignment. 99 begins in subdomain X and continues to the C-terminus of the protein. In this 124 amino acid region there is 46% identity between MIPK and human p38, versus a 65% identity in the entire protein. In the final 36 amino acids, identity drops to 28%, with the last 6 residues in p38 missing from MIPK. While it is clear that MIPK is a member of the p38 family of serine/threonine protein kinases, MIPK does not appear to be the seastar homologue of p38 kinase. 3.3.4 Comparison of seastar MIPK with other proteins. MIPK was clearly a member of the p38 family of protein kinases based on the TGY sequence in the activation loop. To more closely investigate the relationship between MIPK and p38, sequence homologies were compared between p38 homologues from a number of different species (Table 2). p38 homologues are distinct in their high degree of identity, 84%-99%. MIPK was found to be 65%-66% identical, and 74-75% conserved, with the p38 homologues. This is a significantly lesser degree of conservation than expected for a p38 homologue. Comparisons were therefore expanded to the other p38-like proteins that have been identified. Four p38-related proteins in the human system are p38, p38(3, p 3 8 y and p388. Although some sequence discrepancies have been noted between different groups, these variations accounted for an insignificant portion of the proteins. Human homologues within the p38 family vary from 5796-71 % identical, 72%-83% conserved (Table 3). MIPK was found to be 54%-65% identical to, 68%-74% conserved with the human p38 family. It was evident in looking at the numbers that MIPK was not more or less closely related to any of the family members. Another source of p38 homologues is the yeast system, with Hogl from S. cerevisiae and Styl from S. pombe both containing the characteristic TGY sequence in subdomain VIII. Even the yeast homologues show 43%-53% identity, 6\%-70% 100 Table 2: Identity/similarity between p38 homologues and MlPK.a MIPK p38 p38 p38 p38 p38 seastar Xenopus carp dog rat mouse p38-human 65/74 85/91 89/92 96/98 95/97 96/97 p38-mouse 66/74 88/93 86/90 99/99 99/100 -p38-rat 66/74 88/93 85/90 98/99 -p38-dog 66/74 88/93 86/90 -p38-carp 65/74 84/89 -p38-Xenopus 66/75 -3 The first number represents the percent identity between the two sequences. The second number represents the percent similarity between the two sequences. Percentages were generated using the AGALIGN program. 101 Table 3. Identity/similarity between p38 family members and MIPK.3 MIPK H o g l S t y l p388 p 3 8 y p38p2 seastar yeast s. pombe human human human p38-human 65/74 50/66 53/70 58/73 62/77 71/83 p38(32-human 62/72 45/63 50/68 57/72 61/74 -p38y-human 56/71 45/61 49/66 63/75 -p388-human 54/68 43/61 44/62 -S t y l - 5 . pombe 48/66 81/88 -Hogl-5. cerevisiae 48/61 -3 The first number represents the percent identity between the two sequences. The second number represents the percent similarity between the two sequences. Percentages were generated using the AGALIGN program. 102 conservation with the human p38 family, and 48% identity to, 6\%-66% conservation with MIPK. MIPK did not appear to be the clear P. ochraceus homologue of any of the currently identified p38 family proteins. This indicated that MIPK represents a potentially novel member of the p38 family. To further investigate the relationship between MIPK and the MAP kinase superfamily, MIPK homology trees were constructed using PHYLIP (Felsenstein, 1993). Amino acid sequences (Figure 16, Panel A) and nucleotide sequences (Figure 16, Panel B) were aligned using clustal, with the accession numbers listed in Table 4. To achieve more reliable comparisons, N- and C-terminal overhangs were removed. PHYLIP was used to build a tree from the species alignments. With this unrooted, additive tree model, the genetic distances are expected to equal the sums of the horizontal branch lengths. The MIPK protein sequence relatedness tree (Figure 16, Panel A), confirmed that the closest relatives of MIPK are the p38 kinases. MIPK appeared on its own branch, with no closely related neighbours. The next closest sequences were the p 3 8 p kinases, with p38y and p385 being slightly less homologous. MIPK was approximately equally distant from the Erk and Sapk families. The homology tree allowed the visualization of the close degree of homology found in the p38, Erk and Sapk families. The MIPK nucleotide sequence relatedness tree (Figure 16, Panel B) contains fewer sequences than the amino acid based tree, but displayed very similar patterns of homology. The MIPK gene was most closely related to the p38 kinases, yet still appeared on a distinct branch. Distances from the other p38 family members appeared significantly higher, and approached the level of the Erk and Sapk families. The nucleotide sequences of the p38 homologues of different species showed a lower degree 103 Panel A erk3 human — mpk2 seastar f- p42 Xenopus erk2 human erk2 mouse erk2 rat erk l rat erk l human — hog l 5. cerevsiae sty l S. pombe sapk4 human p385 human sapk3 rat erk5 human erk6 human p38y human sapk3 human MIPK seastar p38(32 human p38-2 human L p38p" human p38 Xenopus • p38 carp p38 human p38p* mouse p38 dog p38 rat p38 mouse 0.1 i i ~ j n jnk2 human jnk3 rat j n k l human Figure 16. Homology tree of MIPK with related kinases. Panel A. Amino acid sequence alignments were used to generate the tree. MIPK can be seen in the centre of the panel. Panel B. Nucleic acid sequence alignments were used to generate the tree. MIPK can be seen near the top of the panel. For both panels, horizontal branch lengths represent the distance matrix scores based on clustal sequence alignments, generated using the PHYLIP scoring algorithm (Felsenstein, 1993). 104 Panel B • p38 Xenopus — p38 carp p38 dog p38 mouse p38 rat MIPK seastar • jnk2 human erkl rat • erkl human p42 Xenopus i- p388 human L sapk4 human — sapk3 rat i — erk6 human I p38y human p38-2 human p38(3 human p38(52 human erk2 human erk2 mouse erk2 rat hogl 5. cerevisiae 1 styl S. pombe 0.1 105 Table 4. Names and accession numbers of kinases listed in the MIPK homology trees. Protein Accession number Identification erkl human humerkla Erkl human erk2 human humerk2a Erk2 human erk2 mouse muserk2 Erk2 - mouse erk2 rat raterk2 Erk2 - rat erk3 human P31152 Erk3 - human erk5 human X79483 Erk6 - human erk6 human hserk6 Erk6 - human hogl 5. cerevisiae yschogla Hogl - Saccharomyces cerevisiae j nk l human A53063 Jnk l - human jnk2 human humjnk2 Jnk2 - human jnk3 rat ratsapkc Jnk3 - rat MIPK seastar n/a MIPK - Pisaster ochraceus mpk2 seastar n/a Mpk - Pisaster ochraceus p38 carp cyimapkp38 p38 - carp p38 dog AF003597 p38 - dog p38 human humcsbpl p38 - human p38 mouse RNU91847 p38 - mouse p38 rat RNU73142 p38 - rat p38 Xenopus xlmpk2k p38 - Xenopus laevis p388 human AF015256 p388 - human p38y human HSU66243 p38y- human p38P human HSU53442 p38(3 - human p38P mouse muscrkl p38P - mouse p38(32 human AF001174 p38p2 - human, brain p38-2 human HSU92268 p38-2 - human, skeletal muscle p42 Xenopus xeb<p42 Erk2 - Xenopus laevis sapk3 human hssapk3 Sapk3 - human sapk3 rat rnsapk3 Sapk3 - rat sapk4 human AF004709 Sapk4 - human sty l S. pombe spknastyl Styl - Schizosaccharomyces pombe 106 of homology than the amino acid sequences, yet still pointed to MIPK being a separate isoform of the p38 family. The strongest proof of this will be the identification of either a p38 homologue in the seastar system, or of a MIPK homologue in a mammalian system. This work, will require the development of MIPK specific probes, as well as a more thorough understanding of the role of MIPK in oocyte maturation and stimulus response pathways. 107 3 .4 Expression and characterization of MIPK 3.4.1 Introduction. The Glutathione S-transferase (GST) Gene Fusion System is an integrated system for the expression, purification and detection of fusion proteins produced in E. coli. The large quantities of pure proteins that can be produced are a valuable tool to be used for characterization. The use of site-directed mutagenesis is another important technique that can be exploited in attempts to further understand the role of this protein in cell signalling. The use of highly concentrated, purified protein samples is also convenient for assessing antibody specificity. Complete characterization of MIPK requires the combination of both protein chemistry and molecular biology techniques. Results 3.4.2 Bacterial expression of MIPK as a GST-fusion protein. The full length open reading frame of the MIPK gene was cloned into a pGEX-4T expression vector. This system allowed for the production of pure MIPK as a GST-fusion protein to be used in further characterizations. Expression of MIPK was tested for several different clones to ensure maximum expression levels. Two different E. coli strains were also assessed to maximize GST-MIPK production, DH5CC (the standard E. coli strain recommended) and UT5600 (a protease-deficient E. coli). Results indicated that UT5600 cells gave the highest levels of MIPK expression (Figure 17), with approximately 30% of the protein present in the soluble fraction. A kinase-dead mutant of MIPK was also generated by site-directed mutagenesis. A mutant primer (KR) was used to convert the conserved lysine residue present in 108 OO DX CM VO I I— CO CX> 1X3 i Z Q_ CO i 1—r n—r VO 00 CT> VO s ) - OO VO CM CM C L _0J la 00 C L _0J o o VO LT, H -a c >^ rd C GJ box-O rB P u u t/i X b O ( J u> u, <*- o 0 M -LO - 3 c tu .2 3 u w X Q .koB rS S | § S'S £ tU — u ^ W rt z: 2 -d "3 u s-<U U rs to C ^ Hj — c *> X- 3 JJ OJ Cl O u UJ o f— < <U c rd Cu U-l c oX CN VO H U "3 C rd Cu 1—1 o tu 3 u3 cn O c o — VO - 3 m s f-- § « ' f nv W J > I-^ C M -rS 'J] O i — | +J — K 2 o vi O. "X3 tu j _ « c o rd CM 5) rs 2 o x uu CU s-[j Uu 0 U cri c rt • cu oi £ "> vq u ^ bo 1 rt in v O 2 w x> U X= CU _D S I r - 1 - 3 O « o o LO LO § 2 1 ^ ~ rt ' VO C L LO CU s > rt jo rd w. tu <AT2 CU _y s ^ J3 u c u C/l CU - 3 CU OJ a c £ O _ 5 g 2 ° C L LO X ' ^ 5 f u £ •5" bo r— '5 C/l o O x LO' g cu be rd > rd U U c cu O CU cu LO U LO O rt X 9 - 3 -u> w -d LO OJ (U C C rt ro I N o -tu : %~ —' M - C/l O tu LO C c ra, o r-i rd >—j tu 00 r t . s a c a t n LO- U rt S cu C L tu u LO rt O tu s > " ° rt X J -subdomain II and required for phosphotransferase activity, to an arginine residue. The mutant, K62R, was tested for expression levels as described for MIPK. GST-K62R gave very similar expression pattern as was found for GST-MIPK (Figure 17). Both fusion proteins were adsorbed to glutathione agarose beads to yield purified preparations. These beads were useful tools for characterizing the intact MIPK protein with respect to phosphotransferase activity, antibody crossreactivity, and upstream activators. For some protocols, including microinjection, thrombin cleavage of the fusion protein was required to obtain a soluble product. To ensure that the MIPK and K62R were not subject to proteolysis by the thrombin, the molecular weight of the cleaved protein was checked by SDS-PAGE. The resulting product consisted of a single 40-kDa protein band (Figure 17). 3.4.3 Antibody profiling of GST-MIPK. Two panels of anti-p38 monoclonal antibodies were available. O n e set was generated against full length mouse p38, while the other set was generated against a peptide fragment. All antibodies had been tested for crossreactivity with p38, with the whole protein antibodies showing the most promising results. Still it was interesting to test all available monoclonal antibodies, regardless of their ability to recognize p38, in a search for antibodies that might cross react with MIPK. A total of 27 monoclonal antibodies were tested for their ability to recognize expressed GST-MIPK on a Western blot. O f these, 18 were generated against the p38 peptide, while 9 were generated against the whole protein. Figure 18 shows the results obtained with the 9 whole protein antibodies. While all of the antibodies easily immunoreacted with GST-p38, none of the antibodies was capable of crossreacting with GST-MIPK. Similar results were obtained with the remainder of the antibodies were 110 tested (data not shown). Seven of the 18 antibodies had been found to recognize p38, yet none of the antibodies could detect GST-MIPK. The high concentration of MIPK and p38 used in these experiments guaranteed the opportunity for detection of even weakly crossreacting antibodies. This indicated that the epitopes made available for antibody generation were distinctly different between p38 and MIPK, and again implied that these proteins were not homologues. The polyclonal p38 antibodies currently available commercially and in-house, were also tested for crossreactivity with MIPK. Although previous Western blot data indicated that these antibodies would not recognize MIPK (Figure 7), the higher concentration of GST-MIPK available for testing could be used to assess lower levels of detection. As predicted, the N- and C-terminal antibodies both failed to crossreact with GST-MIPK (data not shown). Equally important, the Cdk5-CT antibody was tested for its ability to recognize GST-p38. This antibody proved to be selective in its detection of MIPK, showing no crossreactivity with p38 (Figure 18, Lane 20). It was interesting to note the vast degree of diversity in the N- and C-termini of MIPK versus p38. The immunizing peptides used to generate p38-NT and p38-CT polyclonal antibodies represent regions of 0% identity (CT) and 22-36% identity (NT, depending on the influence of the 8 residue insert in MIPK) (Table 5). In contrast, the Cdk5-CT immunizing peptide was 60% identical with the equivalent peptide in MIPK, while matching only 40% of the amino acid sequence in human p38. It can therefore be concluded that these antibody probes seem to be very specific in their ability to recognize p38 or MIPK, but not both enzymes. This suggests that the use of the currently available probes for detecting p38 would not uncover any MIPK-related proteins in other species. I l l Figure 18. Immunoreactivitv comparisons of GST-MIPK and GST-p38. Monoclonal antibodies generated against p38 were tested for immunoreactivitv with GST-MIPK (lanes 1-9) and GST-p38 (lanes 11-19). The presence of GST-MIPK was confirmed through Western blotting with Cdk5-CT (lane 10). GST-p38 was tested for immunoreactivity with Cdk5-CT (lane 20). Reference numbers for the monoclonal antibodies are shown below the panel. The migration of GST-MIPK and GST-p38 are indicated by the arrow. Migration of Mr standards is shown to the left of the panel. Table 5. Alignment of immunizing peptides with MIPK and p38 sequences Antibody Sequence Alignment Percent Match Cdk5-CT MIPK p38-human NPVQRISAEEALQHP DVDKRITAEEALSHP DSDKRITAAQALAHA 60% 40% p38-NT MIPK MSQE RP - TFYRQELC MNNPVTGSGETLSDDGYHRYELC 22%-36% p38-CT MIPK DEVISFVPPPLDQEEMES QLIQAVRHQSRR 0% M I P K - C T p38 KELTFQLIQAVRHQSRR KSLTYDEVISFVPPPLD 18% 3.4.4 Western blotting comparisons of MIPK and p38 in P. ochraceus. To this stage it appeared that MIPK was not the seastar homologue of p38, although this could not be confirmed without actual cloning of the seastar p38 gene. The anti-p38 antibodies available did not immunoprecipitate any proteins from the seastar. Cytosolic extracts from P. ochraceus oocytes were therefore fractionated on a ResourceQ column before being subjected to SDS-PAGE. Duplicate gels were Western-blotted with Cdk5-CT, p38-CT, or both antibodies (Figure 19). Clean bands were found in both individual Western blots, although both proteins appeared in the same fraction from the ResourceQ column. To clarify this result, one of the membranes was probed with both antibodies. Two proteins could be resolved using this technique, although differing by only a few kDa, with MIPK representing the larger of the two proteins. This result clearly indicated that two p38-like proteins are expressed in seastar oocytes. One of these proteins, MIPK, was recognized only by the Cdk5-CT antibody, while p38-CT could only detect the lower molecular weight protein. Phosphotyrosine Western blots indicate that only MIPK was phosphorylated in the immature oocyte blocked at prophase, based on the detection of a single band. Further characterization of this second p38-like protein will require more information about the primary structure, and ideally cloning of the cDNA encoding this protein. This may be possible through the use of degenerate oligonucleotides modelled after the N- and C-termini of p38. 3.4.5 Design of MIPK specific antibodies. To determine whether MIPK homologues existed in other species, it was necessary to create MIPK-specific probes. Through analysis of the MIPK sequence, it was clear that the regions of highest diversity between MIPK and p38 family members involved the N- and C- termini. Peptides were chosen from these regions for 114 26 -4 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Fraction number Cdk5-CT Western Blot p38-CT Western Blot Cdk5-CT/p38-CT Western Blot Figure 19. Identification of p38 isoforms in P. ochraceus oocytes by Western blotting. Cytosolic extract was fractionated by ResourceQ chromatography and further separated by SDS-PAGE. Identical membranes were Western-blotted with Cdk5-CT (panel A) and p38-CT (panel B), and both antibodies (panel C). MIPK migration is indicated by the open arrow ( —> ), a second p38 isoform by the closed arrow ( —•> ). Panel C shows that while the two isoforms cofractionate over ResourceQ, the two proteins resolve by SDS-PAGE. ResourceQ fractions are shown beneath the blots, and migration of Mr standards indicated to the left of the blots. 1 1 immunization of rabbits and antibody generation. Unfortunately, only one of the peptides could be synthesized, the C-terminal peptide corresponding to amino acids 348-363 (Table 5). This peptide was not only unique to MIPK versus the p38 isoforms, but also showed no homology with any other proteins in the protein data bases, making it a very good candidate for a specific MIPK probe. Preliminary results have shown that the antibody crossreacts with MIPK from seastar, but does not crossreact with similar molecular weight proteins in rat tissues (data not shown). The antibody is still in development, with potential for higher affinity antibodies being generated in the future. In summary, starting with limited protein sequence data, important information has been obtained with regard to the primary structure of MIPK. This has resulted in the cloning and sequencing of the complete MIPK gene from seastar cDNA, and production of a recombinant fusion protein for expression in bacteria. A dominant negative, kinase-dead mutant of MIPK has also been generated. This information has so far been used to identify related proteins from other species. MIPK was found to be closely related to p38 kinases, yet no homologue could be identified. Tools were therefore created to allow screening for MIPK homologues in other systems, and to allow further characterization of MIPK in the oocyte system. 116 3.5 M I P K ac t i v i t y in the P. ochraceus s y s tem 3.5.1 Introduction. As stated in Section 3.3.4, MIPK has been identified as a novel p38-like protein kinase in the P. ochraceus oocyte system. The purified kinase from the seastar and the recombinant protein from bacteria are important tools in determining the role of MIPK in the oocyte as well as the potential role of homologous enzymes in other cell systems. Previous characterization of the MIPK protein could be exploited in an attempt to understand the effect of MIPK activity on oocyte maturation, as well as other potential routes for the activation of MIPK. This included the search for potential targets for MIPK phosphorylation, and potential upstream activators of the MIPK pathway. With the lack of a method for assessing MIPK kinase activity, MIPK activation was estimated by its level of tyrosine phosphorylation, based on the assumption that tyrosine phosphorylation on MIPK would be activating. Results 3.5.2 Time course of activation bv osmotic shock treatment. The close homology between MIPK and the p38 family of stress activated protein kinases prompted the analysis of the activity of MIPK in response to stress conditions. Seastar oocytes were subjected to high osmolarity conditions to assess the activity of MIPK during osmotic shock. Natural seawater has a concentration of salt of approximately 0.5 M. Oocytes were placed in seawater of 1 M , 1.5 M, 2 M and 3 M NaCI and aliquots were tested for MIPK activation. The highest medium concentration that permitted survival of the oocytes was 1 M NaCI. The results indicated a dramatic 117 activation of MIPK in response to osmotic shock, based on 4G10 immunoreactivity (Figure 20, Panel A). This was despite an extreme decrease in the amount of MIPK protein found in the oocyte (Figure 20, Panel B). Results indicated an approximately constant level of MIPK tyrosine phosphorylation in the cell after addition of NaCI, despite this decrease in protein. This indicated that the tyrosine phosphorylation, and potentially activation, of MIPK contributed to the osmotic shock response and that it was required at a minimum threshold level, resulting in a higher percentage of MIPK being phosphorylated in the cell. This may indicate that a pool of active MIPK was protected from proteolysis during the osmotic shock response. To ensure that this tyrosine phosphorylated protein was MIPK and not the other p38 isoforms in the oocyte, the immunoprecipitations from the time course were Western-blotted with a p38-CT antibody. Results confirmed that there was no p38 present in the immunoprecipitations (data not shown). 3.5.3 Time course of activation by heat shock treatment. Heat shock time courses were also tested for MIPK activation. Normal conditions for oocyte maturation ranges from 10°C-14°C (Fraser et al, 1981). Based on this information, heat shock temperatures of 25°C, 35°C and 45°C were tested (Figure 21). At 45°C, the oocytes appeared to melt, likely due to the jelly coat surrounding the cell, and the time course was found to be unreliable. At 25°C, a small activation of MIPK was apparent after 60 minutes incubation. At 35°C, MIPK activation was visible within 20 min and continued increasing through 30 min. After 30 min the total tyrosine phosphorylation remained constant, while the amount of MIPK protein began to drop. As with the osmotic shock time course, it appeared that the MIPK was activated to a threshold level, and that this level was maintained despite a net decrease in MIPK protein. 118 kDa 4G10 Cdk5-CT 0 0.5 10 20 30 60 Time (min) E i.«H ^ l o Q. on O 0.5H l r 5 f h i 1 1 1 r 0 0.5 10 20 30 60 Time (min) Figure 20, Phosphorylation of MIPK in homogenates of a P. ochraceus oocyte osmotic shock time course. P. ochraceus oocyte osmotic shock time course homogenates were immunoprecipitated with Cdk5-CT and Western-blotted with either 4G10, antiphosphotyrosine antibody (panel A), or Cdk5-CT (panel B). The numbers below the blots show the time course of osmotic shock in minutes. Migration of Mr standards is shown to the left of the blots. Alkaline phosphatase signals on the Western blots were quantitated by densitometric analysis and phosphotyrosine levels corrected for MIPK protein levels (panel C). Data is the average of 3 independent experiments±S.E.M. 119 4G10 Cdk5-CT 0 10 20 30 60 0 10 20 30 60 Time (min) 25°C 35°C SO-LO o 3H -2 E % 2 > 'Lo o H 0 10 20 T 30 r 60 0 10 20 30 60 Time (min) 25°C 35°C Figure 21. Phosphorylation of MIPK in homogenates of a P. ochraceus oocyte heat shock time course. P. ochraceus oocyte heat shock time course homogenates were immunoprecipitated with Cdk5-CT and Western-blotted with either 4 G 1 0 , antiphosphotyrosine antibody (panel A), or Cdk5-CT (panel B). The numbers below the blots show the time course of heat shock in minutes, with the temperatures used listed below the time points. Migration of Mr standards is shown to the left of the blots. Alkaline phosphatase signals on the Western blots were quantitated by densitometric analysis and phosphotyrosine levels corrected for MIPK protein levels (panel C). Data is the average of 3 independent experiments±S.E.M. 3.5.4 Postfertilization activation of MIPK. In the seastar oocyte system, the majority of research information is limited to the events surrounding meiotic maturation, with very little data on the time following fertilization. It was therefore interesting to assess a potential role for MIPK in regulating the development of the seastar embryo. Seastars were induced to spawn through injection with 1-methyladenine, resulting in the shedding of mature, fertilizable oocytes. These oocytes were fertilized and the embryos followed for two days, at which point they had developed to an early gastula stage. Extracts from various post-fertilization time points were assessed for MIPK tyrosine phosphorylation levels, and the levels compared with those found in immature and mature oocytes. Results showed that the very low level of phosphorylation that exists at the time of fertilization decreased to below detection within the first 6 h post-fertilization (Figure 22). From 12-20 h, the tyrosine phosphorylation of MIPK increased dramatically. This level dropped by 24 h and appeared to stay low through 48 h. MIPK protein levels began to drop by the 2 day embryo, potentially due to an increase in the protease levels in the sample. The early development of P. ochraceus embryos is characterized by an initial rapid increase in cell number to the 256-cell stage. At 12°C, this period of synchronous cleavage lasts approximately 14 h. After that, the individual cells within the embryo assume independent division rates, and there is a flattening of the developmental curve (Figure 23,- Fraser et al, 1981). It appears that the activation of MIPK is coincident with the transition from synchronous cell cleavages to differential cleavages. The most likely explanation for the potential activation of MIPK post-fertilization can be predicted by taking into account previous characterization of the enzyme. Earlier studies of maturation and stress time courses indicated that MIPK was tyrosine phosphorylated in 121 31 I M 3 6 12 24 48 Time (h) I M 3 6 12 24 48 Time (h) Figure 22. Phosphorylation of mipk in homogenates of a P. ochraceus oocyte fertilization time course. P. ochraceus oocyte time course homogenates were immunoprecipitated with anti-Cdk5-CT and Western-blotted with either 4G10, antiphosphotyrosine antibody (panel A), or Cdk5 -CT (panel B). Detection of p44mpk is indicated by the solid arrow ( — ) , p40m iP' c is indicated by the open arrow (^— ). Immature oocytes are represented by "I", mature oocytes represented by "M", and the numbers below the blot show the time course of fertilization in hours from 0 (mature oocytes) to 48 hours post-fertilization. Migration of Mr standards (kDa) are shown on the left. Alkaline phosphatase signals on the Western blots were quantitated by densitometric analysis and phosphotyrosine levels corrected for MIPK protein levels (panel C) . This data is representative of 3 independent experiments. 122 10000 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Time post-fertilization (h) at 12°C Figure 23. P. ochraceus embryo development time course. The number of cells per seastar embryo as a function of time post-fertilization. (Adapted from Fraser eta!., 1981) 123 cells which were arrested in the cell cycle. Under these circumstances, MIPK may be acting as a cytostatic factor. The clear decrease in the rate of cell division would require activation of enzymes which promote quiescence and cell cycle blockages, in preparation for differentiation. It therefore follows that the role of MIPK post-fertilization may be involved in the cells exiting from the cell cycle. 3.5.5 Assessment of kinase activity of purified MIPK. To further understand the role of MIPK in the oocyte system and to confirm previous assumptions about activation and tyrosine phosphorylation, it was necessary to detect potential downstream targets. The partially purified MIPK protein from immature oocyte extract was used to assess MIPK substrate specificity. The kinase was purified as described in Section 3.2.2 including the final concentration step on a ResourceQ column. This preparation was then assessed for phosphotransferase activity towards a panel of common protein kinase substrates (Figure 24). The migration of MIPK on the column was assessed through Western blotting with Cdk5 - C T , and was found to elute in fractions 24-26. When kinase assays were performed on the ResourceQ fractions, activity was found towards a variety of substrates. Unfortunately, these kinase activities were found to elute in fractions 27-30, distinct from the elution profile of MIPK. This confirmed that the assays were done under correct conditions for measuring phosphotransferase activity, and indicated that the substrates tested were not appropriate for MIPK. One potential problem with this technique for measuring kinase activity was the length of time required for the purification of MIPK. The lack of a substrate resulted in the need to follow MIPK through Western blotting of the column fractions. This process took one day per column run, with the entire process lasting one week. There was a 124 Figure 24. Assessment of kinase activity of partially purified MIPK. MIPK was purified from immature oocytes with the final step being concentration on ResourceQ. Fractions from the ResourceQ were tested for phosphotransferase activity towards a panel of common substrates, shown to the right of the graph. The fraction numbers are indicated on the x-axis, the amount of [y-32P]ATP incorporated into the substrates (cpm/10 min) are indicated on the y-axis. The fractions containing MIPK immunoreactivity are indicated by the grey bar. 125 possibility that the kinase activity of MIPK was not stable through this time period, despite its tyrosine phosphorylation. Myelin basic protein (MBP) and histone Hi (HHl) were tested as substrates at earlier column steps, and MBP did show some potential as being a weak substrate for MIPK (data not shown). This activity was either resolved from MIPK or the activity was not stable through the subsequent columns steps. There was some evidence to indicate the potential requirement for additional components for MIPK kinase activity. Initial studies were performed using a MonoQ column. This column has similar binding characteristics to ResourceQ, but does not show identical elution profiles for all proteins. When immature and mature extracts were resolved on a MonoQ column, MIPK was found to elute in the flowthrough fractions in the immature extract, but bound to the column in the mature extract (data not shown). While this could be attributed to the change in tyrosine phosphorylation state, this fluctuation in elution profile has not been observed for other MAP kinase isoforms undergoing changes in tyrosine phosphorylation. Gel filtration data has not confirmed the presence of a second subunit, but this has only been used after purification or after ResourceQ fractionation, both of which have the potential of stripping off a required element from the MIPK protein. It is also possible that MIPK is complexed with its upstream activating kinase in the mature extracts, since in mammalian systems inactive E r k 2 is bound to Mekl (Fukuda etal, 1997). A second method was used to assess MIPK phosphotransferase activity. Extracts from immature, mature, 3 h post-fertilization, and 18 h post-fertilization were resolved on a ResourceQ column, and the peak MIPK fractions identified. These fractions were incubated with [y-32P]ATP for one hour, followed by immunoprecipitation with C d k 5 -CT. The complexed proteins were analyzed by Western blotting and autoradiography. Figure 25 shows the autoradiogram as well as the corresponding phosphotyrosine levels 126 kDa 9 7 -6 7 -4 6 -26-^ <J— p40miPk p40mipk I M 3 h 18 h Figure 25. Autophosphorvlation of partially purified MIPK. Cytosolic extracts from immature (I), mature (M), 3 hour fertilized (3 h), and 18 hour fertilized (18 h) P. ochraceus oocytes were fractionated on ResourceQ and the MIPK peak fractions pooled. The proteins were then autophosphorylated before immunoprecipitation with Cdk5-CT. Panel A, autoradiograph of Western blot,- Panel B, Western blot probed with 4G10 antibody. Migration of MIPK is indicated by the open arrow, and Mr standards shown to the left of the autoradiograph. 127 of MIPK at these stages. A band of phosphorylation was identified which comigrated with MIPK as identified using the Western blot (data not shown). This phosphorylation could be due to autophosphorylation of MIPK, or phosphorylation of MIPK by an upstream kinase. Other major bands of phosphorylation migrated with antibody chains. What was clear was an increased phosphorylation level in the immature and 18 h post-fertilization samples, coincident with the increased phosphotyrosine level of MIPK. 3.5.6 Assessment of phosphotransferase activity of bacteriallv expressed MIPK. GST-MIPK was tested for phosphotransferase activity towards a panel of common substrates including MBP, histone H i , Atf2 and c-Jun. GST-MIPK-K62R was used as a negative control in all assays, to ensure that the activity measured was not due to contaminating bacterial proteins. MIPK did not show phosphorylating activity towards any of the substrates tested (data not shown). Often bacterially expressed proteins are inactive, either due to improper 3-dimensional structure, or due to a lack of activating phosphorylation. T o assess the level of tyrosine phosphorylation on the recombinant protein, the relative 4G10 levels of GST -MIPK, GST-MIPK-K62R, and immunoprecipitated endogenous MIPK from various time course samples. Unfortunately, fresh immature seastar extracts were not available for use in this experiment, and the osmotic and heat shock samples had to represent endogenous MIPK phosphorylation levels. Taking MIPK from the osmotic shock time course as the maximum potential tyrosine phosphorylation, heat shock activated MIPK possessed 22% of the maximum tyrosine phosphorylation, GST-MIPK contained only 3.4% of the maximum phosphorylation, and GST-MIPK-K62R was phosphorylated at less than 1 % of the maximum potential level (Figure 26). It was therefore unlikely that the expressed GST-MIPK would possess sufficient activity to be 128 Osmotic Shock Heat Shock GST-MIPK GST-K62R Figure 26. Levels of tyrosine phosphorylation in endogenous and bacteriallv expressed MIPK. Endogenous MIPK protein was purified by immunoprecipitation from 30 sec osmotic shock, and 60 min heat shock time course samples, and compared to bacterially expressed proteins. Samples were electrophoresed on duplicate gels and Western blotted with 4G10 antiphosphotyrosine antibody and C d k 5 - C T to quantitate protein levels. Immunoreactivity was measured by densitometric analysis and phosphotyrosine levels corrected for MIPK protein. Maximum phosphorylation was equated to the level of phosphorylation in the osmotic shock sample. 129 measured in these assays. It may be necessary to express MIPK in an activatible system, such as transient transfection systems, in order to obtain an active preparation of the enzyme. 3.5.7 Examination of in vivo effects of MIPK in the oocyte system by microinjection. Several attempts were made to assess the role of MIPK in the oocyte system. Initial studies involved oocyte maturation in response to 1-methyladenine, to determine whether MIPK was acting as a cytostatic factor in the immature oocyte. Oocytes were injected with kinase-dead MIPK-K62R, in anticipation that this protein would act as a dominant-negative in the MIPK signalling pathway. If MIPK was responsible for the cell cycle block in G2, it was predicted that inactivation could result in induction of maturation in the absence of 1-methyladenine. However, microinjection of kinase-dead MIPK did not induce maturation. A second possibility was that knocking out MIPK could increase the rate of maturation in response to 1-methyladenine. Therefore, oocytes that had been injected with MIPK-K62R were treated with 1-methyladenine and the rate of maturation observed. Unfortunately, oocyte maturation occurs quite rapidly under normal conditions, with GVBD apparent within 60 min. No significant differences could be measured in the rate of onset of GVBD when MIPK-K62R injected oocytes were compared with control oocytes injected with PBS. It was not possible to measure the ability of MIPK-K62R to block endogenous MIPK activity, and experiments were therefore based on the assumption that the kinase-dead mutant could act as a dominant negative factor. It may be possible to observe more markers to compare the rates of maturation, such as cytoskeletal rearrangements. This may identify more subtle variations in the maturation process. 130 A second method used to inactivate MIPK in the oocyte was injection of antisense oligonucleotides. It was hoped that incubation of the oocyte would result in blockage of protein synthesis of MIPK, resulting in a decrease in MIPK present in the cell. Two oligonucleotides were designed. Antisense-1 was created to block the start codon transcript, however its GC content was low and it was unclear whether it would bind well to the mRNA. Antisense-2 was created to bind downstream of the start codon, and contained a higher GC content. Control oligonucleotides were Sense and Missense oligonucleotides modelled after Antisense-1. The oligonucleotides were injected into immature oocytes and incubated 22-24 h to allow protein turn-over to occur. Oocytes were observed for spontaneous maturation in response to the loss of MIPK activity. Again, no significant maturation was observed post-injection. The maturation process is very complex, with the activation of many kinase cascades required. It was therefore not surprising that the loss of MIPK was not sufficient to induce maturation. It could be that the combination of MIPK inactivation with injection of a maturation-activated kinase such as Mek l , would cause maturation of the oocyte in the absence of 1-methyladenine. It is also possible that 22-24 h incubation was not sufficient to allow protein turn-over to be complete, and therefore maternal MIPK may have been present throughout the experiment. Further attempts to assess the role of MIPK in the seastar oocytes exploited the post-fertilization activation of MIPK. Immature oocytes were initially injected as described above, with both the kinase-dead MIPK-K62R or the antisense oligonucleotides. When no maturation could be observed, oocytes were treated with 1-methyladenine in preparation for fertilization. Once fertilized, development of the embryo was carefully observed over a 24 h time period. In the case of MIPK-K62R injection, the microinjected oocytes appeared normal up to the point of fertilization. 131 After fertilization, the oocytes appeared to undergo some form of cell death within 5 h (Figure 27). All control cells survived for the full 24 h post-fertilization. It is unclear what the meaning of this observation is at this time, and further experiments will have to be performed using additional controls, including the use of other recombinant protein, rather than PBS, as negative controls. Injection of the oligonucleotides into the seastar oocytes resulted in an overall decrease in the rate of development of the embryo. This was observed for both the antisense oligonucleotides and the control missense primer (Figures 28/29). This may reflect the influence of the buffer components in the preparation of the primers. At 10 h post-fertilization, cell division rates of the control H 2 0 injected cells and antisense-2 cells appeared very similar (Figure 28). The missense and antisense-1 injected cells were very delayed in their division rates, at this same time point. By 22 h post-fertilization, significant development to the blastula stage had occurred in the control H 2 0 injected cells (Figure 29). A very clear ring of cells on the outer edge of the embryo was visible, although the quality of the embryos was questionable. In the antisense-2 injected oocytes, the cells had not undergone any differentiation. It was difficult to assess the relative cell numbers at this stage. The antisense-1 and missense injected cells had developed very poorly, with divisions appearing to be very asymmetrical. These results indicated the probable interference of the oligonucleotide buffer with the development of the embryo. Therefore, these experiments remain inconclusive, and will have to be repeated with fresh oocytes next season. It may be necessary to dialyse these solutions to remove the interfering components. These studies do have the potential for confirming the role of MIPK in the regulation of embryo development at the transition between synchronous cleavages and differential cleavages. 132 5 h Post-fertilization 22 h Post-fertilization Figure 27. Microinjection of kinase-dead MIPK and p38 inhibitor into P. ochraceus oocytes. Immature seastar oocytes were injected with either kinase dead K62R-MIPK (B,E) or p38 inhibitor compound (C,F) in PBS. Panels A and D show the control oocytes injected with PBS. Oocytes were matured in 1 -methyladenine for 4 h prior to fertilization. Embryonic development was monitored for 22 h, with photographs taken at 5 h (A-C) and 22 h (D-F) post-fertilization. 133 c v v « t« E J : O >N , <U o o iu E fc ^3 O 3 w ^ •5 * ^ ra ra Q P - ° c E 3 E E y w o o OH O -M c c _o u <U o OJ u 3 c o "o V cn c cu <l oo r s V fc c o "d — '% c O CU 3 -0 "3 £ Q t^ c o cu c ra C u be c c o <u • cn . C fc. +J iu C -fc. IU £ P o -d cu u £ 0 — CU CU cn 3.5 u CO • » m A A tu in c tu E a. OJ T f OJ "t* CN > , tu u 1 -d 8 o .y OJ _ l E it. ^3 o •E: +i E T ro m o P - ° c E 3 E _E "to "'"c3 .2 CL, O tu '-C> o JU (J 3 C o o tu in C tu in '3 C < oS r s tu ro •*-> c> S tu U; "d — O tu tU M -3 X l o"° S> % <3 J2 r s - J -oj in i— c o c i tu 'c I re C i_ «-> U tu E aa 00 c c4 1- +-> OJ Cl O . <U c OJ Cl ^ 2 +-> CL H J = J-* r s £ r s Cl *3 — OJ OJ Cl • . in OJ in m ro U ! U 04 0 S4 136 1 3 7 A final set of microinjection experiments examined the influence of the p38 inhibitor compound on oocyte maturation and embronic development. Oocytes were injected with the inhibitor, as the jelly coat of the oocyte would likely not allow the compound access to the cell. The oocytes were observed for -24 h post-injection for the appearance of spontaneous maturation. There was no significant occurrence of maturation in the absence of 1-methyladenine, when the cells were treated with the p38 inhibitor. The oocytes were then treated with 1-methyladenine, again with no significant difference in the rate of maturation observed. Post-fertilization, the embryonic development matched that of the control cells (Figure 27). The p38 inhibitor was therefore not capable of interfering with the signalling mechanisms involved in oocyte maturation and embryonic development. Although it will be important to assess the affect of this inhibitor on MIPK activity in vitro once a substrate for the kinase can be found, these results indicate that MIPK activity was not decreased significantly in the presence of the p38 inhibitor. 3.5.8 Identification of an upstream MIPK kinase. Preliminary experiments were performed to determine the existence of upstream kinases of MIPK, or MIPKK. Extracts from the 0 and 30 sec 1 M NaCl osmotic shock time course were first fractionated through a 1 ml ResourceQ column. Using GST-MIPK as a substrate, the column fractions were assayed for MIPKK activity in the presence of [y-32P]ATP. The GST-MIPK was electrophoresed on an SDS-polyacrylamide gel and Western blotting with 4GlO antiphosphotyrosine antibody. Under these conditions, no increase in tyrosine phosphorylation were observed, and no radioactive label was incorporated into the GST-MIPK (data not shown). 138 A more promising assay system is based on the evidence that MAP kinases associate tightly with their upstream Mek (Errede and Ge, 1996,- Bardwell and Thorner, 1996). GST-MIPK agarose beads were used as an affinity matrix to bind the potential upstream kinase. After careful washes, the beads were incubated with radiolabeled ATP to measure bound phosphotransferase activity towards GST-MIPK. The mixture was then analyzed using SDS-PAGE, autoradiography, and Western blotting with an anti-Ste7-VIII antibody. This antibody was modelled after the S. cerevisiae Mek homolog Ste7, and has been found to crossreact with many different Mek isoforms. Although preliminary, the results indicate the presence of a MIPKK which was able to bind to the GST-MIPK beads and phosphorylate the enzyme. Western blotting showed several crossreactive species, the most prominent at 48 kDa (Figure 30). Further experiments will be required to confirm the identity of this MIPKK, and to determine the specificity of its phosphotransferase activity. Still, this method shows great potential for identifying the MIPKK in the seastar system. 139 Ste7-VIII Western blot Autoradiography 0 60 120 0 60 120 Time (min) GST-MIPK MKK? Figure 30, Identification of an upstream MIPK kinase in the P. ochraceus oocyte. GST-MIPK beads were used as an affinity matrix to bind proteins in the immature (0 min), maturing (60 min) and mature (120 min) seastar oocyte cytosol. Proteins were allowed to complex with MIPK for 2 h, followed by low-stringency washed. The beads were then subjected to a phosphotransferase assay in the presence of [y- 3 2P]ATP. The mixture was separated by SDS-PAGE. Bound proteins were visualized through Western blotting with Ste7-VIII (Panel A), and radiolabeled proteins were visualized by autoradiography (Panel B). Migration of GST-MIPK is indicated by the open arrow ( ^ — ) ; migration of a potential M K K isoform in indicated by the closed arrow ( — ) . Migrations of Mr standards are shown to the left of the Western blot. CHAPTER 4 DISCUSSION/FUTURE DIRECTIONS 4.1 Discussion. A 40 kDa protein from the P. ochraceus oocyte system has been identified as a novel member of the p38 family of MAP kinases. This protein was first identified as major tyrosine phosphorylated protein in the immature oocyte, arrested in late G 2 phase of meiosis I. Further characterization of this protein uncovered crossreactivity with an antibody made against the C-terminus of a cyclin-dependent kinase, C d k 5 . This protein underwent dephosphorylation in response to 1-methyladenine treatment in a time course which mirrored the pattern of phosphorylation of a known seastar MAP kinase, p 4 4 m p k . Despite its immunoreactivity with Cdk5 - C T , this protein lacked many features that would indicate that it belonged to the cyclin-dependent kinase family. Many of the other antibodies known to crossreact with Cdk5 failed to detect the 40 kDa protein. Instead, the protein showed indications of belonging to the MAP kinase family, known to be activated by threonine and tyrosine phosphorylation. Therefore, in consideration of the distinctive dephosphorylation during oocyte maturation, the protein became known as maturation-inhibited protein kinase, MIPK.. MIPK was partially purified from oocyte cytosol, after which micropeptide sequencing could be performed. Six peptides were sequenced, four of which identified MIPK not only as a member of the MAP kinase family, as predicted, but also as a specific relation to p38 MAP kinase. Using this information to design degenerate oligonucleotides, the full length cDNA was cloned from seastar mRNA using multiple PCR steps. The sequence was found to contain the eleven kinase subdomains, and amino acids conserved in all protein kinases, and MIPK was defined as a serine/threonine protein 141 kinase. A characteristic Thr-Gly-Tyr (TGY) sequence in its activation loop, between subdomains VII and VIII, confirmed M I P K as a member of the M A P kinase family, and as a definite relative of p38 M A P kinase. Closer analysis of the sequence of M I P K , in comparison with p38, uncovered major differences in primary structure. The kinase domains were very similar, with the exception of subdomains II and X . Subdomain II contains the important lysine residue, which is essential for kinase activity. This residue is often mutated in the formation of a kinase-dead enzyme. The amino acids surrounding this conserved lysine showed a low level of conservation. The key importance of this lysine in the phosphotransferase reaction indicates that this region is likely to influence substrate preference. The difference in sequence in Subdomain II may therefore imply a unique active site conformation for M I P K . Subdomain X is the most variable of the kinase subdomains, and it was therefore not surprising to see the difference in amino acid sequence. Comparisons of p38 homologues from several species showed that subdomain X has not been highly conserved evolutionarily (Figure 15). Despite this, the degree of discrepancy of the M I P K sequence appeared to be significantly higher than that observed between the other p38 kinases. This again may translate to differences in substrate specificity. Overall M I P K shared 7 1 % identity in its kinase domain with human p38, and 65% identity in the entire protein. This compares to Xenopus p38, which is 88% identical in the kinase domain when aligned with human p38, and 85% identical in the entire sequence,-carp p38, which is 93% identical in the kinase domain, and 89% identical in the entire sequence,- and mouse and rat p38, which are 95% identical to human p38 in the kinase domain and overall (see also Table 2). It is evident by these numbers that the p38 kinases 142 are very well conserved, from Xenopus through to human, and that MIPK does not show the same level of identity with p38 as do the known p38 homologues with each other. The differences are even stronger when looking outside the kinase domain, at the N- and C-terminal regions of the proteins. MIPK showed no similarity to the p38 kinase sequences at either terminus. The N-terminus of MIPK contained an 8 amino acid insert, not seen in any of the p38 kinases. Even excluding this insert, the surrounding residues exhibit no correlation with the sequence found in p38 kinase. Similarly, the C-terminus of MIPK was drastically different from p38, sharing only 1 6 % sequence identity. This included a deletion of 6 amino acids at the terminus of MIPK. It is especially important to note these differences in consideration of the probes being used to detect p38 in many systems. Antibody probes are normally generated against N-terminal or C-terminal peptide sequences, in an attempt to create immunoprecipitating tools. These antibodies would be useless in recognizing MIPK, making the detection of MIPK in other systems very difficult. Three other subfamilies of p38 kinases have also been identified and MIPK was therefore subjected to sequence comparisons with these other family members. MIPK showed closest homology, 6 5 % , to the original p38, with 6 2 % identity to p38B, 5 6 % identity to p38y, and 5 4 % identity to p388 (Table 3). With the high degree of conservation also apparent within these subfamilies, it seems clear that MIPK does not represent the seastar counterpart of any known p38 family members. This argument was strengthened with the discovery of a second p38-like protein in the seastar system which could be resolved from MIPK by SDS-PAGE (Figure 19). Although showing very similar size and elution profile from a ResourceQ column, this second protein specifically immunoreacted with a p38-CT antibody. Because the variation seen within the novel members in the p38 family was most apparent in the N- and C-termini, this 143 antibody crossreactivity strongly supported the presence of a seastar homologue of mammalian p38, which was distinct from MIPK. This p38 kinase did not appear to undergo regulation in response to 1-methyladenine stimulation or during embryonic development, as evidenced by a lack of change in tyrosine phosphorylation, or by the absence of h s p 2 7 kinase activity of Mapkapk2 in the oocyte system (D. Lefebvre, personal communication). These sequence variations suggest that MIPK and p38 have different upstream kinases and downstream targets. The sequences required for substrate recognition by various serine/threonine protein kinases were assessed by Songyang et al. (1996). By comparing the sequences of Cdkl, C d k 2 , C d k 5 and Erkl to p38 kinases and MIPK it was possible to generate a table of amino acids predicted to interact with the peptide substrate (Table 6). The substrate binding pocket involves residues from many of the conserved kinase subdomains, contacting the side chains of peptide substrates from the -4 to +3 positions relative to the phosphorylatable residue. The p38 homologues from the different species tested show identical predicted substrate specificity using these guidelines. The only residue not conserved was involved in the +1 position. The definition of MAP kinases as proline-directed kinases implies that this discrepancy in sequence is insignificant. MIPK showed very high conservation of sequence in the residues predicted to interact with the peptide substrate. Only one amino acid was different, but was involved in the recognition of the +2 and +3 positions of the substrate. The other p38 family members show a relatively higher sequence variance in the binding pocket. However, substrate specificity analyses have shown similar phosphorylating potentials between the p38 family (Kumar etal, 1997,- Goedert etal, 1997), indicating that these residues may represent conserved changes with respect to overall phosphotransferase potential. The p38 family members do show differences in these 144 L U O J o Q_ ro C M X 1 — 1 o 0 0 C M O Lu CM i« i <_> 1—1 ro o H H 1—1 O > C M o CM o > r->. O J O I O - > CM C_) o Q_ I— L O C Q I D i^l g o o ^ = oo co. Co O ZD L U Q _ on oi O O 0 3 O O >- o i >-_ i < _j u u u o o o o Q i CrT Q i _ Q- CL . CL. O \ \ ^ C£ oo oo oo Q-Q_ <C Q_ Q-o a o oo 0 0 U 0 U 0 > -I—I I—I I—I I X I I u a a a oo Q a Q Q oo oo oo c t : S 5 >• i Q L U 0 0 —I 0 0 0 0 r - i CM LTl - _ i j £ A : ^ v - a - a - a i -U U U U I D ZD < < < < O L3 O U I— < i -f I— 0 0 O O 0 3 0 3 0 0 O O 0 3 0 3 I E I— CL . O . DZ) |— Q-oo oo oo oo oo ro ro ro Q . Q . Q . Q . 0 3 oo ro + O J H H O ' Q . oo CM + in (0° I IS o Q _ x C M in o C M 1—1 CM 1—1 1—1 o > C M 0 0 T-H 1—1 |-» 1—1 > oo Q i O O C O 0O ° ^ ZD fc! O i5 t i <=> 2 oo co to O ZD L U CL. oo Q i 03 oo oo o Q i >- !=! Q i < - J DZ Q_ 0 3 0 3 0 3 L U L U L U O O O I— O ZC 0 3 0 3 0 3 > -Q i o i oi —I < < < 0 3 > > > >-L U L U L U O > - > - > - > -o o o o Qi Qi Qi Qi CL . CL . Q. Q-Qi Qi Qi Qi _1 _ l _ l Qi CM L O _ | 32 -a -a >_ O J O J L U oo oo oo oo < < < < o o o o Qi Qi Qi Qi a . a_ a_ Q_ o i o i o i Qi Q i Q i Q i Q i C 2 ?- to oo oo oo oo ro ro ro oo a . Q. Q. Q. 0 0 o Q i Q_ Q ; Q_ 1 45 residues in comparison to other proline-directed kinases, indicating a unique substrate pool for the p38 kinase family. Some of the most important revelations regarding MIPK have resulted from time course studies of MIPK tyrosine phosphorylation in response to various stimuli. 1-Methyladenine time courses uncovered the first indications that MIPK regulation was very distinct from that of the seastar MAP kinase, p44 r a p k. With the high sequence homology within the p38 family, it followed that MIPK might be activated in response to stress stimuli. The most obvious stimulus to test was response to high osmolarity media. MIPK was dramatically activated by osmotic shock, as assessed by phosphotyrosine levels. Within 30 sec of treatment, maximal total tyrosine phosphorylation was reached. With time, however, the MIPK protein levels decreased significantly, yet the relative phosphotyrosine levels in the cytosol remained constant, indicating a potential role for MIPK activity in the response to high osmolarity. Responses of MIPK to heat shock were also tested. While not as dramatic as the osmotic shock activation, MIPK did become tyrosine phosphorylated in response to heat. These results confirm MIPK as a stress-activated protein kinase. It was also interesting to assess the potential role of MIPK in embryonic development. Preliminary results showed a lack of involvement of MIPK in the early post-fertilization events. However, approximately at 12 h post-fertilization, MIPK appeared to become activated based on the tyrosine phosphorylation state. This time point correlated with the transition point in embryonic development between synchronous cell division, and differential cleavages. At this time point, the overall rate of cell division decreases (Figure 23). This may indicate a role for MIPK in the regulation of cell cycle blocks, such as that present in the immature oocyte and those occurring post-fertilization. Cellular responses to stress could also include a departure from the 146 cell cycle while the cell repairs any damages. MIPK may potentially be acting as a cytostatic factor, involved in a cell's progression through the cell cycle. 4.2 Future directions. Further characterization of MIPK has been attempted, yet limited conclusive results have arisen. Preliminary experiments involving microinjection techniques were performed to assess the role of MIPK in the oocyte system. A kinase-dead mutant of MIPK was used as a dominant-negative, while antisense oligonucleotides were used to block MIPK protein synthesis. The results for both sets of experiments were inconclusive and will require further investigation. Neither of the methods of reducing MIPK phosphotransferase activity was sufficient to induce maturation of the oocyte, in the absence of 1-methyladenine. It may be useful to find more sensitive markers of the maturation process, such as through staining of cytoskeletal proteins, to observe subtle changes in the oocyte's response to hormonal stimulation. The loss of MIPK activity may not, on its own, be sufficient to induce maturation. It may be necessary to combine microinjection of the kinase-dead mutant with that of a maturation activating factor, such as active Mekl. The combination of the two factors may cause a dramatic effect on the regulation of the cell cycle block, and allow the release from G 2 in the absence of 1-methyladenine. Fertilization studies of MIPK by microinjection may be of key importance in understanding the role of MIPK in the regulation of cellular processes. The time course studies indicated the activation of MIPK around the transition to differential cell divisions, which is coupled to an overall decrease in the rate of cell divisions. By interfering with the activation of MIPK during embryonic development, it would be expected that the embryo would not reach the blastula stage, where clear differentiation has occurred. 147 The dividing embryo would be expected to take on a more homogeneous appearance, and the rate of cell division expected to be unregulated. The preliminary experiments, although promising, will have to be repeated, with more specific controls, in order to assess the affects of MIPK inactivation of the development of the seastar embryo. Difficulties were faced in assessing the kinase activity of MIPK. As purified from the immature oocyte, MIPK phosphotransferase activity could not be detected. These problems were also a factor in tests of the bacterially expressed fusion-protein, GST-MIPK. Clearly these techniques are insufficient for the measurement of substrate preference. To obtain an active preparation of MIPK, it may be necessary to express the recombinant protein in a different cell system, such as transient transfection systems or baculovirus expression systems. These systems would be sensitive to stimulation by a variety of factors, including heat shock, allowing purification of an active kinase. This active preparation could then be used for measurements of in vitro kinase activity, and therefore substrate specificity. These systems would also allow a prediction of the activation potential of MIPK in response to a variety of stimuli. A second method that could be exploited in the characterization of MIPK is 2-hybrid screening in yeast. This method has proven very powerful not only in the identification of potential downstream targets, but also in capturing potential upstream activators. This information is essential in defining the MIPK signalling cascade. Identification of substrates would allow true measurements of MIPK phosphotransferase activity. MAP kinases are known to require threonine phosphorylation as well as the tyrosine phosphorylation. Phosphorylation at negative regulatory sites may also play a role in the regulation of MIPK. Based on this, measurement of phosphotyrosine levels may not be a true determinant of activation. Observations of phosphorylation on 148 tyrosine, while being a marker of potential activation, do not ensure that the kinase is in its active state. The use of GST-MIPK as an affinity matrix also shows potential for allowing identification of upstream activating kinases. Preliminary data confirmed that GST-MIPK does have the capacity to bind kinase activity from the seastar cytosolic extracts. Larger resin beds could therefore be used to concentrate larger quantities of these kinase activities, and allow further identification and characterization to occur. Active preparations of the fusion protein may also be valuable as an affinity matrix for downstream substrates. The ability to radiolabel the bound proteins will allow specific recognition of the potential substrates. This may act as a partial purification step, potentially sufficient to obtain micropeptide sequencing data. The ultimate goal of this research is the identification of a mammalian counterpart of MIPK. Using the antibody probe available, Cdk5 - C T , to detect potential MIPK-like proteins, rat tissues were screened for immunoreactive proteins (Figure 31, Panel A). A major 40 kDa immunoreactive protein, was detected mainly in the rat heart, but it was also visible in brain and very slightly in kidney, liver and lung. C d k 5 - C T failed to immunoprecipitate this protein, and as yet no evidence of tyrosine phosphorylation has been noted. Further analysis of the developing heart revealed that this 40 kDa protein was present only in the non-dividing adult tissue, not in the embryonic or neonatal heart (Figure 31, Panel B). More specific MIPK probes, including the MIPK-CT antibody, will be useful in confirming the relation of this 40 kDa protein to MIPK. To confirm that C d k 5 - C T was not simply crossreacting with the known p38 homologues, the rat tissues were also screened for proteins immunoreactive with p38-CT (Figure 31, Panel C). p38 - C T crossreacted with a ubiquitously expressed 38 kDa protein in the rat tissues. This immunoreactivity was so strong, relative to the p 3 8 149 tu o tu -d tu Jr 3 O « t: . ! - tu a rs P I — I— ^ OO cn *d c <u * 2 CU rt O C co rt tu Bc_c O . 3 rt tu o "d fc c ro O rt o rt O •43 X C +3 tU c ah tu "d -d CJ £ | u > CU c rt Cu Cu ro cu LU C flj tu 3 D. co O S£ tu ~ £ g ^ tu -d rt •— co •d >, .£ 'rt CO g rt C +- > •- o JJ a e °-o ^ rt U o "*3 _ c C ro O CL cu X. +-> >, -d 3 g 1 a • - tu <£ JC C M _ a " tu U 0 rt ~ tu c 5 Z c 2 3 jC E rd .5 E DD P E OX X3 O >> -d tu c tu • i-u CQ rt Cu " cu C/l X tu CO J C J '•d ~d •4-> tu S- •(-> rt re tu u CO OC — C C a .2 CO v> tu "d CO re re c -d -* rt s l -E-o 1 1 O re "•g & E E tu c JC tu +J f— c rt cu co jd .S o -d a i re 3 co c re re [—i bo U -E > & o o §tu --d E CO ro 150 isoform found in seastar, that no p38 protein could be detected in the seastar cytosolic extract. Relative intensities indicated that p38 was not highly expressed in the heart tissue, the major source of Cdk5-CT reactive protein. Based on this data, there is a strong probability that a MIPK counterpart, unique from the other p38 family members, exists in the mammalian systems. This protein shows the potential for being specifically expressed in non-dividing tissues. The low degree of homology in the N- and C-termini of the p38 kinases can be exploited when searching for a mammalian counterpart to seastar MIPK. By using oligonucleotide primers based on the 5' and 3' ends of the sequence, along with more generic primers from internal sequences, it may be possible to use PCR to amplify a MIPK clone from a cDNA source. Northern blot analyses will be useful in determining tissue distribution of a mammalian MIPK,- however, the specificity of the probe to be used is in question. There must be allowances made for degeneracy and species diversity, while ensuring that the probe will not crossreact with other p38 family members. The EST data base may be searched for sequences matching the terminal amino acids of MIPK. The high degree of conservation observed for the p38 family should lend to the discovery of mammalian homologues, despite the degree of separation of the species Based on the available Western blotting data, work is underway to screen a human adult heart cDNA library with a portion of the MIPK coding region, in an attempt to identify a human MIPK sequence. Again, it is possible that the probes being used will not be specific for the MIPK homologue, and may therefore identify other p38 family members. This should not be a problem, as thorough, higher stringency second screenings should eliminate some of the non-specific hybridizations. The combination of 151 techniques available should allow the identification and cloning of a mammalian counterpart of seastar MIPK. 4.3 Conclusions. A maturation-inhibited protein kinase, MIPK, has been identified in the P. ochraceus oocyte system. This enzyme was found to be closely related to the p38 family of MAP kinases, yet it did not appear to be the seastar homologue of any of the known p38 kinases. MIPK may therefore define a new p38 kinase isoform. MIPK was found to be tyrosine phosphorylated in the immature oocyte, blocked in G2 phase of meoisis I. Stimulation with the natural hormone 1-methyladenine resulted in the dephosphorylation and presumably inactivation of MIPK. MIPK was also tyrosine phosphorylated and potentially activated in cells responding to high osmolarity and heat shock, indicating that MIPK can act as a stress-activated protein kinase. MIPK regulation was observed in developing seastar embryos, with tyrosine phosphorylation evident at a time point correlating with the conversion from synchronous cell divisions to differential regulated cell cycles. The MIPK activity may be associated with the overall decrease in the rate of cell divisions. MIPK may represent a novel p38 kinase, involved as a cytostatic factor in the seastar oocyte system. Future experiments will involve the identification of upstream kinases and downstream targets in the MIPK signalling cascade. The ultimate goal of this research is the identification and cloning of a mammalian homologue to MIPK. Preliminary Western blot analyses indicate the presence of a 40 kDa immunoreactive protein which is highly expressed in non-dividing tissues such as heart and brain. MIPK is a potentially important target for therapeutic treatments of degenerative diseases related to the heart and nervous system. Reduction of the MIPK protein kinase activity 152 may provide a mechanism for inducing cell division in normally non-dividing tissues. 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Science, 270:2008-2011. Zhou, G., QinBao, Z., and Dixon, J.E. (1995) Components of a new human protein kinase signal transduction pathway. J. Biol. Chem., 270:12665-12669. Zhou, Z., Gartner, A., Cade, R., Ammerer, G., and Errede, B. (1993) Pheromone-induced signal transduction in Saccbaromyces cerevisiae requires the sequential function of three protein kinases. Mol. Cell. Biol., 13:1069-1080. Zhu, A.X., Zhao, Y., Moller, D.E., and Flier, J.S. (1994) Cloning and characterization of p 9 7 m a p k , a novel human homolog of rat ERK.3. Mol. Cell. Biol., 14:8202-8211. 166 APPENDIX I DESCRIPTION OF ANTIBODIES anti-Cdc2 Kinase-C-Terminus (Cdc2-CT) Immunogen - 36 residue synthetic peptide C-FLSKMLVYDPAKRISGKMALKHPYFDDLDNQIKKM Based upon the C-terminal residues 263-297 of the mouse 34 kDa cdc2-encoded protein kinase (Cisek & Corden, 1989). Recognizes the 34 kDa protein encoded by the c d c 2 gene in various mammalian species. In rat tissues it cross reacts with 46 and 70 kDa proteins. Recognizes p 3 4 c d c 2 from mouse, rat, sheep and human. anti-Cdc2 Kinase-IX (Cdc2-IX) Immunogen - 25 residue synthetic peptide PLFHGDSEIDQLFRIFRALGTP-GGC Based upon residues 202-223 of the mouse 34 kDa ctic2-encoded protein kinase (Cisek & Corden, 1989) in catalytic subdomain IX region (Hanks et al, 1988). Recognizes the 34 kDa protein encoded by the cdc2 gene in diverse species. Recognizes p 3 4 c d c 2 from sea star, mouse, rat, sheep and human. anti-Cyclin-Dependent Kinase 5-C-Terminus (Cdk5-CT) Immunogen-16 residue synthetic peptide CNPVQRISAEEALQHP Based upon the C-terminal residues 268-283 of the human 31 kDa C D K 5 - (or PSSALRE kinase) encoded protein kinase (Hanks, 1987,- Meyerson et al, 1992). Recognizes the 32 167 kDa protein purified from bovine brain (Lew et al, 1992), which corresponds to cyclin-dependent protein kinase 5. Recognizes CDK.5 from cow and human. anti-Erkl-C-Terminus (Erkl-CT) Immunogen-38 residue peptide CGGPFTFDMELDDLPKERLKELIFQETARFQPGAPEAP Based upon residues 333-367 of the rat 43 kDa Erkl MAP kinase (Boulton et al, 1990), which corresponds to the C-terminus of the protein. Recognizes the 43 kDa MAP kinase encoded by the Erkl gene, 42 kDa MAP kinase encoded by the Mapk (Erk2) gene and the 44 kDa MAP kinase encoded by the seastar Mpk gene. It also appears to recognize other putative MAP kinases in the range of 40 to 70 kDa. Recognizes the various MAP kinases in cytosolic and nuclear extracts from sea star, clam, frog, chicken, mouse, rat, sheep and human. anti-Fus3-C-terminus (Fus3-CT) Immunogen-25 residue synthetic peptide CGG-DHHKEALTTKDLKKLIWNEIFS Based upon residues 332-353 of the Saccbaromyces cerevisiae 40 kDa Fus3 MAP kinase (Elion eta!., 1990), which corresponds to the C-terminus of the protein. Recognizes the 40 kDa MAP kinase encoded by the recombinant Fus3 gene expressed in E. coli. It does not cross-react with sea star MAP kinase (Mpk), mammalian p42mapk (Erk2) nor with 168 anti-p38 High Osmolarity Glycerol Kinase-C-Terminus (Hog-CT) Immunogen-19 residue synthetic peptide C D E V I S F V P P P L D Q E E M E S Corresponds to the C-terminal amino acids of human p38 Hog. Should not cross-react with Erk l , Erk2, Erk3, Erk5 or Sapk. Is known to crossreacts with p38 from rat and human. anti-p38 High Osmolarity Glycerol Kinase-N-Terminus (Hog-NT) Immunogen-14 residue synthetic peptide M S Q E R P T F Y R Q E L C Based upon the N-terminal residues 1-13 of the murine p38 Hog (Han et al., 1994). Western blots recombinant rat Hog expressed in E. coli. Recognizes a single 38 kDa band in various rat tissues including brain, liver, spleen, thymus and lung. Recognition of mouse, rat and other mammalian p38 Hog is expected. anti-Cdc2 Kinase-Subdomain III-PSTAIRE region (PSTAIRE) Immunogen-19 residue synthetic peptide EGVPSTAIREISLLKE-GGC Based upon residues 42-57 of the human 34 kDa Cdc2-encoded protein kinase (Lee et al., 1987) in kinase subdomain III region according to the classification of Hanks et al. (1988). With putative protein kinases of -32 kDa and - 58 kDa, which may be encoded by cdc2-like genes. Recognizes p 3 4 c d c 2 from fission yeast, Paramecium, Dictyostelium, sea star, frog, chicken, mouse, rat, sheep and human 169 anti-Maturation Inhibited Protein Kinase-C-Terminus (MIPK-CT) Immunogen-19 residue synthetic peptide CG-KELTFQLIQAVRHQSSR Based upon residues 347-363 of seastar p40 MIPK. Recognizes recombinant expressed MIPK and purified seastar MIPK. anti-Ste7-subdomain VIII (Ste7-VIII) Immunogen-14 residue synthetic peptide FVGTSTYMSPERIC Based upon residues 434-446 of Saccharomyces cerevisiae Ste7 (Teague et al., 1986) in kinase subdomain VIII region according to the classification of Hanks etal. (1988). This antibody reacts with recombinant S. cerevisiae S t e 7 expressed in E. coli purified 46 kDa MAP kinase kinase from skeletal muscle. anti-Cyclin Dependent Kinase 5-C-Terminus (anti-Cdk5-CT-SC) (Santa Cruz-C8) Immunogen-8 residue synthetic peptide YFSDFCPP Based upon last 8 amino acids of the human C d k 5 sequence (Meyerson et al., 1992). anti-Cyclin Dependent Kinase 5-monoclonal (Cdk5-monoclonal) (Santa Cruz-DC 17) Immunogen-human recombinant Cdk5 protein Recognizes human Cdk5 by immunoprecipitation, Western blotting and cell staining. 170 APPENDIX II DESCRIPTION OF OLIGONUCLEOTIDES S1A P 5' - TT(T/C) CCI GGI TA(T/C) ATI GAA - 3' S l B P 5' - TT(T/C) CCI GGI TA(T/C) ATI GAG - 3' Degenerate sense oligonucleotides based on the MIPK peptide sequence FPGYIE in peptide 1 sequenced from the purified protein. 18 bp. S2A P 5' - GA(T/C) GA(A/G) CA(T/C) GTI CA(A/G) TTT - 3' S2B P 5' - GA(T/C) GA(A/G) CA(T/C) GTI CA(A/G) TTC - 3' Degenerate sense oligonucleotides based on the MIPK peptide sequence DEHVQF in peptide 2 sequenced from the purified protein. 18 bp. S2C P 5' - AA(T/C) TGI AQA/G) TG(T/C) TC(A/G) TC - 3' Degenerate antisense oligonucleotide based on the MIPK peptide sequence DEHVQF in peptide 2 sequenced from the purified protein. 17 bp. S 4 A P 5' - CCI GTI CA(A/G) TA(T/C) CA(A/G) AAA - 3' S4B P 5' - CCI GTI CA(A/G) TA(T/Q CA(A/G) AAG - 3' Degenerate sense oligonucleotides based on the MIPK peptide sequence PVQYQK in peptide 4 sequenced from the purified protein. 18 bp. 171 S5A P 5' -• TTI GCI AQA/G) TAI GGA TG - 3' S5B P 5' - TTI GCI AC(A/G) TAI GGG TG - 3' Degenerate antisense oligonucleotides based on the MIPK peptide sequence HPYVAK in peptide 5 sequenced from the purified protein. 17 bp. S 4 C P 5' - AAA CTC TCC GCA GTG GGA GC - 3' Based on MIPK sequence KLSAVGA, aa 35-41. 20 bp. Sense primer for 3' RACE reactions. S 5 D P 5' - GAT GAC TCA GTG CCT CTT CAG - 3' Based on MIPK sequence AEEALSHP, aa 308-315. 21 bp. Antisense primer for 5' RACE reactions. ATGE P 5' - CCG AAT TCC ATG AAC AAC CCA GTA ACA GG - 3' Based on MIPK sequence MNNPVTG, aa 1-7. 29 bp. Sense primer for amplification of MIPK coding region for cloning into pGEX-4T expression vector. CCGAATTCC EcoRI cloning site. STOPS P 5' - AGC GTC GAC TAT TTA CCT TCT GCT T T G ATG TC - 3' Based on MIPK sequence RHQSSR*, aa 358-363. 32 bp. Antisense primer for amplification of MIPK coding region for cloning into pGEX-4T expression vector. AGCGTCGACTAT Sail cloning site. 172 S 4 G P 5' - AAG C T T TCT CGA CCA TTT CAG - 3' Based on MIPK sequence KLSRPFQ, aa 63-69. 22 bp. Sense primer for site directed mutagenesis (K62R) of MIPK. This oligonucleotide was used as mutagenesis primer 3 with primer 4 (STOPS), to create a PCR fragment to ligate with the ATGE - KR fragment. KR P 5' - G C G GAT AGC AAT CTT TAT G C C AG - 3' Based on MIPK sequence TGIKIAIK, aa 55-62. 24 bp. Antisense primer for site directed mutagenesis (K62R) of MIPK. The lysine codon A A G (nt 184-186) is changed to CGC (arginine). This oligonucleotide was used as mutagenesis primer 1 with primer 2 (ATGE) to amplify a PCR fragment to ligate with the S 4 G -STOPS fragment. Antisense 1 5' - T C C TGT TAC TGG GTT GTT CAT - 3' Based on MIPK sequence MNNPVTG, aa. 1-7. 21 bp. Antisense primer used for microinjection experiments. Antisense2 5' - CTG AAC CGG CAC C T C CCA - 3' Based on MIPK sequence WEVPVQ, aa. 26-32. 18 bp. Antisense primer used for microinjection experiments. Sense 1 5' - ATG AAC AAC CCA GTA ACA GGA - 3' Based on MIPK sequence MNNPVTG, aa. 1-7. 21 bp. Sense primer used as negative control for microinjection experiments. 173 M i s s e n s e 2 5' - T C C AGC CAC GAC GCA CTC - 3' Based on MIPK sequence WEVPVQ, aa. 26-32. 18 bp. Missense primer used as negative control for microinjection experiments. S2D P 5' - CAA GTA CAT TCA TTC AGT TGG T G - 3' Based on MIPK sequence LKYIHSVGV, aa 147-153. 23 bp. Sense primer for 3' RACE reactions and sequencing. S2E P 5' - AGA TTT TGG TCT TGC T C G TCA AG - 3' Based on MIPK sequence LDFGLARQA, aa 176-184. 23 bp. Sense primer for 3' RACE reactions and sequencing. S2F P 5' - ACC TGT CAT CTC ATC ATC AGC TTG - 3' Based on MIPK sequence QADDEMTG, aa 183-190. 24 bp. Antisense primer for 5' RACE reactions and sequencing. S2G 5' - ACG CTA TTT CCT GGA TCG GAT CAC - 3' Based on MIPK sequence TLFPGSDHI, aa. 230-238. 24 bp. Sense primer for sequencing. S2B' 5' - TCT CTG ATG AAC ATG TGC AGT TCC - 3' Based on MIPK sequence LSDEHVQFL, aa. 131-139. 24 bp. Sense primer for sequencing. 174 S4D P 5' - GAT CCG TGT GCT CAT CCT TAA AC - 3' Based on MIPK sequence GSVCSSLN, aa 45-52. 23 bp. Sense primer for 3' RACE reactions and sequencing. S4E P 5' - CAT GTA TAC GTC CCG AAA GCT T G - 3 Based on MIPK sequence SSFRDVYM, aa 106-1 13. 23 bp. Antisense primer for 5' RACE and sequencing. S4F P 5' - GGT CGA GAA AGC TTC TTG ATA GC - 3 Based on MIPK sequence AIKKLSRP, aa 60-67. 23 bp. Antisense primer for sequencing. S4H 5' - ATG CTG TAG AAG T C G AAG CTC T C G -Based on MIPK sequence RELRLLQH, aa. 79-86. 24 bp. Antisense primer for sequencing. S5E P 5' - CTC CAG AAG GTC AAC AGC AAT TTC -Based on MIPK sequence EIAVDLLE, aa 288-295. 24 bp. Antisense primer for 5' RACE and sequencing. S5F P 5' - CGA AAT TGC TGT TGA CCT TCT GG - 3 Based on MIPK sequence NEIAVDLLE, aa 287-295. 23 bp. Sense primer for sequencing. APPENDIX III C L O N I N G A N D S E Q U E N C I N G OF A SHORTER MIPK TRANSCRIPT During the cloning of MIPK, a lower band was observed in the PCR reactions. To further characterize this band, a panel of internal primers were used in a series of PCR reactions (Figure 32). Four out of six pairs of primers resulted in the amplification of two bands by PCR, with a size difference of approximately 200 bp. The primer combinations which did not give two products covered the 3' half of the gene, and a small 5' fragment, and were separated by a 189 bp region in which the deletion must lie. To assess whether a second gene was involved in generating this shorter product, it was necessary to clone and sequence the cDNA. To specifically amplify the lower band, an initial PCR reaction was electrophoresed on an agarose gel (Figure 33, panel A). The leading edge of the PCR product was carefully cut away from the main product. This was used as a template for a second round of PCR and the product checked for specificity (Figure 33, panel B). This product was clearly less than 1 kb in size, indicating that the lower band had been specifically amplified. This band was cloned into pGEX-4T3 and sequenced as shown in Figure 34. The entire sequence of the lower band was identical to the MIPK sequence. A single 168 bp sequence from residue 275 to 444 was deleted in the lower band sequence. This indicated that the lower band was a product of the same gene as MIPK, and was due to an alternative splice or is an artifact of the cDNA synthesis. To assess whether the lower band clone was able to produce a protein product, the pGEX-4T3 vector was used to express the protein in E. coli. When expressed under equivalent conditions as the MIPK clone, the lower band was found to express an intact protein of expected size (Figure 35). This protein was expressed in similar amounts as 176 4C 4D 4E 2D 2E 2F 5E 5D MIPK Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 4C-5E 4C-2F 4C-4E 4C-5D 4D-5D 2E-5D 770 bp 467 235 839 809 415 1 2 3 4 5 6 B y w W . < ( w m • — i . i * iwKft i mm -Figure 32. Identification of a shorter transcript of MIPK by PCR amplification. Several oligonucleotides were designed to be used in PCR reactions. These primers, if working properly, give a range of PCR products from 235 bp to 839 bp (panel A). These primers were tested internally on MIPK cDNA, two bands were found to be amplified in some, but not all, of the reactions. Panel B is an ethidium bromide stained agarose gel of the PCR products. Lane 1, S4C-S5E,- lane 2, S4C-S2F,- lane 3, S4C-S4E; lane 4, S4C-S5D,-lane 5, S4D-S5D,- lane 6, S2E-S5D. Standards are shown on either side of the gel. 177 Figure 33. Specific PCR amplification of a shorter transcript of MIPK. MIPK was amplified under the standard conditions, and the reaction products separated on an agarose gel (panel A). The shorter product was carefully separated from the major band and subjected to a second round of PCR (panel B). The standards are shown in the first lane of both panels. 178 Panel A ATGAACAACCCAGTAACAGGATCAGGAGAAACGTTATCTGATGACGGGTATCATCGATATGAACTGAATAAA 72 ATGAACAACCCAGTAACAGGATCAGGAGAAACGTTATCTGATGACGGGTATCATCGATATGAACTGAATAAA ACTACATGGGAGGTGCCGGTTCAGTACCAAAAACTCTCCGCAGTGGGAGCTGGTGCATATGGATCCGTGTGC 144 ACTACATGGGAGGTGCCGGTTCAGTACCAAAAACTCTCCGCAGTGGGAGCTGGTGCATATGGATCCGTGTGC TCATCCTTAAACACAAAAACTGGCATAAAGATTGCTATCAAGAAGCTTTCTCGACCATTTCAGTCTGCGATT 216 TCATCCTTAAACACAAAAACTGGCATAAAGATTGCTATCAAGAAGCTTTCTCGACCATTTCAGTCTGCGATT CATGCCAAGAGAACGTACCGAGAGCTTCGACTTCTACAGCATATGGATCATGAAAACATCATCAGTCTACTA 288 CATGCCAAGAGAACGTACCGAGAGCTTCGACTTCTACAGCATATGGATCATGAAAAC GATGTGTTTTGTAGAGGAGATACCTTATCAAGCTTTCGGGACGTATACATGGTGACACATTTGATGGGTGCA 360 GATCTGAATAGTATTACAAAAACACAGAAACTCTCTGATGAACATGTGCAGTTCCTTGTGTATCAAATACTT 432 CGTGGGCTCAAGTACATTCATTCAGTTGGTGTAATCCATCGTGATCTGAAGCCCAGTAACTTGGCTGTGAAT 504 TACATTCATTCAGTTGGTGTAATCCATCGTGATCTGAAGCCCAGTAACTTGGCTGTGAAT GAAGACTGCGAATTGAGGATACTAGATTTTGGTCTTGCTCGTCAAGCTGATGATGAGATGACAGGTTACGTA 57 6 GAAGACTGCGAATTGAGGATACTAGATTTTGGTCTTGCTCGTCAAGCTGATGATGAGATGACAGGTTACGTA GCTACACGATGGTATAGAGCACCAGAAATCATGCTGAATTGGATGCATTACACCAATACTGTGGATATGTGG 648 GCTACACGATGGTATAGAGCACCAGAAATCATGCTGAATTGGATGCATTACACCAATACTGTGGATATGTGG TCTGTTGGATGTATAATGGCAGAACTTCTCACAGGTAAAACGCTATTTCCTGGATCGGATCACATTGATCAG 720 TCTGTTGGATGTATAATGGCAGAACTTCTCACAGGTAAAACGCTATTTCCTGGATCGGATCACATTGATCAG TTGAGTCGCATCATGGATCTAACTGGTACACCTGATGATGAAATCCTTGCCAAAATCCAGAGTGAAGATGCA 792 TTGAGTCGCATCATGGATCTAACTGGTACACCTGATGATGAAATCCTTGCCAAAATCCAGAGTGAAGATGCA CGGAACTTTGTTAAATCTCAACCTAAAACTAAGAAAAAAGATTTTCGTGGATATTTTGCTGGAGCAAACGAA 864 CGGAACTTTGTTAAATCTCAACCTAAAACTAAGAAAAAAGATTTTCGTGGATATTTTGCTGGAGCAAACGAA ATTGCTGTTGACCTTCTGGAGAAAATGCTTCTGTTGGATGTAGACAAGCGTATCACTGCTGAAGAGGCACTG 936 ATTGCTGTTGACCTTCTGGAGAAAATGCTTCTGTTGGATGTAGACAAGCGTATCACTGCTGAAGAGGCACTG AGTCATCCTTATGTTGCCAAATATCATGATGAAAGTGATGAGCCTATTGGTAAGCAGTTTGATGATTCCTTT 1008 AGTCATCCTTATGTTGCCAAATATCATGATGAAAGTGATGAGCCTATTGGTAAGCAGTTTGATGATTCCTTT GAACAGCAAGACTTGACTGTGCAGCAGTGGAAAGAGCTTACTTTTCAGCTGATTCAAGCAGTAAGACATCAA 1080 GAACAGCAAGACTTGACTGTGCAGCAGTGGAAAGAGCTTACTTTTCAGCTGATTCAAGCAGTAAGACATCAA AGCAGAAGGTAA 1089 AGCAGAAGGTAA 179 PanelB I MNNPVTGSGETLSDDGYHRYELNKTTWEVPVQYQKLSAVGAGAYGSVCSSLNTKT 5 5 MNNPVTG S GETL S DDG YHR YELNKTTWEVPVQ YQKL S AVGAGAYGSVC S S LNTKT 5 5 n m iv GIKIAIKKLSRPFQSAIHAKRTYRELRLLQHMDHENIISLLDVFCRGDTLSSFRD 110 GIKIAIKKLSRPFQSAIHAKRTYRELRLLQHMDHEN 91 V VI VYMVTHLMGADLNSITKTQKLSDEHVQFLVYQILRGLKYIHSVGVIHRDLKPSNL 165 YIHSVGVIHRDLKPSNL 108 vn vm ix AVNEDCELRILDFGLARQADDEMTGYVATRWYRAPEIMLNWMHYTNTVDMWSVGC 22 0 AVNEDCELRILDFGLARQADDEMTGYVATRWYRAPEIMLM#1HYTNTVDMWSVGC 163 X IMAELLTGKTLFPGSDHIDQLSRIMDLTGTPDDEILAKIQSEDARNFVKSQPKTK 275 IMAELLTGKTLFPGSDHIDQLSRIMDLTGTPDDEILAKIQSEDARNFVKSQPKTK 218 XI KKDFRGYFAGANEIAVDLLEKMLLLDVDKRITAEEALSHPYVAKYHDESDEPIGK 3 3 0 KKDFRGYFAGANEIAVDLLEKMLLLDVDKRITAEEALSHPYVAKYHDESDEPIGK 273 QFDDSFEQQDLTVQQWKELTFQLIQAVRHQSRR* 3 63 QFDDSFEQQDLTVQQWKELTFQLIQAVRHQSRR* 3 03 Figure 34. Sequence alignment of MIPK with the shorter transcript of MIPK. Panel A. The nucleotide sequences of MIPK and the shorter transcript of MIPK were aligned manually. The nucleotides sequenced are numbered for MIPK, starting at" 1" for the first residue of the start codon. Panel B. The predicted amino acid sequences for MIPK and the shorter transcript of MIPK were aligned manually. The Roman numerals indicate the kinase subdomains, while amino acids which are conserved kinases are shown in bold. 180 MIPK MIPK-LB MIPK MIPK-LB Figure 35. Expression of the shorter transcript of MIPK in E. coli. GST-MIPK and GST-MIPK-lower band were expressed in DH5(X cells and levels assessed by Western-blotting (Lanes 1-4) or by Coomassie-staining (Lanes 5-8). Lanes 1,3,5,7 show the soluble fractions "S" of the lysates,- Lanes 2,4,6,8 show the insoluble pellets "P" from the lysates. 181 GST-MIPK, with a similar solubility profile. This protein could be recognized by the Cdk5-CT antibody, confirming that the entire protein was being synthesized. It is therefore clear that this lower band clone is an in frame, 168 bp deletion in the MIPK gene which is able to produce a protein 59 amino acids shorter than MIPK. Closer analysis of the region missing in the lower band sequence shows that the deletion is in subdomain IV and V of the kinase domain. This region is important in "substrate recognition" and is likely to be essential for kinase activity. Also deleted in this clone is the region containing peptide S2 from the purified kinase. This was conclusive evidence that the clone was not the MIPK protein being followed in the purification. To determine whether or not it is the result of a cloning artifact, the PCR amplification will have to be repeated with a different preparation of RNA. Western blots from ResourceQ fractionated oocyte extract were also analyzed for the presence of a minor lower band immunoreacting with Cdk5-CT (data not shown). The protein would be approximately 7 kDa smaller than MIPK so would clearly resolve by SDS-PAGE. No protein was identified by Western blotting of the estimated 33 kDa. It was therefore concluded that this clone is likely to be an artifact created during cDNA synthesis. 182 

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