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Investigation of CDC2-independent mitotic protein kinases Gowdy, Patrick Michael 1997

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I N V E S T I G A T I O N O F C D C 2 - I N D E P E N D E N T M I T O T I C P R O T E I N K I N A S E S by P A T R I C K M I C H A E L G O W D Y B . S c , Queen's University, 1994 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F Master of Science in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Department of Biochemistry and Molecular Biology We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A January 1997 © Patrick Michael Gowdy, 1997 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 TjQ<jr\&\A\STP-i AA>£> rAOi£.c\lLryL KlnLOt-,^ The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Mi tos i s is a h ighly coordinated event wh i ch ensures the equal segregation o f the cytoplasmic and genetic material into two daughter cells. A key regulatory process in ensuring the temporal f idel i ty of mitosis involves reversible protein phosphorylat ion mediated by protein kinases and phosphatases. The major k n o w n protein kinase required for tr iggering mitosis is the cyc l in-dependent kinase, C d c 2 . Recent evidence suggests that Cdc2-independent protein kinase s ignal ing pathways are also invo lved i n mitosis. The murine mammary tumour ce l l l ine , F T 2 1 0 , has a temperature-sensitive mutation i n the cdc2 gene that causes ce l l cyc le arrest i n late G 2 at the non-permissive temperature. Th is arrest can be overcome, and mitot ic events induced, by protein phosphatase inhibitors. Th is experimental system al lows dist inct ion between Cdc2-independent and -dependent pathways at mitosis. Th is a l lows for identif icat ion and investigation of the roles of protein kinases in Cdc2-independent mitot ic pathways. I have used an in-gel protein kinase renaturation assay to identify two kinases that are activated independently o f Cdc2 at mitosis: a novel 45 k D a mitot ic protein kinase and a ce l l cycle-regulated novel 60 k D a histone-specific mitotic kinase. The 45 k D a protein kinase, p45 , was active only at mitosis. p45 kinase activity was detected in a l l ce l l l ines tested suggesting an essential role in ce l l d iv is ion. The p45 kinase was determined to be unrelated to the known 46 k D a kinases, J N K 1 and M A P K A P kinase-2. The histone kinase was also active only at mitosis. The identif icat ion of this histone-specific mitot ic kinase conf irms the existence of Cdc2-independent pathways for chromosome condensat ion at mitosis. i i I also investigated the regulation of the mitotic kinase, polo-like kinase-1 (P lk l ) , in C d c 2 mutant cells. P l k l was inactive at the non-permissive temperature and therefore requires Cdc2-dependent signaling pathways for activity. P l k l is involved in regulating chromosome segregation during mitosis. These results further strengthen the role of C d c 2 in the regulation of mitotic microtubule dynamics. iii Table of Contents Abstract i i Table of Contents iv List of Tables vi i i List of Figures ix List of Abbreviations xi Acknowledgments xii i Chapter 1 Introduction 1 1.1 Cel l cycle 1 1.1.1 The stages of mitosis 1 1.2 Protein phosphorylation in regulating mitosis 5 1.3 The ro l eo fCdc2 5 1.3.1 Regulation of Cdc2 7 1.4 The roles of other mitotic protein kinases 10 1.4.1 M P M - 2 epitope kinases 10 1.4.2 N I M A 11 1.4.3 P K C p I I 15 1.5 Serine/Threonine protein phosphatases 16 1.5.1 Physiological roles of protein phosphatases 17 1.5.2 Serine/threonine protein phosphatase inhibitors 18 1.5.3 Okadaicacid 19 iv 1.5.4 Fostr iec in 19 1.6 The F T 2 1 0 ce l l l ine 21 1.6.1 Protein phosphatase inhibitors overcome the G 2 arrest 21 at the non-permissive temperature 1.6.2 Protein kinases are required to overcome the G 2 arrest 23 mediated by phosphatase inhibitors 1.7 Research objectives 24 Chapter 2 Materials and Methods 25 2.1 C e l l culture and synchronization of F T 2 1 0 cells 25 2.2 F l o w cytometr ic analysis 26 2.3 In-gel renaturation protein kinase assays 26 2.4 J N K 1 micropur i f i cat ion and Western blott ing 27 2.5 P l k l immunoprecip i tat ion, 3 2 P- l abe l l i ng and activity assays 28 2.6 M i t o t i c E L I S A 29 2.7 Fract ionat ion and puri f icat ion o f histones 30 2.8 Partial pur i f icat ion of p45 kinase 30 2.9 Preparation o f Ets-1(29-139) 31 Chapter 3 Identification of a novel 45 kDa mitotic kinase activated 33 by protein phosphatase inhibitors 3.1 A 45 k D a mitot ic protein kinase is activated by protein 33 phosphatase inhibitors independently of C d c 2 in F T 2 1 0 cel ls 3.2 The p45 kinase is found in a l l ce l l l ines 36 v 3.3 Myel in basic protein is preferred over Ets-1(29-139) as a 38 substrate for p45 kinase 3.4 The p45 kinase is not J N K 1 38 3.5 The p45 kinase is not M A P K A P kinase-2 40 3.6 The effects of the protein kinase inhibitors staurosporine and 44 chelerythrine chloride on p45 kinase activity 3.7 Partial purification of the p45 kinase 50 Chapter 4 Identification of a histone-specific kinase activity in 52 Cdc2 mutant FT210 cells 4.1 Fractionation of histones from mixed histone samples 53 4.2 Identification of a histone kinase in FT210 cells 53 Chapter 5 Investigation of the activation of polo-like kinase-1 58 in Cdc2 mutant FT210 cells 5.1 P l k l activity and abundance increases in a cell cycle- 59 dependent manner 5.2 P l k l activation is dependent upon Cdc2 59 Chapter 6 Discussion 62 6.1 The identification of a novel mitotic 45 kDa kinase 62 6.2 Identification of a Cdc2-independent mitotic histone kinase 65 vi 6.3 P l k l activity is dependent upon Cdc2 activity: implications 68 for mitotic microtubule dynamics 6.4 Conclusions 72 Bibliography 74 vii List of Tables Table 1. The Serine/Threonine Protein Phosphatase Family 17 Table 2. Serine/Threonine Protein Phosphatase Inhibitors 19 Table 3. Inhibition of Protein Kinases by Staurosporine 23 vii i List of Figures Figure 1. The eukaryotic cell cycle 2 Figure 2. The stages of mitosis 3 Figure 3. The opposing effects of regulatory protein kinases and 6 phosphatases on target proteins Figure 4. The regulation of Cdc2 8 Figure 5. Regulation of Cdc25 phosphatase activity by the M P M - 2 12 reactive kinase P l x l Figure 6. Regulation of mitotic entry requires the activity of both Cdc2 14 and N I M A protein kinases in Aspergillus nidulans Figure 7. The structures of the protein phosphatase inhibitors 20 okadaic acid and fostriecin Figure 8. G2 arrest of FT210 cells at the non-permissive temperature 22 Figure 9. Cel l cycle synchronization of FT210 cells 34 Figure 10. A 45 kDa mitotic kinase is activated by phosphatase 35 inhibitors independently of Cdc2 Figure 11. Detection of p45 kinase activity in various cell lines 37 Figure 12 Myel in basic protein is the preferred substrate of p45 kinase 39 Figure 13. The p45 mitotic kinase is not J N K 1 41 Figure 14. Effect of anisomycin on p45 kinase activity in FT210 cells 42 at the non-permissive temperature Figure 15. Effect of anisomycin on entry into mitosis in Cdc2 mutant 43 FT210 cells at 39°C Figure 16. The effect of staurosporine on p45 kinase activity 46 ix Figure 17. The effect of staurosporine on mitosis 47 Figure 18. The effect of chelerythrine chloride on entry into mitosis 48 Figure 19. Chelerythrine chloride inhibits p45 kinase activation 49 Figure 20. Partial purification of p45 kinase 51 Figure 21. Fractionation and purification of histones 54 Figure 22. Identification of a cell cycle-dependent histone kinase 55 in FT210 cells Figure 23. Specificity of the histone kinase in FT210 cells 56 Figure 24. P l k l activity and abundance in FT210 cells at the 60 non-permissive temperature Figure 25. Model of the role of P l k l in chromosome segregation 71 x List of Abbreviations A D P adenosine diphosphate A T P adenosine triphosphate B S A bovine serum albumin C A K cdk-activating kinase c - A M P cyclic-adenosine monophosphate cdk cyclin-dependent kinase D M E M Dulbecco's modified Eagle medium D T T dithiothreitol E D T A ethylenediaminetetraacetic acid E G F epidermal growth factor E G T A ethylene glycol-bis(P-aminoethylether) N , N , N ' , N ' -tetraacetic acid E L I S A enzyme-linked immunosorbent assay F B S fetal bovine serum F P L C fast protein liquid chromatography G S T glutathione S-transferase H E P E S /Y-2-Hydroxyethylpiperazine-A^-2-ethanesulphonic acid H P L C high performance liquid chromatography Hsp heat shock protein IC50 50% inhibitory concentration I G F insulin-like growth factor I P T G isopropylthiogalactopyranoside xi J N K 1 c-Jun N-terminal kinase 1 M A P mitogen-activated protein M A P K A P kinase-2 M A P kinase-associated protein kinase-2 M B P myelin basic protein M E M minimum essential medium M K L P - 1 mitotic kinesin-like protein-1 M O P S 3-(./V-morpholino)propanesulphonic acid M W L molecular weight limit N I M A never in mitosis A N M A F nuclear membrane-activating factor NP-40 Nonidet P-40 O A okadaic acid P A G E polyacrylamide gel electrophoresis P B S phosphate-buffered saline P K C protein kinase C P l k l polo-like kinase 1 P M S F phenylmethylsulfonyl fluoride PP1 protein phosphatase 1 PP2 protein phosphatase 2 S D S sodium dodecyl sulfate Ser Serine T B S Tris-buffered saline T C A trichloroacetic acid Thr Threonine Tris Tris(hydroxymethyl)aminomethane T P A tetradecanoyl phorbol acetate Tyr Tyrosine xi i Acknowledgments Welcome to the one and only page that many of you wi l l actually read. I would like to take this opportunity to thank the many people who have influenced my currently brief stint in biochemistry. Thanks to Peter Greer and Marty Petkovich for giving me my first break into the world of science and encouraging me to continue on. Just what was slipped into those pitchers at the Grad Club I'll never know but it got me hooked. I would also like to thank Michel Roberge for his seemingly endless encouragement, guidance and patience throughout this study. Your enthusiasm and excitement for this work certainly provided a great impetus to keep on going. Many others deserve to be noted in making the last two years extremely enjoyable. Thanks Deb for all those enlightening coffee breaks and great music taste. Y o u were a constant entertainment even when you didn't want to be. Geoff, L i l i , Duncan and Hilary, you all provided many memorable moments and helped make my time here a fantastic experience. Thank you very much. Cobes, Anand, Logan, Phil , Manish and all the rest of my friends in the department many thanks. Your friendships wi l l not be forgotten and you all certainly made the last couple of years entertaining to say the least. I would like to thank my parents for their unwavering support in encouraging me to pursue my dreams. Lastly, I would like to thank Renee for her continuous love and support. Y o u truly picked me up when I was down and believed that this would get done even when I was a skeptic. I could not have done this as easily without you. Anand, I told you I'd beat you out of here! xiii Chapter 1 Introduction 1.1 Cell cycle The eukaryotic cell cycle is the highly coordinated process by which cells grow and propagate. Revealing the intricacies of the cell cycle is fundamental to our understanding of the basis of life. The cell cycle is tightly regulated in order to faithfully transmit genetic information. Failure to consistently regulate the cell cycle can result in uncontrolled cellular growth, genomic abnormalities and cellular death. The cell cycle can be generally divided into two phases: interphase and mitosis (Fig. 1) (reviewed in Murray and Hunt 1993). Interphase can be further subdivided into the G l , S and G2 phases. The D N A is faithfully replicated during S phase while both the G l and G2 phases are characterized by cellular growth in preparation for cell division. Mitosis is the process by which a complete copy of the genome is segregated to each daughter cell. Mitosis can be divided into four distinct phases: prophase, metaphase, anaphase and telophase (Fig. 2). 1.1.1 The stages of mitosis Prophase During prophase, the nuclear envelope breaks down, abolishing the distinction between nucleus and cytoplasm. Furthermore, the chromosomes dramatically condense to form visible chromosome structures composed of two chromatids held together at centromeres. The duplicated centrosome splits and the individual centrosomes move toward 1 INTERPHASE Figure 1. The eukaryotic cell cycle The eukaryotic cell cycle can be divided into four phases. Mitosis is the process by which a parental cell divides into two daughter cells. Interphase constitutes the gap phases G l and G2 , and S phase during which chromosome replication occurs. 2 Chromosome Interphase Interphase Figure 2. The stages of mitosis D u r i n g prophase, the chromosomes condense, the nuclear envelope breaks d o w n and the dupl icated centrosome splits into ind iv idua l centrosomes w h i c h migrate to opposite poles o f the ce l l establ ishing the spindle poles. A t metaphase, the chromosomes are al igned at the equatorial plate. Sp indle f ibers reach f r o m pole to pole and attach to the kinetochores o f chromatids. Anaphase is character ized by the separation o f the two sister chromatids into ind iv idua l chromosomes. The ce l l elongates, as do the pole to pole spindles, and cytokinesis begins w i th the appearance o f the cleavage furrow. F ina l l y , at telophase, the nuclear envelope starts to re fo rm around the new daughter nuc le i , the chromosomes decondense and cytok ines is is completed fo rming two new daughter cel ls . 3 opposite poles of the cell establishing the bipolarity of the cell. Each centrosome consists of a pair of centrioles oriented at right angles and connected by fibrils which organize the mitotic microtubule array necessary for proper chromosome segregation. Microtubules extend across the cell from pole to pole and to the kinetochore of each chromatid. Metaphase Metaphase is characterized by the microtubule-attached sister chromatids moving toward and aligning at the equatorial plane of the cell. Anaphase The sister chromatids separate into independent chromosomes at anaphase. Each chromosome is attached through a microtubule to a spindle pole. The chromosomes move along the microtubules to opposite poles of the cell. The pole to pole microtubules lengthen resulting in elongation of the cell. Cytokinesis is the physical process of splitting the parent cell into two daughter cells and marks the end of mitosis. The onset of cytokinesis occurs at anaphase with the invagination of the plasma membrane at the equatorial plane of the cell establishing a cleavage furrow. Telophase Cytokinesis is completed with an actomyosin contractile ring pinching the cell into two new daughter cells. The nuclear membrane starts to reform around the new daughter nuclei, chromosomes begin to decondense and the nucleolus becomes visible again. The spindle apparatus disappears as the microtubules depolymerize. The centrosome becomes the microtubule organizing centre and initiates the return to an interphase microtubule pattern. 4 1.2 Protein phosphorylation in regulating mitosis Revers ib le protein phosphorylat ion is a key regulatory process i n tr iggering entry into, and progression through, mitosis wh i ch requires both a protein kinase, w h i c h transfers the y-phosphate group f rom an A T P molecule to a specif ic site on a target protein, and a protein phosphatase, wh i ch removes the phosphate group f rom the site (F ig. 3). The leve l o f phosphorylat ion o f target proteins can be altered by changing the activity o f either the specif ic regulatory protein kinase or the protein phosphatase. Target proteins are often themselves protein kinases and phosphatases, wh i ch i n turn act on other targets. Du r ing mitosis, a control led and concerted cascade o f phosphorylat ion events occurs, forming a signal ing pathway wh i ch ult imately el icits the characteristic events o f mitosis, such as phosphorylat ion of the nuclear lamins w h i c h leads to their depolymer izat ion at mitosis. A single protein kinase or phosphatase may have mult ip le target proteins and thus affect numerous s ignal ing pathways. 1.3 The role of Cdc2 The serine/threonine protein kinase C d c 2 is required for mitosis and its activation induces various mitot ic events (reviewed by N i g g 1993; K i n g et al. 1994). C d c 2 direct ly phosphorylates the nuclear lamins leading to nuclear envelope breakdown. C d c 2 hyperphosphorylates histone H I and this correlates w i th chromosome condensation. The format ion o f the mitot ic microtubule array and disassembly o f the nucleolus have also been shown to be control led by C d c 2 . The members o f the C d c 2 s ignal ing pathways invo lved in these h igh ly diverse mitot ic processes remain unidentif ied. 5 Activity +/-Activity +/-regulatory kinase eg: Weel regulatory phosphatase eg: Cdc25 target protein eg: Cdc2 Figure 3. The opposing effects of regulatory protein kinases and phosphatases on target proteins Regulation of the activity of a protein kinase often requires a balance between the opposing inhibitory/activating effects of a kinase and phosphatase. Perturbation of this delicate balance by activating or inhibiting the regulatory kinases or phosphatases can have profound effects on the activity of the target protein. 6 1.3.1 Regulation of Cdc2 C d c 2 is a member o f the cyclin-dependent kinase fami ly ( C D K ) whose activity requires association w i th a cyc l i n regulatory subunit (reviewed by Pines 1993). C D K s , i n conjunct ion w i th their cyc l in partners, function to regulate ce l l cyc le transitions. C d c 2 w i th its cognate cyc l in partners, c yc l in A and cyc l in B, regulates entry into mitosis. Ac t i va ted Cdc2-cyc l in B triggers entry into mitosis and the t imely inactivation o f both Cdc2-cyc l in complexes is required for exit f rom mitosis (Dunphy 1994; K i n g et al. 1994). The regulation of C d c 2 activity is an excellent example of the delicate interplay between protein kinases and protein phosphatases. C d c 2 activity is both posi t ive ly and negatively regulated by phosphorylat ion at three distinct sites: T h r l 6 1 , T h r l 4 and T y r l 5 (F ig . 4) (Norbury et al. 1991). Phosphorylat ion o f T h r l 6 1 by CDK-ac t i va t ing kinase ( C A K ) is required for C d c 2 activity (So lomon 1994). B y analogy to the equivalent phosphory lat ion o f T h r l 6 0 in C d k 2 , phosphorylat ion o f C d c 2 T h r l 6 1 in combinat ion w i th c yc l i n b ind ing moves a loop containing T h r l 6 1 away f rom the substrate b ind ing cleft (DeBondt et al. 1993). C A K can only phosphorylate C d c 2 when it is associated w i th its c y c l i n partner. Th is phosphorylat ion can be removed by protein phosphatase 2 A ( P P2A ) in vitro (Gou ld et al. 1991; Lee et al. 1991) and the addit ion o f P P 2 A to Xenopus oocyte extracts inhibits T h r l 6 1 phosphorylat ion (Fel ix et al. 1990; Lee et al. 1994). It has not yet been determined i f P P 2 A acts directly on C d c 2 in vivo or inactivates some other component o f the C A K activating pathway. Cdc2-cyc l in B phosphorylated at T h r l 6 1 accumulates dur ing G 2 but remains inact ive due to phosphorylat ion at T y r l 5 and T h r l 4 wh i ch overlap the ATP-b ind ing site. In f iss ion yeast, on ly T y r l 5 is phosphorylated. B o t h the W e e l and M i k l protein kinases can phosphorylate T y r l 5 of C d c 2 (Featherstone and Russe l l 1991; Parker et al. 1992). W e e l homologues have been found in various eukaryotes ranging f rom yeast to humans (Igarashi et al. 1991; Booher et al. 1993; Tang et al. 1993). 7 INACTIVE PP2A Histone HI nuclear lamins spindle proteins Thrl4 PP2A INACTIVE ACTIVE Figure 4. The regulation of Cdc2 Both phosphorylation of Thrl61 by C A K and dephosphorylation of T y r l 5 and T h r l 4 residues by Cdc25 are required for activation of cyclin-bound Cdc2 at mitosis. Cdc2-cyclin B activity is inhibited during interphase by phosphorylation of T h r l 4 and T y r l 5 by the W e e l and M y t l protein kinases. 8 W e e l is inhibited at mitosis by phosphorylation of its carboxy-terminal catalytic domain by the N i m l protein kinase in S. pombe (Coleman et al. 1993; Parker et al. 1993; W u and Russell 1993). A second protein kinase, W i s l , has also been identified that plays a role in the negative regulation of W e e l (Warbrick and Fantes 1991). Neither N i m l nor W i s l homologues have been identified in higher eukaryotes. Human W e e l is inhibited by hyperphosphorylation at mitosis by an unidentified protein kinase, possibly an M P M - 2 epitope kinase which is distinct from Cdc2-cyclin B (Coleman and Dunphy 1994). Throughout interphase, W e e l is maintained in an active state by a PP2A-l ike protein phosphatase (Tang et al. 1993). Recently, M y t l was identified as a dual-specificity protein kinase that phosphorylates Cdc2 on both Thr l4 and T y r l 5 in Xenopus oocyte extracts (Mueller et al. 1995). The reason for the dual phosphorylation of Cdc2 in vertebrates is unclear as Cdc2 mutant studies have indicated that either Thr l4 or Ty r l5 is sufficient for negative regulation (Krek and Nigg 1991; Norbury et al. 1991; Pickham et al. 1992). The dual phosphorylation may be necessary for the proper coordination of Cdc2 activity by Cdc25 and to prevent untimely dephosphorylation by protein tyrosine phosphatases. Cdc25 is the dual specificity protein phosphatase that dephosphorylates the inhibitory sites, Thr l4 and T y r l 5 and triggers Cdc2-cyclin B activity at mitosis (Dunphy and Kumagai 1991; Mi l la r et al. 1991; Lee et al. 1992). In S. pombe, there is only one isoform of Cdc25 whereas in mammals three have been identified: Cdc25A, Cdc25B and Cdc25C (Sadhu et al. 1990). The isoforms are highly related in their carboxy-terminal catalytic domains but diverge greatly in their amino-terminal regulatory domains. Each human Cdc25 isoform is active at different stages of the cell cycle with Cdc25C being required for the G 2 - M transition (Sadhu et al. 1990). Cdc25 is activated at mitosis by phosphorylation of key residues in the amino-terminal regulatory domain. A newly discovered protein kinase, P l x l , phosphorylates 9 many of these sites and activates Cdc25 in vitro (Izumi and Mailer 1995; Kumagai and Dunphy 1996). Cdc2 can also phosphorylate and activate Cdc25 (Galaktionov and Beach 1991; Strausfeld et al. 1994). Thus, Cdc25 phosphorylation and activity is maintained throughout mitosis by association with active Cdc2-cyclin B in vivo (Izumi et al. 1992; Izumi and Mailer 1993). Cdc25 is dephosphorylated by a PP2A-l ike phosphatase that is active throughout interphase and inactive during mitosis (Clarke et al. 1993). 1.4 The roles of other mitotic protein kinases Recent studies have identified mitotic protein kinases downstream of Cdc2 as well as protein kinases that are independent of Cdc2. These kinases include members of the M P M - 2 epitope kinase family, the Aspergillus nidulans N I M A kinase and the (311 isoform of protein kinase C (PKC) amongst others. 1.4.1 MPM-2 epitope kinases M P M - 2 antigens are a discrete set of phosphoproteins that react with the monoclonal antibody M P M - 2 (Davis et al. 1983). The M P M - 2 antibody was raised against a mitotic HeLa cell extract and selected by its preferential reactivity with mitotic versus interphase cells. The M P M - 2 antigens are synthesized in S phase and modified by phosphorylation at the G 2 / M transition (Kuang et al. 1989). The M P M - 2 antibody recognizes the consensus phosphorylated epitope L T P L K (Westendorf et al. 1994). The kinases phosphorylating the M P M - 2 epitope are largely unknown. Cdc2 has been shown to be an M P M - 2 kinase (Kuang et al. 1989; Kuang et al. 1991). However, in the absence of Cdc2, M P M - 2 kinase activity is not eliminated, suggesting there are other unidentified M P M - 2 kinases (Kuang et al. 1994; personal observations P. Gowdy and M . Roberge). Many M P M - 2 antigens represent proteins that regulate mitosis, such as Cdc25, P l x l and W e e l , some of which may be M P M - 2 kinases themselves. Active Cdc25 is an 10 M P M - 2 antigen even in the absence of Cdc2 (Kuang et al. 1994). This suggests that Cdc25 activity may be regulated by a Cdc2-independent M P M - 2 kinase. P l x l phosphorylates Cdc25 on key mitotic regulatory sites which are M P M - 2 reactive (Kumagai and Dunphy 1996). Phosphorylation of Cdc25 by P l x l stimulated Cdc25 activity in vitro. This suggests that P l x l is an M P M - 2 kinase. Similarly, the activity of W e e l is inhibited at least partly by phosphorylation at an M P M - 2 epitope (Tang et al. 1993). This M P M - 2 kinase has not been identified although it is tempting to speculate that P l x l could phosphorylate both Cdc25 and W e e l , thereby functioning as a trigger of mitosis (Fig. 5). 1.4.2 NIMA In the filamentous fungus Aspergillus nidulans, a second protein kinase, N I M A , is also required for mitotic progression (Osmani et al. 1991a). N I M A is a 79kDa serine/threonine kinase with an amino-terminal catalytic domain and a carboxy-terminal regulatory domain. N I M A activity is low in G l , but increases through S and G 2 and peaks at mitosis (Osmani et al. 1991b). N I M A is degraded at mitosis and this degradation may be required for exit from mitosis (Pu and Osmani 1995). This is analogous to the requirement for cyclin B degradation for exit from mitosis. M m , for never in mitosis, mutants were initially identified in genetic screens of A. nidulans for mutations which resulted in cells arrested in G2 (Morris 1976). Mutations in N I M A caused cell cycle arrest despite normal activation of Cdc2. Conversely, mutation of the A. nidulans Cdc25 resulted in arrest in G2 despite N I M A partial activity (Osmani et al. 1991a). This suggested that both kinases are required for entry into mitosis in A. nidulans. The N I M A pathway is not parallel and independent from the Cdc2 pathway but rather converges with it (Ye et al. 1995). The activation of N I M A occurs in two steps. N I M A is initially partially activated either by autophosphorylation or by an unknown kinase (Osmani et al. 1991b). N I M A is further hyperphosphorylated and fully activated in 11 Plxl v J Figure 5. Regulation of Cdc25 phosphatase activity by the MPM-2 reactive kinase Plxl P l x l phosphorylates and activates Cdc25 which in turn triggers mitosis through activation of Cdc2 by dephosphorylation of T h r l 4 and T y r l 5 . The activity of the Cdc2 inhibitory kinase, W e e l , which phosphorylates T y r l 5 and Thr l4 , is partially inhibited by an M P M - 2 kinase, possibly P l x l . 12 a Cdc2-dependent manner either by phosphorylation by Cdc2 itself or by an intermediate kinase (Ye et al. 1995) (Fig. 6). The carboxy-terminal regulatory domain of N I M A contains 7 putative Cdc2 phosphorylation sites (Ye et al. 1995). It remains to be demonstrated i f N I M A can act on substrates in the partially activated state. N I M A overexpression results in premature chromatin condensation in A. nidulans, the fission yeast S. pombe, Xenopus oocytes and human cells (O'Connell et al. 1994; L u and Hunter 1995). This is accompanied by nuclear envelope breakdown and the disappearance of the nuclear lamins in S. pombe, Xenopus oocytes and human cells (Lu and Hunter 1995). It is unknown i f lamina depolymerization is due to phosphorylation as in mitosis, or degradation as in apoptosis (O'Connell et al. 1994). A. nidulans, as well as S.cerevisiae, are unique in that both have a closed mitosis in which the nuclear envelope does not breakdown (Murray and Hunt 1994). There are no signs of mitotic spindle formation in S. pombe, Xenopus oocytes and human cells (O'Connell et al. 1994; L u and Hunter 1995). It is unclear what substrates N I M A directly phosphorylates in eliciting its mitotic phenotypes. Conversely, overexpression of dominant-negative mutants of N I M A can induce G2 arrest in HeLa cells (Lu and Hunter 1995). This evidence suggests the existence of N I M A - l i k e pathways in higher eukaryotes. Several partial c D N A s of NIMA-related kinases or Neks have been isolated in mammals (Letwin et al. 1992; Schultz and Nigg 1993; Schultz et al. 1994; L u and Hunter 1995). The NIMA-related kinase Nek2, shares the greatest sequence similarity with N I M A , sharing 47% identity over the catalytic domain (Schultz et al. 1994). The cell cycle-dependent protein levels and activity of Nek2 and N I M A are similar, although differences exist (Fry et al. 1995). The most notable difference is that NEVIA kinase activity peaks at mitosis in X . nidulans whereas Nek2 activity peaks at S/G2 phase and is low in mitosis in mammalian cells (Fry et al. 1995). Overexpression of Nek2 does not result in any detectable mitotic phenotype (Lu and Hunter 1995). This suggests that Nek2 may function early in the cell cycle and that the functional mitotic 13 INACTIVE PARTIALLY ACTIVE FULLY ACTIVE MITOSIS INACTIVE ACTIVE Figure 6. Regulation of mitotic entry requires the activity of both Cdc2 and NIMA protein kinases in Aspergillus nidulans A model for the two step activation of N I M A . The first phosphorylation of N I M A occurs by an undetermined mechanism, either by autophosphorylation or by an unknown kinase. The second hyperphosphorylation is Cdc2-dependent, results in fully activated N I M A , and provides a convergence of the two mitotic entry pathways. 14 homologue of N I M A still remains to be identified. 1.4.3 PKCpII The twelve isoforms of the protein kinase C (PKC) family are involved in signaling pathways for many diverse events controlling mitogenic growth and cellular differentiation. P K C has been implicated in the regulation of mitosis in species ranging from yeast to man (Levin et al. 1990; Abe et al. 1991; Usui et al. 1991; Hocevar et al. 1993; Goss et al. 1994; Hofmann et al. 1994). Genetic evidence indicates that the S. cerevisiae P K C homologue PKC1, is required for cell cycle progression (Levin et al. 1990). Deletion of PKC1 leads to a mutant cell cycle phenotype and arrest in G2 phase. P K C 1 participates in a mitotic checkpoint which appears to regulate aspects of osmotic stability required for cell division and is independent from the S. cerevisiae Cdc2 (Levin and Bartlett-Heubusch 1992). The downstream targets of P K C 1 include the yeast homologues of M A P K and M A P K kinase (Irie et al. 1993; Lee et al. 1993). During G2, PKCpI I is translocated to the nucleus and activated by an unknown mechanism (Hocevar et al. 1993; Goss et al. 1994). The nucleus has a distinct phosphoinositide cycle that is responsive to cell cycle regulation (Divecha et al. 1991; Banfic et al. 1993; Divecha et al. 1993a; Divecha et al. 1993b). Nuclear phosphoinositide turnover increases during G2, leading to an increase in diacylglycerol levels which result in the nuclear translocation and activation of PKCpI I (Banfic et al. 1993). Additionally, a nuclear membrane lipid factor, N M A F , has been demonstrated to selectively activate P K C p I I at the nuclear membrane (Murray et al. 1994). The disassembly of the nuclear lamina is a hallmark of mitosis. Lamin B1 has been shown to be an in vivo physiological substrate of P K C P U (Hocevar et al. 1993; Goss et al. 1994). P K C P I I phosphorylates lamin B l at a prominent mitotic phosphorylation site, 15 Ser405. This phosphorylation is prevented by treatment of cells with the highly selective P K C inhibitor chelerythrine chloride (Thompson and Fields 1996). Inhibition of PKC (311 activity by chelerythrine chloride arrests cells at the G 2 / M transition (Thompson and Fields 1996). This arrest is characterized by interphase decondensed chromatin with nucleolar structures and an intact nuclear envelope. Surprisingly, Cdc2 is fully active in these cells (Thompson and Fields 1996). This indicates an essential role of PKCpII in triggering mitosis, possibly in a pathway distinct from Cdc2. 1.5 Serine/Threonine protein phosphatases Research on the control of target protein activity by phosphorylation has focussed mostly on protein kinases due to the misguided concept that protein phosphatases simply act constitutively to reverse the action of regulatory protein kinases. It has become apparent that the regulation of target protein activity by protein phosphatases is essential in regulating various cellular processes. Activity of the dual specificity phosphatases, Cdc25A, B and C is critical for progression through the eukaryotic cell cycle (Millar et al. 1991; Kumagai and Dunphy 1992). Similarly in Drosophila, mutations in the P P 2 A B-type regulatory subunit result in abnormal progression through anaphase with chromatids lagging at the metaphase plate (Mayer-Jaekel et al. 1993) and the PTP1C gene is mutated in the mouse motheaten mutant phenotype (Shultz et al. 1993; Bignon and Siminovitch 1994). The serine/threonine protein phosphatase family is split into two broad categories; type 1 and type 2 (Ingebritsen and Cohen 1983) (Table 1). Type 1 phosphatases, PP1, are inhibited by two heat-stable protein inhibitors, 1-1 and 1-2, and preferentially dephosphorylate the (3-subunit of phosphorylase kinase. Conversely, the type 2 phosphatases dephosphorylate the a-subunit of phosphorylase kinase and are insensitive to 1-1 and 1-2 . The type 2 phosphatase family is further subdivided into three subclasses P P 2 A , 2B and 2C. 16 Table 1. The Serine/Threonine Protein Phosphatase Family Name Catalytic subunit Regulatory subunit Structure T Y P E 1 A T P - M g 2 + dependent Glycogen/SR-associated Myofibril-associated Nuclear T Y P E 2 A Phosphatase 2A2 2A1 2 A 0 P C S M T Y P E 2B T Y P E 2C Mg 2 + -activated C I a , y l , y2, 5 C2 a , p A a , p, y C a , p Rl-2 R G L R M Y N I P P - l A a , p B a , p, y R1-2-CI R G L - C 1 R M Y - C 1 R N - C 1 A - C 2 A - B - C 2 B ' a , p, y, 5 A - B ' - C 2 B " B A - B ' - C A - B - C a M Calmodulin none C 1.5.1 Physiological roles of protein phosphatases The physiological pathways in which PP1 and PP2A play a critical regulatory role are extremely varied (Depaoli-Roach et al. 1994). Adding to the difficulty of studying the specific roles of PP1 and P P 2 A is that many protein phosphatase subtypes are able to functionally compensate for the inhibition of others. It has been determined that PP1 is involved in glycogen metabolism, calcium transport and muscle contraction (Hubbard and Cohen 1993). A role for P P 2 A has been suggested in growth factor signaling pathways, especially in the inactivation of the E r k / M A P kinases and MAP-kinase kinases (Gomez and Cohen 1991). From the point of view of this project, the most interesting role of PP1 and P P 2 A is in the regulation of the cell cycle. The activity of PP1 increases at both the onset of S phase and mitosis (Walker et al. 1992). PP2A has been shown to be involved in the regulation of Cdc2. Treatment of cells with okadaic acid, a potent PP2A inhibitor, results in 17 activation of Cdc2 activity as determined by in vivo histone H I hyperphosphorylation and the induction of mitosis (Goris et al. 1989; Yamashita et al. 1990). In Xenopus oocyte extracts, a PP2A-like activity, I N H , inhibits the formation of active Cdc2-cyclin B (Lee et al. 1991). Both I N H and purified PP2A catalytic subunit dephosphorylate the C A K -mediated activating phosphorylation of Cdc2 Thrl61 in vitro (Lee et al. 1994). The involvement of P P 2 A in entry into mitosis has been confirmed by genetic studies in S. pombe where disruption of the pp2a+ gene results in reduced cell size and slower growth (Kinoshita et al. 1990). Similar genetic studies in Drosophila and S. cerevisiae have shown that mutations in the B subunit of PP2A results in abnormal cell division and problems with chromosome disjunction (Healy et al. 1991; Mayer-Jaekel et al. 1993). Furthermore, both Cdc25 and Wee l are maintained in a dephosphorylated state throughout interphase by a PP2A-like phosphatase activity (Clarke et al. 1993; Tang et al. 1993). This suggests an essential role of P P 2 A and PP1 in the proper timing and triggering of mitosis. 1.5.2 Serine/threonine protein phosphatase inhibitors Many microbial toxins are inhibitors of serine/threonine protein phosphatases demonstrating the critical importance of protein phosphatases in cellular processes (Table 2). Most such molecules are structurally unrelated although all apparently bind to the same site on PP1 or P P 2 A (MacKintosh and MacKintosh 1994). Protein phosphatase inhibitors are indispensable in the study of the roles of reversible protein phosphorylation in cellular processes. Many of these compounds cause hyperphosphorylation of proteins that are not direct targets of protein phosphatases by activating protein kinases that are themselves positively regulated by phosphorylation. This is an important observation that was critical for the development of this project. 18 Table 2. Serine/Threonine Protein Phosphatase Inhibitors. Inhibitors Phosphatase Inhibition ICso(nM) Chemical Nature Okadaic acid Tautomycin Calyculin A Microcystin-LR Inhibitor-1 Inhibitor-2 Fostriecin PP1 PP2A 15 0.1 0.2 1.0 0.5-2 0.1-1 0.15 0.15 0.45 0.8 400 40 polyether carboxylic acid polyketide phosphorylated polyketide cyclic peptide heat-stable protein heat-stable protein phosphate ester 1.5.3 Okadaic acid Okadaic acid is a polyether carboxylic acid produced by several marine dinoflagellates which is a potent inhibitor of PP2A (IC50 O. lnM) and a strong inhibitor of PP1 (IC50 10-15 nM) (Tachibana et al. 1981; Goris et al. 1989; Cohen et al. 1990; Felix et al. 1990) (Fig. 7). Treatment of cells with okadaic acid alters many diverse phosphorylation events that affect transcriptional activation, cellular shape changes and interestingly, causes a lethal pseudomitosis (Yamashita et al. 1990). Okadaic acid treated cells are rounded up with condensed chromosomes, depolymerized nuclear lamins and separated spindle poles. This is characteristic of a phenotypically normal mitosis although the cells are arrested at a prometaphase-like stage with no separation of chromatids or cytokinesis (Yamashita et al. 1990). 1.5.4 Fostriecin The Streptomyces pulvraceus subspecies fostreus produces a protein phosphatase inhibitor, fostriecin, which has shown promise as an antitumour drug (Stampwala et al. 1983;Tunacef al. 1983; Leopold etal. 1984; Jackson et al. 1985; Scheithauer etal. 1986). Fostriecin is a much more potent inhibitor of PP2A ( IC50 40nM) than PP1 ( IC50 400nM) 19 Figure 7. The structures of the protein phosphatase inhibitors okadaic acid and fostriecin A. Okadaic acid and B. fostriecin are both potent inhibitors of P P 2 A and PP1, and were isolated from marine dinoflagellates and Streptomyees pulveraceus, respectively. 20 (Roberge et al. 1994). Fostriecin is a water-soluble phosphate ester with a conjugated triene system (Fig. 7). Similar to the effect of okadaic acid, cells treated with fostriecin enter mitosis even when arrested at the G2 checkpoint or with incompletely replicated D N A (Roberge et al. 1994). Furthermore, fostriecin was found to induce chromosome condensation in the absence of Cdc2 activity and histone H I hyperphosphorylation (Guo et al. 1995). This suggested the existence of novel Cdc2-independent pathways for entry into mitosis and formed the basis of this project. 1.6 The FT210 cell line The FT210 cell line is a temperature-sensitive mutant of the mouse mammary carcinoma F M 3 A cell line. FT210 cells have a temperature-sensitive lesion in the cdc2 gene. The cdc2 gene product becomes inactivated and degraded at the non-permissive temperature of 39°C. This results in cell cycle arrest in late G2 (Th'ng et al. 1990) (Fig. 8). D N A sequence analysis revealed two point mutations in highly conserved coding regions of the gene resulting in an isoleucine to valine change in the P S T A I R region, and a proline to serine change in the C-terminal region (Th'ng et al. 1990). There is no other significant degradation of other proteins at the non-permissive temperature. 1.6.1 Protein phosphatase inhibitors overcome the G2 arrest at the non-permissive temperature The G2 arrest at the non-permissive temperature can be overcome by the protein phosphatase inhibitors okadaic acid and fostriecin, resulting in a phenotypic lethal mitosis in which cells become arrested in a pseudometaphase-like state (Guo et al. 1995). The induction of mitosis was determined by nuclear lamina depolymerization and chromosome condensation. Chromosome condensation in the absence of Cdc2 was correlated with hyperphosphorylation of histones H3 and H 2 A (Guo et al. 1995). This is in contrast to 21 Figure 8. G2 arrest of FT210 cells at the non-permissive temperature FT210 cells were incubated for 16 hours in isoleucine-deficient RPMI-1640 supplemented with 10% heat-inactivated dialyzed FBS, and released into complete medium. The released cells were incubated in aphidicholin for 9 hours at 32°C. Following release from the aphidocholin block, the cells were incubated at the non-permissive temperature of 39°C for 16 hours. Flow cytometric analysis of A. Cycling FT210 cells. B. FT210 cells after 16 hour incubation at 39°C. 22 normal mitosis in which histone H I is hyperphosphorylated by Cdc2 and histone H3 is phosphorylated at one site (Gurley et al. 1978). Both cyclins A and B were degraded in FT210 cells treated with phosphatase inhibitors (Guo et al. 1995). Degradation of cyclins A and B is required for exit from mitosis. Therefore, the untimely premature degradation of cyclins may cause the lethal mitotic block in FT210 cells at 39°C. The mechanism by which phosphatase inhibitors trigger cyclin degradation is unknown but is known to require the proteasome. 1.6.2 Protein kinases are required to overcome the G2 arrest mediated by phosphatase inhibitors Entry into mitosis triggered by phosphatase inhibitors at the non-permissive temperature in FT210 cells can be antagonized by the general protein kinase inhibitor staurosporine, indicating a requirement for protein kinase activity. Staurosporine has been demonstrated to inhibit both histone H I and H3 kinase activity and to cause G l and G 2 / M phase arrest (Abe et al. 1991; Th'ng et al. 1994). Staurosporine recognizes and binds to the kinase's A T P binding site. Staurosporine effectively inhibits a number of kinases including P K C , cyclic-adenosine monophosphate (cAMP)-dependent protein kinase, phosphorylase kinase, S6 kinase, pp60 v ~ s r c , epidermal growth factor (EGF) receptor/kinase and insulin-like growth factor (IGF) receptor/kinase (Hidaka and Kobayashi 1992 and see Table 3). Table 3: Inhibition of Protein Kinases by Staurosporine. Protein kinase Protein kinase C c-AMP-dependent protein kinase pp60v-src E G F receptor/kinase IC50(nM) 2.7 8.2 6.4 630 23 1.7 Research objectives The identities of the protein kinases involved in the regulation of the events of mitosis remains largely a mystery. These protein kinases have an essential role in cell growth and division and potentially represent realistic targets for antitumour drugs. The goal of this project was to further our understanding of mitotic control by identifying mitotic protein kinases that are activated by protein phosphatase inhibitors and are regulated independently of Cdc2. In this work, I have identified a novel mitotic 45 kDa protein kinase and a histone-specific 60 kDa mitotic protein kinase that are activated by phosphatase inhibitors in FT210 cells in the absence of Cdc2 activity. The 45 kDa kinase was shown to be tightly cell cycle-regulated, and is active only at mitosis. Furthermore, I determined that the kinase was not the known 46 kDa kinases M A P K A P kinase-2 or J N K 1 . 1 further characterized the effects of known mitotic inhibitors on the activity of the kinase and partially purified the 45kDa mitotic kinase. Subsequently, I investigated the activity of another mitotic kinase, P l k l , in FT210 cells at the non-permissive temperature. I determined that P l k l was inactive although abundantly present in mutant FT210 cells even when induced into mitosis by treatment with phosphatase inhibitors. This suggests that mitotic P l k l activity is regulated by an uncharacterized Cdc2-dependent pathway. P l k l is thought to have an essential role in the microtubule dynamics involved in chromosome segregation at anaphase. Therefore, inactivity of P l k l in FT210 cells induced to enter mitosis with protein phosphatase inhibitors, may prevent progression past metaphase. 24 Chapter 2 Materials and Methods 2.1 Cell culture and synchronization of FT210 cells Asynchronous populations of mouse mammary tumour FT210 cells were grown in suspension at 32°C in a humidified 5% CO2 atmosphere in RPMI-1640 (Gibco) supplemented with 10% F B S (Gibco), 50 units/ml penicillin and 50 |LLg/ml streptomycin at an initial density of 5 x l O 5 cells/ml. Human Jurkat T-cell lymphoma cells were grown as a suspension in RPMI-1640 supplemented with 10% F B S , 50 units/ml penicillin and 50 |lg/ml streptomycin at 37°C and humidified 5% CO2, at an initial density of 5 x l O 5 cells/ml. Human breast carcinoma M C F 7 cells expressing a dominant-negative mutant p53 cloned into a p C M V plasmid (Fan et al. 1995) were cultured as monolayers in D M E M supplemented with 10% F B S , 2 m M L-glutamine, 50 units/ml penicillin, 50 p:g/ml streptomycin, 1 m M sodium pyruvate, M E M non-essential amino acids, 1 (ig/ml bovine insulin, 1 | lg/ml hydrocortisone, 1 ng/ml human epidermal growth factor and 1 ng/ml (3-estradiol at 37°C in humidified 5% CO2. The human Alzheimer primary fibroblast cell line 4148A, was cultured as monolayers in D M E M supplemented with 3.5 g/1 D-glucose, 15% F B S , 50 units/ml penicillin and 50 ug/ml streptomycin at 37°C in humidified 10% CO2. In order to synchronize FT210 cells to S and early G2 phases of the cell cycle, cells were treated for 16 hours with aphidicholin (1.25 |ig/ml) to arrest them at the G l / S boundary. The cells were released from the aphidicholin block for 9 hours and incubated again with aphidicholin for another 16 hours. Cells were released from the second aphidicholin block for 3 hours or 6 hours and designated the S and S/G2 populations respectively. 25 Late G2 and mitotic cell populations were obtained by incubating cells in isoleucine deficient R P M I - 1640 supplemented with 10% heat-inactivated, dialyzed F B S (Gibco) for 16 hours, followed by release into complete medium. A mitotic population was obtained by incubating released cells in the presence of nocodazole (50 ng/ml) for 16 hours. To obtain a late G2 population lacking Cdc2, released cells were incubated in aphidicholin (1.25 |ig/ml) for 9 hours at 32°C. Following release from the aphidicholin block, the cells were incubated at 39°C for 16 hours. Nocodazole and aphidicholin were obtained from Sigma Chemical Co. 2.2 Flow cytometric analysis FT210 cells synchronized to various points of the cell cycle were collected by centrifugation and fixed in 1 ml of 0.5% paraformaldehyde in P B S for 15 minutes at 4°C. The cells were centrifuged and permeabilized in 1 ml of 0.1% Triton X-100 in P B S for 3 minutes on ice. To prepare the cells for D N A staining, the cells were incubated with 1 mg/ml R N A s e A for 30 minutes at 37°C. The D N A was stained with 50 |_lg/ml propidium iodide (Sigma) in P B S at 4°C in the dark for at least 30 minutes. A minimum of 25 000 cells were subjected to flow cytometry using a Coulter Epics E S P flow cytometer (Coulter corp.) equipped with an ion laser (Coherent Inc.). Laser excitation was at 488 nm. Propidium iodide fluorescence was measured using a 610 nm longpass filter. Scatter parameters were used to gate out cell debris and doublets. 2.3 In-gel renaturation protein kinase assays Synchronized FT210 cells were collected and washed with cold PBS . The cells were either solubilized directly in SDS sample buffer or lysed on ice for 30 minutes in 5 0 m M T r i s - H C l p H 8.0, 1% NP-40, I m M D T T , 2 m M E D T A , 5 m M E G T A , 2 5 m M NaF, I m M N a V C ^ , I m M P M S F , 2 (ig/ml leupeptin, 2 (ig/ml aprotinin, 2 |lg/ml pepstatin, 30 | ig/ml Dnasel and 30 |Xg/ml RNaseA. Cellular debris was removed by 26 centrifugation at 16 000 x g for 20 minutes. Protein concentration was determined using the Bradford assay (Bradford 1976). Equal amounts of protein were separated by SDS polyacrylamide electrophoresis. Protein kinase substrates were polymerized directly into the separating gel at a concentration of 0.5 mg/ml. Following electrophoresis, SDS was removed by washing the gel 3 times for 20 minutes each in 50mM Tr i s -HCl p H 8.0, 20% 2-propanol. Proteins were completely denatured by incubating in 50mM Tr i s -HCl p H 8.0, 6 M guanidine-HCl for 1 hour. Renaturation of the separated proteins was achieved through successive washes with 5 0 m M Tr i s -HCl p H 8.0, 0.4% Tween-40 and 5 m M (3-mercaptoethanol at 4°C for 16 hours. The gel was preincubated in kinase buffer, 4 0 m M H E P E S p H 8.0, 5 m M M g C i 2 , 2 m M D T T , for 10 minutes. The assay was started by adding 10 | l C i / m l y [ 3 2 P ] - A T P to the kinase buffer and the gel was incubated for 1 hour with gentle shaking. Excess radioactivity was removed with extensive washing in 5% (w/v) trichloroacetic acid, 1% sodium pyrophosphate until radioactivity in the wash buffer was negligible. The gel was then dried and the phosphorylated bands viewed and quantitated using a Phosphorlmager (Molecular Dynamics). 2.4 JNK1 micropurification and Western blotting Following collection and washing in cold PBS , cells were lysed for 30 minutes on ice in 2 0 m M M O P S p H 7.2, 1% NP-40, I m M D T T , 2 m M E D T A , 5 m M E G T A , 2 5 m M NaF, I m M N a V 0 4 , I m M P M S F , 2 | ig/ml leupeptin, 2 (lg/ml aprotinin, 2 | ig /ml pepstatin, 30 flg/ml Dnasel and 30 fig/ml RNaseA, and cellular debris was removed by centrifugation. Cel l lysates were incubated with c-Jun(l-169) fused to glutathione S-transferase (GST) and bound to glutathione agarose (UBI) for 6 hours at 4°C on a rocking platform. 27 The c-Jun-agarose bead complexes were washed 3 times in 12.5mM M O P S p H 7.2, 1% NP-40, 7 .5mM M g C l 2 , 250mM N a C l , I m M D T T , I m M N a V 0 4 ) 5 m M NaF, 5 m M E D T A , 5 m M E G T A , I m M P M S F , 2 | lg/ml leupeptin, 2 u.g/ml aprotinin, 2 u.g/ml pepstatin. SDS sample buffer was added to the c-Jun beads and incubated at 37°C for 20 minutes. For Western analysis, the whole sample including the c-Jun agarose beads was resolved by 10% S D S - P A G E , transferred to nitrocellulose (Gibco) and blocked for 16 hours in P B S and 10% non-fat dry milk. The nitrocellulose sheet was incubated with a J N K 1 specific antibody (Santa Cruz) in PBS , 0.1% Tween-20. Antigen-antibody complexes were detected using a horseradish peroxidase-conjugated anti-rabbit IgG antibody and enhanced chemiluminescence (SuperSignal, Pierce). 2.5 Plkl immunoprecipitation, 32P-labelIing and activity assays Synchronized FT210 cells were collected, washed once with ice-cold PBS and lysed in 5 0 m M H E P E S p H 7.4, 1% NP-40, lOOmM N a C l , I m M D T T , 2 m M E D T A , 5 m M E G T A , 2 5 m M NaF, I m M N a V 0 4 , I m M P M S F , 2 |Xg/ml leupeptin, 2 [ig/ml aprotinin, 2 Ug/ml pepstatin, 30 (ig/ml Dnasel and 30 (ig/ml RNaseA for 30 minutes on ice and cellular debris was removed by centrifugation. Ce l l lysates were precleared by incubation with rabbit pre-immune serum for 30 minutes at 4°C on a rocking platform. Immunoprecipitation of cleared lysates was performed with the P l k l antibody, R32 (1:100; vol/vol), for 1 hour at 4°C. This was followed by incubation with protein A-agarose beads (Gibco) for 30 minutes. The beads were then washed 3 times in lysis buffer. P l k l kinase activity was determined by incubating the immunecomplexes in kinase buffer (20mM H E P E S p H 7.4, 150mM KC1, l O m M M g C l 2 , I m M E G T A , 0 .5mM D T T , 5 m M NaF) for 15 minutes at room temperature. The P l k l activity assay was performed by incubation of the beads in 20 | i l of activation buffer at 30°C for 15 minutes. The activation buffer was the kinase buffer supplemented with 0.2 uCi /u l y [ 3 2 P ] - A T P , 10 u M A T P , 0.5 28 mg/ml dephosphorylated casein (Sigma). Reactions were stopped by the addition of an equal volume of 2 x SDS sample buffer, heated at 95°C for 5 minutes and separated by S D S - P A G E . Phosphorylated casein was quantitated using a Phosphorlmager (Molecular Dynamics). For [ 3 2P]-labelling experiments, FT210 cells were synchronized to various phases of the cell cycle and incubated in normal media supplemented with 200 | i C i / m l [3 2P]-orfhophosphate (Dupont) for 3 hours. P l k l was immunoprecipitated as described above and the immunoprecipitated proteins were separated by S D S - P A G E and visualized by autoradiography. 2.6 Mitotic ELISA FT210 cell lysates were prepared by lysing cells in T B S , 1% NP40, I m M E G T A , 25 m M NaF, I m M P M S F , 2 u.g/ml leupeptin, 2 |Xg/ml aprotinin, 2 |lg/ml pepstatin. Supernatants were transferred to Polysorb E L I S A plates (Nunc) and dried. The samples were blocked for at least 1 hour in T B S , 3% non-fat dry milk (Carnation) at room temperature and then incubated for 16 hours with the TG3 antibody, diluted 1:50 (vol/vol), in blocking solution at 4°C. The plate was washed five times with 10 m M Tr i s -HCl p H 7.4, 0.02% Tween-20 wash buffer. The antigen-antibody complexes were detected by incubating with a horseradish peroxidase-conjugated anti-mouse I g M antibody (Southern Biotechnology Associates) for more than 2 hours at 4°C followed by washing in wash buffer. TG3 antigen-antibody complexes were visualized by the addition of 120 m M Na2HP04, 100 m M citric acid p H 4.0 containing 0.5 mg/ml 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.01% hydrogen peroxide for 1 hour at room temperature and read at 405 nm with a Biotek plate reader. 29 2.7 Fractionation and purification of histones Calf thymus histones ( ICN Biomedical) were resolved using a continuous elution preparative protein S D S - P A G E apparatus (Model 491, BioRad). Briefly, 10 mg of mixed histones were solubilized in SDS sample buffer and applied to a 4% polyacrylamide stacking and a 15% polyacrylamide separating gel at 4°C. A constant current of 45 m A was used to resolve the histones. A BioRad Econo System was used to collect 1.5 ml fractions as soon as the bromophenol blue tracking dye front had exited the gel. Fractions were analysed for histone content by conventional S D S - P A G E and Coomassie Blue staining. 2.8 Partial purification of p45 kinase Greater than 1 x 10 9 FT210 cells were blocked in mitosis with 50 ng/ml nocodazole for 24 hours. The cells were lysed with 10 strokes of a Dounce homogenizer in 6 ml of 5 0 m M T r i s - H C l p H 8.0, 1% NP-40, I m M D T T , 2 m M E D T A , 5 m M E G T A , 2 5 m M NaF, I m M N a V 0 4 , I m M P M S F , 2 0 0 | l M fostriecin, 2 (ig/ml leupeptin, 2 | lg /ml aprotinin, 2 (ig/ml pepstatin, 30 | lg/ml DNase l and 30 | lg/ml RNaseA and incubated on ice for 30 minutes. Cellular debris was removed by centrifugation at 35 000 x g for 30 minutes followed by filtration through a 0.8 \lm syringe filter (Nalgene). The lysate was loaded onto a Poros Q column attached to a Dionex H P L C apparatus and eluted with a linear gradient up to I M N a C l in a buffer of 50mM Tr i s -HCl p H 8.0 with a flow rate of 1.0 ml/min over 30 minutes. 20 fil of each fraction was assayed for p45 kinase activity in an in-gel kinase assay containing 0.5 mg/ml of Ets-1(29-139). Fractions containing p45 kinase activity were pooled and concentrated with a Mill ipore Ultrafree-CL, 5000 M W L concentrator. The eluate was washed repeatedly and concentrated in 50mM Tr i s -HCl p H 6.0 to change the p H and lower the N a C l concentration. 30 The eluate was subsequently loaded on a Poros HS column and eluted with a linear gradient up to 1 M N a C l in a buffer of 50mM Tr i s -HCl p H 6.0 with a flow rate of 1.0 ml/min over 30 minutes. A n in-gel kinase assay was used as previously described to determine fractions with p45 kinase activity. The eluate was concentrated and the p H adjusted to 8.0. The eluate was incubated with Ets-1(29-139) conjugated to agarose for 4 hours at 4°C on a Nutator. The Ets-1(29-139) was conjugated to agarose by a disulfide linkage using the Sulfolink system (Pierce). Briefly, 500 | l l of SulfoLink gel was equilibrated in 6 volumes of 5 0 m M T r i s - H C l p H 8.5, 5 m M E D T A - N a . 10 mg of partially purified Ets-1(29-139) was applied to the gel slurry and mixed on a rotating platform at room temperature for 15 minutes followed by a 30 minute incubation with no mixing. The gel was washed with 3 volumes of 5 0 m M Tr i s -HCl p H 8.5, 5 m M E D T A - N a and then mixed with 1 volume of 5 0 m M T r i s - H C l p H 8.5, 5 m M E D T A - N a , 50mM cysteine for 15 minutes at room temperature. The gel was further incubated for 30 minutes without mixing. The gel was washed extensively with 16 volumes of 1 M N a C l followed by 16 volumes of degassed 0.05% sodium azide and stored at 4°C. Just prior to use an aliquot of gel required for the experiment was incubated in 6 M guanidine chloride for 45 minutes to denature the Ets-1(29-139) substrate. The gel was washed 6 times to remove all the guanidine chloride and incubated with the eluate. Following incubation the beads were washed 4 times with 2 5 m M Tr i s -HCl p H 8.0, 250mM N a C l , 1% NP-40 and incubated in SDS sample buffer for 15 minutes at 37°C. The entire sample including the beads was resolved by S D S - P A G E and an in-gel kinase assay was performed. 2.9 Preparation of Ets-1(29-139) The Ets-1(29-139) plasmid was a generous gift of L . Donaldson. A n exponentially growing culture of BL21(DE3) (genotype: E. coli B F" dcm ompT hsdS{v^'m^') 31 ga//L(DE3)) E.Coli transformed with the Ets-1(29-139) plasmid were induced with I m M I P T G at an ODgoo of approximately 1.0. The culture was grown for an additional 3 hours and the bacteria were lysed by sonication in a buffer of 50 m M Tr i s -HCl p H 8.5, 2 | ig/ml leupeptin, 2 |0.g/ml aprotinin, 2 (ig/ml pepstatin, 10 mg/ml D T T for 4 minutes at 4°C. Cellular debris and unlysed cells were removed by centrifugation at 35 000 x g for 30 minutes and the supernatant was filtered through a 0.8 um syringe filter (Nalgene). The supernatant was loaded on a Mono Q column attached to a Pharmacia F P L C system and eluted with a linear gradient up to 500 m M N a C l in Tr i s -HCl p H 8.5 with a flow rate of 4 ml/min. Fractions were analyzed for Ets(29-139) by S D S - P A G E and Coomassie Blue staining. 32 Chapter 3 Identification of a novel 45 kDa mitotic kinase activated by protein phosphatase inhibitors 3.1 A 45 kDa mitotic protein kinase is activated by protein phosphatase inhibitors independently of Cdc2 in FT210 cells In an effort to identify mitotic kinases activated independently of Cdc2 by protein phosphatase inhibitors, various substrates were used in an in-gel kinase assay (Hutchcroft et al. 1991; Heider et al. 1994). Briefly, FT210 cells were synchronized to various phases of the cell cycle (Fig. 9). These cells were treated with or without 150 | l M fostriecin or 0.5 j i M okadaic acid for 2 hours. The cells were either lysed and nuclei and other cellular debris removed by centrifugation or solubilized directly in SDS sample buffer. Both methods of sample preparation gave identical results in the in-gel kinase assay. The proteins were separated by SDS polyacrylamide electrophoresis in a gel containing substrate proteins at a concentration of 0.5 mg/ml. After sequential denaturation and renaturation of the proteins in the gel matrix, the gel was incubated in a kinase assay buffer containing y [ 3 2 P ] - A T P . Renatured kinases that can phosphorylate the included substrate allow for detection of substrate-specific protein kinase activity. The gel was washed extensively to remove unincorporated radiolabel, dried and exposed to a Phosphorlmager screen to visualize the phosphorylated bands (Fig. 10). This assay provides a "snapshot" of the protein kinases active at a particular phase of the cell cycle and allows for direct comparison of the cell cycle-regulated activity of 33 Relative DNAcnntcnt Figure 9. Cell cycle synchronization of FT210 cells To synchronize cells in S and early G2 phases of the cell cycle, cells were treated for 16 hours with aphidicholin (1.25 fig/ml) to arrest them at the Gl/S boundary. The cells were released from the aphidicholin block for 9 hours and incubated again with aphidicholin for 16 hours. Cells were released from the second aphidicholin block for 3 hours or 6 hours and designated the S and S/G2 populations respectively. Late G2 and mitotic cell populations were obtained by incubating cells in isoleucine-deficient RPMI-1640 supplemented with 10% heat-inactivated dialyzed FBS for 16 hours; followed by release into complete medium. A mitotic population, M, was achieved by incubating released cells in the presence of nocodazole (50 ng/ml) for 16 hours. To obtain a late G2 population, released cells were incubated in aphidicholin (1.25 jig/ml) for 9 hours at 32°C. Following release from the aphidicholin block, the cells were incubated at 39°C for 16 hours resulting in a late G2 population of cells designated; 39°C. Cell cycle synchronization was monitored by FACS analysis. Briefly, cells were fixed with paraformaldehyde, DNA was stained with propidium iodide, and analysed by flow cytometry. 34 MW S S/G2 39°C M 106 -69 -44 -kDa - +OA - +OA - +OA 1»W -• p w w -p60 #-p40/42 Figure 10. A 45kDa mitotic kinase is activated by phosphatase inhibitors independently of Cdc2 In-gel kinase assay of renatured proteins from FT210 cell total lysates synchronized to various stages of the cell cycle. Cells were left untreated or treated with 0.5 | i M okadaic acid (OA) for 2 hours. The in-gel kinase assay contained 0.5 mg/ml Ets-1(29-139) as substrate. The positions of the p40/42, p45 and p60 kDa kinases are indicated by arrows. 35 various unknown kinases. The in-gel kinase assay is limited to only those protein kinases which do not require a regulatory subunit for activity, and which can become active following denaturation and renaturation. Some kinases were active at all stages of the cell cycle, such as p40/42 in Fig . 10. Other kinases, such as p60, were active only upon phosphatase treatment, but displayed no cell cycle stage specificity. One in-gel kinase substrate consisted of amino acids 29-139 of the Ets-1 transcription factor in which the leucine residue at position 36 had been mutated to a proline to create a consensus M A P kinase site, P X T P (personal communication L . Donaldson and L . P . Mcintosh). Incorporation of this Ets-1(29-139) substrate in an in-gel kinase assay identified a 45 kDa protein kinase (p45) as a cell cycle-regulated mitotic kinase (Fig. 10). p45 kinase activity was observed only in cells blocked in mitosis with nocodazole. The kinase was not observed at all other stages of the cell cycle in synchronized FT210 cells. Interestingly, p45 kinase was activated in FT210 cells, which express a temperature-sensitive Cdc2 mutant at the non-permissive temperature of 39°C, by treatment with the protein phosphatase inhibitors okadaic acid (Fig. 10) and fostriecin (not shown). This demonstrates that p45 kinase can be activated independently of the major mitotic kinase Cdc2. 3.2 The p45 kinase is found in all cell lines It was determined that p45 kinase was active during mitosis in all cell lines tested (Fig. 11). These included a human breast cancer cell line, M C F 7 ; a human T cell line, Jurkat; and a human Alzheimer primary fibroblast cell line, 4148A. The ubiquity of the p45 kinase suggests an essential role in mitosis. 36 MW Cell lines kDa 199 -106 -69 -44 -2 9 -4148A Jurkat MCF7 FT210 * • •1 f 111' • 3 m Figure 11. Detection of p45 kinase activity in various cell lines In-gel kinase assays of mitotic total cell extracts from FT210, M C F 7 , Jurkat and Alzheimer skin fibroblasts, 4148A, cells. The position of the p45 kinase is indicated by the arrow. The in-gel kinase assay contained 0.5 mg/ml Ets-1(29-139) as substrate. 37 3.3 Myelin basic protein is preferred over Ets-1(29-139) as a substrate for p45 kinase In-gel kinase assays were also used to characterize the in vitro substrate specificity of p45 kinase. The standard protein kinase substrates: casein, histones and myelin basic protein as well as Ets-1(29-139) were polymerized directly into SDS gels at 0.5 mg/ml concentration. p45 kinase autophosphorylation activity was determined using bovine serum albumin as the in-gel substrate. Kinase activity was measured using a Phosphorlmager and was standardized relative to p45 kinase's activity with Ets-1(29-139) (Fig. 12). Mye l in basic protein (MBP) was the preferred substrate for p45 kinase, being approximately 4 times more highly phosphorylated than the Ets-1(29-139) substrate. Neither casein nor histone substrates were significantly phosphorylated above p45 kinase autophosphorylation levels. The substrate preference can be summarized as follows: myelin basic protein > Ets-1(29-139) > casein = histone = B S A . 3.4 The p45 kinase is not JNK1 A candidate p45 mitotic protein kinase was the c-Jun N-terminal kinase 1, J N K 1 . J N K 1 , is a 46 kDa member of the M A P kinase superfamily that was initially identified by its ability to bind to the transcription factor c-Jun (Kyriakis et al. 1994). Other members of this family include the 55 kDa J N K 2 and p 3 8 / H O G l (Han et al. 1994). J N K 1 is activated in response to cellular stress such as U V radiation and heat shock, and is involved in ceramide initiated stress-induced apoptosis (Adler et al. 1995; Westwick et al. 1995; Verheij et al. 1996). Activation of the JNK1 pathway by the Rho, Rac and Cdc42 GTPases has been demonstrated during cell cycle progression through G l phase (Olson et al. 1995). The role of J N K 1 activation during cell cycle progression has not been determined. 38 5 4H > 3H •s 2H — ets-1 3__E 3 _ E BSA histone casein MBP Figure 12. Myelin basic protein is a preferred substrate of p45 kinase In-gel kinase assays were performed on mitotic FT210 total cell lysates in gels containing 0.5 mg/ml of histones, dephosphorylated casein, Ets-1(29-139), B S A or M B P . p45 kinase activity was quantitated using a Phosphorlmager. p45 kinase activity was standardized relative to Ets-1(29-139) activity. 39 It had been previously reported that two kinases of 46 and 55 kDa were activated in cells treated with the translational inhibitor anisomycin and that this activity was enhanced by treatment with okadaic acid (Cano et al. 1994). The kinases were originally identified using an in-gel kinase assay with c-Jun as a substrate. Based on this observation it was proposed by Cano et al. that the 46 kDa kinase was J N K 1 and the 55 k D a kinase was J N K 2 . Therefore, I wanted to determine whether the mitotic p45 kinase was in fact J N K 1 . Precipitations of cell lysates from mitotic FT210 cells using a c-Jun N-terminal polypeptide (residues 1-169) fused to G S T and conjugated to agarose failed to deplete the p45 kinase (Fig. 13). Moreover, precipitated JNK1 was inactive in the in-gel kinase assay, although it was readily detected by immunoblotting with J N K 1 antibodies (Fig. 13). Anisomycin is a protein synthesis inhibitor which induces chromatin-associated protein phosphorylation and immediate-early gene induction similarly, to epidermal growth factor (EGF) and tetradecanoyl phorbol acetate (TPA) (Mahadevan et al. 1991). Anisomycin stimulates a subset of the responses elicited by E G F and T P A at protein synthesis subinhibitory concentrations (Edwards and Mahadevan 1992). Most importantly, anisomycin has been demonstrated to be a specific activator of J N K 1 (Kyriakis et al. 1994; Cano et al. 1995). Anisomycin failed to activate p45 kinase in Cdc2 mutant FT210 cells blocked in late G2 at the non-permissive temperature (Fig. 14) and did not induce mitotic events (Fig. 15). Therefore, the p45 kinase is probably not J N K 1 . 3.5 The p45 kinase is not MAPKAP kinase-2 Recently, Cano et al. (1996) reported that the anisomycin-activated 46 and 55 kDa kinases identified by in-gel kinase assay were the murine isoforms of M A P kinase-activated protein kinase-2 ( M A P K A P kinase-2). M A P K A P kinase-2 is activated by 40 A.» In-sel kinase assay -B* In-gel kinase assay -13 • JNK1 IP Western blot cell post IP IP lysate supt. 1 2 3 Figure 13. The p45 mitotic kinase is not JNK1 A. In-gel kinase assay o f total lysates f rom nocodazole-arrested cel ls before, lane 1, and after precipitat ion w i th c-Jun(l-169)-GST conjugated to agarose, lane 2. Lane 3 is the precipitated J N K 1 . The gel contains 0.5 mg/ml Ets-1(29-139) as substrate. B. Western blot analysis using a J N K 1 antibody o f proteins immunoprec ip i ta ted w i th c-Jun(l-169) beads. The arrow indicates the pos i t ion o f the p45 kinase. 41 39°C M — Anis Anis +OA — +OA kDa +OA wKM mmSWrn x. 29 -Figure 14. Effect of anisomycin on p45 kinase activity in FT210 cells at the non-permissive temperature Synchronized FT210 cells at the non-permissive temperature (39°C) were treated with or without 50 | lg/ml of anisomycin (Anis) for 4 hours and with or without 0.5 | i M okadaic acid (OA) for 2 additional hours. Nocodazole-arrested mitotic cells (M) were used as a positive control. The arrow indicates the position of p45 kinase. The in-gel kinase assay contained 0.5 mg/ml Ets-1(29-139) as substrate. 42 0 0.05 0.5 10 20 Mitotic Anisomycin concentration Hg/ml Figure 15. Effect of anisomycin on entry into mitosis in Cdc2 mutant FT210 cells at 39°C Mitotic ELISA was performed on total cell lysates from synchronized FT210 cells at 39°C treated for 4 hours with increasing concentrations of anisomycin. Mitotic activity was detected with the TG3 antibody. TG3 reactivity was expressed relative to that of mitotic cells blocked with nocodazole. The Bradford assay was used to confirm equal protein concentrations for each sample. 43 phosphorylation by p 3 8 / H O G l and phosphorylates Hsp25/27 (Rouse et al. 1994; Cuenda et al. 1995). M A P K A P kinase-2 was demonstrated to bind to the amino terminus of c-Jun (Cano et al. 1996). As shown previously, the p45 kinase has no affinity for the amino terminus of c-Jun (Fig. 13). Furthermore, in murine cells, there are two isoforms of M A P K A P kinase-2 of 45 and 55 kDa respectively, whereas in human cells, M A P K A P kinase-2 exists solely as a 50 kDa isoform (Cuenda et al. 1995; Huot et al. 1995; Cano et al. 1996). The identified mitotic p45 kinase displays no change between its migration in murine FT210 cells, and human cells, Jurkat and M C F 7 cells (Fig. 11) and in this respect is distinct from M A P K A P kinase-2. 3.6 The effects of the protein kinase inhibitors staurosporine and chelerythrine chloride on p45 kinase activity Protein kinase inhibitors are valuable tools to identify possible candidates for an unknown kinase and to reveal putative regulatory pathways. The protein kinase inhibitor staurosporine inhibits effectively a number of kinases (discussed in section 1.6.2). Previous studies have demonstrated that staurosporine induces cell cycle arrest in both G l and G 2 / M (Abe et al. 1991). Staurosporine has been shown to antagonize the induction of mitosis by okadaic acid and fostriecin and to cause chromosome decondensation in mitotic cells (Th'ng et al. 1994; Guo et al. 1995). Chelerythrine chloride is a highly selective P K C inhibitor in vitro (IC50 0.66 f lM) exhibiting little inhibition of cAMP-dependent protein kinases, Cdc2-cyclin B kinase, Ca2+/calmodulin-dependent kinases and tyrosine kinases (Herbert et al. 1990). Chelerythrine chloride has been shown to block cells at the G 2 / M transition with high Cdc2-cyclin B activity (Thompson and Fields 1996). This may suggest an essential role of P K C in mitosis. The effects of these two inhibitors on mitosis are well characterized and 44 therefore I was interested in determining the effects of these protein kinase inhibitors on the activity of mitotic p45 kinase. Synchronized Cdc2 mutant FT210 cells at 39°C were treated with 100 ng/ml staurosporine for 3 hours and subsequently with or without 0.5 [ i M okadaic acid for 2 hours to induce mitotic events. Similarly, nocodazole-blocked mitotic FT210 cells were treated with 100 ng/ml staurosporine for 2 and 4 hours. In-gel kinase assays performed on total cell lysates revealed that staurosporine did not inhibit the activation of p45 kinase by okadaic acid but did inhibit p45 kinase activity in mitotic cells (Fig. 16). Furthermore, E L I S A demonstrated that staurosporine did not antagonize the reactivity of the mitotic antibody TG3 in cells treated with okadaic acid but caused decreased TG3 reactivity in mitotic cells treated with staurosporine (Fig. 17). These results indicate that although the p45 kinase is sensitive to staurosporine in mitotic cells, the activation of p45 kinase by the potent protein phosphatase inhibitor okadaic acid is not affected by staurosporine. Similar experiments were performed with the in vitro P K C inhibitor chelerythrine chloride. Chelerythrine chloride blocked cycling cells in G2 and antagonized the induction of mitosis by okadaic acid in FT210 cells at 39°C (Fig. 18). Chelerythrine chloride also inhibited p45 kinase activation (Fig. 19). This was shown by treating FT210 cells at 39°C with 20 \iM chelerythrine chloride for 4 hours and with or without 0.5 \iM okadaic acid for 2 hours and carrying out an in-gel kinase assay on the cell lysates. Conversely, nocodazole-blocked mitotic FT210 cells treated with chelerythrine chloride for 2 and 4 hours showed no decrease in p45 kinase activity (Fig. 19). These results show that the p45 kinase is not directly inhibited by chelerythrine chloride and suggest a role for the target of chelerythrine chloride in the induction of mitosis. 45 MW kDa 199 -106 -69 -44 -29 -39°C M +STSP +STSP — 100ng lOOng +OA+OA +STSP +STSP lOOng lOOng 2hr 4hr Figure 16. The effect of staurosporine on p45 kinase activity In-gel kinase assay of synchronized FT210 cells at the non-permissive temperature (39°C) treated with 100 ng/ml staurosporine (STSP) for 1 hour and with or without 0.5 u M okadaic acid (OA) for 2 additional hours. Nocodazole-blocked mitotic FT210 cells (M) were treated with 100 ng/ml staurosporine for 2 and 4 hours respectively. The arrow indicates the position of the p45 kinase. The in-gel kinase assay contained 0.5 mg/ml Ets-1(29-139) as substrate. 46 1.25 • H T w £3 0.75-^ a > 0.5 H 0.25 H X T +S +S +OA +OA T +S 2hr +S 4hr 39°C Mitotic cells Figure 17. The effect of staurosporine on mitosis FT210 cells were synchronized and blocked in G2 at 39°C, treated with 100 ng/ml staurosporine (S) for 3 hours and with or without 0.5 ( i M okadaic acid (OA) for 2 hours. Similarly, FT210 cells were blocked in mitosis by treating with 50 ng/ml nocodazole for 16 hours and were treated with 100 ng/ml staurosporine for 2 and 4 hours. Mitotic E L I S A was performed on total cell lysates using the mitotic indicator antibody, T G 3 , as previously described. TG3 immunoreactivity is expressed relative to that of nocodazole-blocked FT210 cells. 47 A +Okadaic acid oi 1 1 1 1 1 oH , 1 1 1 1 O 10 20 30 40 50 0 10 20 30 40 50 Chelerythrine chloride concentration (jxM) Chelerythrine chloride concentration ((iM) Figure 18. The effect of chelerythrine chloride on entry into mitosis A. Asynchronously growing MCF7 cells were incubated with increasing concentrations of chelerythrine chloride and nocodazole (50 ng/ml) to block the cells in mitosis. After 16 hours, mitotic ELISA was performed on total cell lysates using the mitotic indicator antibody, TG3 as previously described. TG3 reactivity was expressed relative to that of cells treated with nocodazole only. B. FT210 cells at the non-permissive temperature were incubated with increasing concentrations of chelerythrine chloride for 4 hours and treated with 0.5 |J.M okadaic acid for 2 hours to induce mitosis. TG3 reactivity was expressed relative to that of cells treated with okadaic acid only. 48 39°C - CC CC -20uM 20uM +OA+OA M CC CC 20uM 20uM 2hr 4hr 44 -29 -Figure 19. Chelerythrine chloride inhibits p45 kinase activation In-gel kinase assay of cell extracts from FT210 cells at the non-permissive temperature (39°C) treated with or without 20 uM chelerythrine chloride (CC) for 4 hours followed by incubation with 0.5 uM okadaic acid (OA) for 2 hours. Nocodazole-blocked mitotic FT210 cells (M) were treated with 20 uM chelerythrine chloride for 2 and 4 hours respectively. The arrow indicates the position of the p45 kinase. The in-gel kinase assay contained 0.5 mg/ml Ets-1(29-139) as substrate. 49 3.7 Partial purification of the p45 kinase It is necessary to completely purify the p45 kinase to generate the powerful molecular biological and immunological tools required to fully identify and investigate the mitotic function of p45 kinase. This section reports the partial purification of p45 kinase. A cellular extract was prepared from nocodazole-blocked mitotic FT210 cells and the insoluble fraction containing mostly nuclei and cytoskeletal elements was removed. The extract was separated using a linear N a C l gradient on an anion exchange column using an H P L C apparatus. After each purification step, an in-gel kinase assay was used to determine the fractions containing p45 kinase activity. The p45 kinase eluted over three fractions, 23, 24 and 25, at a N a C l concentration of approximately 850 m M (Fig. 20). These fractions were pooled, concentrated and dialyzed to lower the pH and the N a C l concentration. The eluate was loaded on a cation exchange column attached to a H P L C apparatus and separated with a linear N a C l gradient. p45 kinase positive fractions eluted at a N a C l concentration of approximately 350 m M (not shown). The Ets-1(29-139) protein was conjugated to agarose and used as an affinity matrix to purify p45 kinase. The p45 kinase in a total cell lysate bound effectively to the column when the conjugated Ets-1(29-139) was first denatured with 6 M guanidine hydrochloride. This precipitation was not reproducible when the p45 kinase fractions from the cation exchange chromatography step were used, even in an identical buffer to that of the total cell lysate. The p45 kinase may not be active following the two ion exchange chromatography purifications. It may be necessary to employ Ets-1(29-139) affinity chromatography as the initial step in the purification of the p45 kinase. The in-gel kinase assay should be a good final purification step which wi l l separate proteins according to their molecular weight. Electroelution of the p45 kinase from the gel matrix wi l l be followed by tryptic digestion of the p45 kinase to obtain peptides for partial sequence analysis. 50 Fraction number M 2 4 6 8 10 12 14 16 18 Fraction number M 20 22 24 26 28 30 FT ^§ ***** aj§$ Figure 20. Partial purification of p45 kinase A cytop lasmic extract o f nocodazole-blocked mitot ic F T 2 1 0 cel ls was separated on an anion exchange co lumn and an in-gel kinase was performed to ident i fy fractions conta in ing p45 kinase. Tota l ce l lu lar extract f rom mitot ic F T 2 1 0 ce l ls , (M) was used as a posi t ive control . The arrows indicate the posi t ion o f the p45 kinase. The in-gel kinase assay contained 0.5 mg/ml Ets-1(29-139) as substrate. 51 Chapter 4 Identification of a histone-specific kinase activity in Cdc2 mutant FT210 cells One of the hallmarks of mitosis is the striking condensation of the dispersed interphase chromatin into metaphase chromosomes. The process of chromosome condensation remains largely an enigma at the molecular level. Phosphorylation of histones H I and H3 is closely correlated with chromosome condensation (Gurley et al. 1978; Bradbury 1992). Histone H I is progressively phosphorylated from 2-3 phosphates in G 2 to 5-6 phosphates at mitosis (Davis et al. 1983). This hyperphosphorylation coincides with a 15 to 20 fold increase in Cdc2-cyclin B activity at mitosis (Bradbury et al. 1974a; Bradbury et al. 1974b). Cdc2-cyclinB has been demonstrated to directly phosphorylate histone H I in vivo. Histone H3 is phosphorylated only at mitosis, on serine 10 in the amino-terminal tail (Gurley et al. 1978). The kinase(s) responsible for this phosphorylation are unknown. Induction of mitotic events by phosphatase inhibitors in FT210 cells at the non-permissive temperature results in a phenotypically normal condensation of chromosomes (Guo et al. 1995). This condensation correlates with increased phosphorylation of histones H3 and H 2 A in the absence of increased histone H I phosphorylation (Guo et al. 1995). This result shows that histone H I hyperphosphorylation is not required for chromosome condensation and suggests an involvement of histone H3 phosphorylation. Identifying the kinase(s) responsible for histone H3 phosphorylation would help to understand the role of histone H3 phosphorylation in the process of mitotic chromosome condensation. The goal of this work was to identify putative histone H3 kinases in FT210 cells lacking Cdc2-cyclin B activity. 52 4.1 Fractionation of histones from mixed histone samples It is very difficult to separate individual histones to purity by conventional chromatographic techniques as all histones are extremely basic proteins and have many of the same physical characteristics. In order to prepare purified histones H3 and H I in the quantities required for in-gel kinase assays, a continuous elution electrophoresis technique was employed. Briefly, purified calf thymus histones were separated using a preparative protein electrophoresis apparatus, Model 491 Prep Cel l (Bio-Rad). Histone samples were solubilized in SDS sample buffer and resolved by continuous elution S D S - P A G E . Fractions were collected and analysed by S D S - P A G E and Coomassie Blue staining. It was possible to completely fractionate and purify each individual histone (Fig. 21). These purified histone fractions were used as substrates in an in-gel kinase assay. 4.2 Identification of a histone kinase in FT210 cells To determine the presence of a histone H3 kinase in Cdc2 mutant FT210 cells, lysates were prepared from cells in various phases of the cell cycle and treated with phosphatase inhibitors or left untreated. These lysates were separated by S D S - P A G E in a gel including 0.5 mg/ml of purified histone H3 as the kinase substrate. A n in-gel kinase assay showed the presence of a 60 kDa histone H3 mitotic kinase activated by phosphatase inhibitors (Fig. 22). The kinase was present, and could be activated by okadaic acid, in all phases of the cell cycle that were examined. Treatment of FT210 cells in S and G2 phases of the cell cycle with phosphatase inhibitors resulted in chromosome condensation (Guo et al. 1995). These results correlate with the observed activity of the 60 kDa histone H3 kinase. Parallel assays in which B S A , casein and Ets-1(29-139) were used as the in-gel substrates failed to show kinase activity (Fig. 23). In order to determine the specificity of the putative histone H3 kinase, other purified histones were used as in-gel kinase substrates. The histone H3 kinase was also active in the presence of histones H I (Fig. 23) and H 2 A (result not shown). Both of these histones are 53 Fraction number 35 40 45 50 55 60 65 70 75 Figure 21. Fractionation and purification of histones Histone samples were resolved by continuous elution S D S - P A G E using a BioRad preparative protein electrophoresis apparatus and fractions were collected Fractions were analyzed by S D S - P A G E and Coomassie Blue staining. Fractions containing purified histones H3 , H 2 A and H I were pooled and used as substrates for in-gel kinase assays. 54 Figure 22. Identification of a cell cycle-dependent histone kinase in FT210 cells In-gel kinase assay of total cell extracts from FT210 cells synchronized to the indicated phases of the cell cycle and treated with or without 0.5 | i M okadaic acid ( O A ) for 2 hours. The arrow indicates the position of an approximately 60 k D a histone-specific kinase. The in-gel kinase assay contained 0.5 mg/ml histone H3 as substrate. 55 Figure 23. Specificity of the histone kinase in FT210 cells In-gel kinase assays of cell extracts from FT210 cells with the following substrates at a concentration of 0.5 mg/ml incorporated in the gel: A . B S A , B. Histone H I , C . Ets-1(29-139) or D. casein. The arrows indicate the position of the histone specific kinase. 56 phosphorylated in eukaryotic cells whereas histone H4 and histone H 2 B are not. This demonstrated that the 60 kDa protein kinase is not specifc to histone H3 but rather a cell cycle-regulated mitotic histone kinase. 57 Chapter 5 Investigation of the activation of polo-like kinase-1 in Cdc2 mutant FT210 cells Chromosome segregation is a highly complex and dynamic process that relies on the assembly and function of a microtubule-based mitotic spindle apparatus (Koshland 1994). Genetic studies have identified several protein kinases required for proper spindle formation and function. Not surprisingly, Cdc2 has been shown to play a role in the regulation of spindle function. During mitosis, Cdc2 is partly localized to the spindle and it is able to modulate microtubule dynamics in Xenopus cell-free extracts (Bailley et al. 1989; Verde et al. 1990). Indeed, it has been recently shown that spindle-associated, kinesin-related motor proteins are likely physiological substrates of Cdc2 (Blangy et al. 1995). Mitotic protein kinases other than Cdc2 have been proposed to have a role in spindle function and chromosome segregation. Mutations in the serine-threonine protein kinase Polo in D. melanogaster and the structurally related Cdc5p in S. cerevisiae lead to abnormal mitotic and meiotic divisions and polyploidy (Llamazares et al. 1991; Kitada et al. 1993). In cells depleted of Cdc5, nuclear division becomes arrested at a point where the nuclei are connected by a thin bridge of chromatin resulting in a dumbbell terminal morphology (Kitada et al. 1993). The human homologue of Polo is a 68kDa protein termed polo-like kinase 1, P l k l (Golsteyn et al. 1994). The activity of P l k l is low in G l , increases during S and G 2 / M and is rapidly reduced after mitosis. P l k l binds to components of the mitotic spindle apparatus during mitosis. During prophase and metaphase, P l k l associates with microtubules at the spindle poles but relocalizes to the equatorial plane at anaphase (Golsteyn et al. 1995). During mitosis, P l k l is phosphorylated on serine by an unknown kinase(s). Cdc2 is localized to the spindles during mitosis and can phosphorylate P l k l in vitro but has little effect on P l k l activity (Hamanaka et al. 1995). At the spindle poles, P l k l has been shown 58 to associate with mitotic kinesin-like protein-1, M K L P - 1 (Lee et al. 1995). Plkl-associated kinase activity phosphorylates and activates M K L P - 1 in vitro. M K L P - 1 has been demonstrated to cross-bridge anti-parallel microtubules which slide over one another in vitro and may be involved in chromosome segregation in vivo (Nislow et al. 1992). I was interested in determining the activity of P l k l in Cdc2 mutant FT210 cells and investigating the activity of P l k l in these cells following induction of mitosis by treatment with phosphatase inhibitors. 5.1 Plkl activity and abundance increases in a cell cycle dependent manner I first examined the activity of mouse P l k l during the cell cycle. P l k l activity in FT210 cells was low in S phase and increased through G2 phase (Fig. 24B,C). The peak activity of P l k l occurred at mitosis. The abundance of P l k l in the cell also increased in a cell cycle dependent manner (Fig. 24A) agreeing with previously published results (Golsteyn et al. 1995) and confirming the presence of potentially activatable P l k l . 5.2 Plkl activation is dependent upon Cdc2 P l k l was immunoprecipitated from cell lysates prepared from FT210 cells synchronized to various phases of the cell cycle and treated with phosphatase inhibitors or left untreated for two hours. P l k l activity was determined to examine the dependence of P l k l activity on the cell cycle position and the presence of Cdc2. Interestingly, P l k l activity was very low at the non-permissive temperature in FT210 cells, 39°C, when Cdc2 was degraded (Fig. 24). The activity was well below the level of activity in S or G2 phase cells (Fig. 24C). Induction of mitosis in Cdc2 mutant FT210 cells by treatment with phosphatase inhibitors did not activate P l k l . Furthermore, immunoblot analysis revealed abundant amounts of P l k l indicating that it was not degraded in FT210 cells at 39°C (Fig. 24A). 59 S S/G2 39°C M A. - + O A - +OA Plkl Western blot B . Plkl Activity C. 150' > 100 H a > M 50 H i 1 1—r S G2G2+OA 39 39+OA M D . 39°C M - +OA - +OA Phosphorylation of Plkl Figure 24. Plkl activity and abundance in FT210 cells at the non-permissive temperature A. Immunoblot of P l k l in cellular extracts from FT210 cells synchronized to various phases of the cell cycle and treated with 0.5 uJVI okadaic acid (OA) for 2 hours or left untreated. Immunoblotting was performed using the anti-Plkl antibody R32. B. Immunoprecipitation of P l k l from cell extracts prepared from FT210 cells synchronized to the indicated phases of the cell cycle. P l k l activity was assayed using dephosphorylated casein as a substrate. The reaction products were analyzed by S D S - P A G E and exposure to a Phosphorlmager. C. Densitometric quantitation of B. D. Incorporation of [ 3 2P]-labelled orthophosphate into P l k l in FT210 cells at 39°C and at mitosis with or without treatment with 0.5 | i M okadaic acid for 2 hours. 60 In vivo [ 3 2P]-labelling of P l k l indicated that P l k l was phosphorylated when it was active at mitosis but was not phosphorylated when inactive at 39°C in the presence or absence of protein phosphatase inhibitors (Fig. 24D). These results suggest that post-translational modification is required for P l k l activity and that P l k l activity is dependent directly upon Cdc2 signaling pathways. P l k l has no consensus Cdc2 phosphorylation sites and is phosphorylated by unknown kinases in vivo, but not directly by Cdc2 (personal communication H . Lane and E . Nigg). 61 Chapter 6 Discussion 6.1 The identification of a novel mitotic 45 kDa kinase The process of cell division is tightly regulated to ensure the correct segregation of genetic and cytoplasmic material into the daughter cells. Protein kinases and phosphatases temporally control the events of mitosis through reversible protein phosphorylation. The major known mitotic kinase is Cdc2, which has been demonstrated to act on a wide variety of substrates in regulating mitotic events (reviewed by Nigg 1993). Recent studies have determined that other protein kinases act independently of Cdc2 at mitosis. In particular, expression of the Aspergillus nidulans kinase N I M A causes the induction of mitotic events in the absence of Cdc2 activity and mammalian cells arrest in G2 with high Cdc2 activity in the presence of the specific P K C inhibitor chelerythrine chloride (O'Connell et al. 1994; Thompson and Fields 1996). Furthermore, the mouse cell line FT210 has a temperature-sensitive lesion in the Cdc2 gene which results in cell cycle arrest in late G2 at the non-permissive temperature. Treatment of these G2-arrested cells with protein phosphatase inhibitors results in the induction of mitotic events (Guo et al. 1995). This result demonstrates the existence of Cdc2-independent mitotic signaling pathways and provides an experimental system for the identification of the protein kinases involved in these pathways. I report the identification of a 45 kDa protein kinase, p45, that is activated by protein phosphatase inhibitors independently of Cdc2. p45 kinase activity was detected only at mitosis in all cell lines tested, FT210, Jurkat, M C F - 7 and 4148A. The ubiquity of 62 p45 kinase suggests an essential, albeit unknown, role in mitosis. It wi l l be very interesting to identify and investigate the function of this protein kinase in mitosis. The use of inhibitors to gain insight into the regulation of p45 kinase was inconclusive. The highly specific in vitro P K C inhibitor chelerythrine chloride arrested cycling cells in G2 and antagonized the induction of mitosis and activation of the p45 kinase by the protein phosphatase inhibitor okadaic acid. Treatment of mitotic cells with chelerythrine chloride did not result in the inhibition of mitotic events as determined by mitotic E L I S A and p45 kinase activity. Although chelerythrine chloride is a specific in vitro and in vivo inhibitor of P K C it has yet to be conclusively determined i f it targets kinases other than P K C in vivo. These results do suggest that a target for chelerythrine chloride, possibly P K C , is essential for entry into mitosis through Cdc2-independent pathways. The effects of staurosporine on p45 kinase activity are contradictory. Staurosporine does inhibit p45 kinase activity in mitotic cells although staurosporine cannot antagonize the activation of p45 kinase by okadaic acid. Okadaic acid is an extremely powerful protein phosphatase inhibitor that may "superactivate" protein kinases. It is possible that staurosporine was not potent enough to counteract the effects of okadaic acid. Interestingly, staurosporine does inhibit okadaic acid-induced chromosome condensation in Cdc2 mutant FT210 cells and treatment of mitotic cells with staurosporine results in chromosome decondensation and nuclear membrane reformation (Th'ng et al. 1994; Guo et al. 1995). These observations suggest that the p45 kinase is not involved in chromosome condensation or in the disassembly of the nuclear envelope at mitosis. A very early event of mitosis that occurs in okadaic acid-treated FT210 cells prior to any detectable condensation of the chromosomes is the formation of the bipolar centrosomes. I have observed that normal spindles are observed in FT210 cells treated with okadaic acid. It has not been determined what effects staurosporine treatment has on the mitotic microtubule array and spindle poles. It is tempting to speculate that p45 kinase may have a role in the 63 formation of the bipolar spindles. The identification of the p45 kinase is necessary to conclusively determine a role of the p45 kinase in mitosis. Very few protein kinases have been identified with a molecular weight of approximately 45 kDa. M y results demonstrate that the p45 kinase is not the c-Jun N -terminal kinase 1, J N K 1 , or the M A P kinase-activated protein kinase-2. Recently, a 46 kDa mitotic kinase was detected in HeLa cells using mixed histones as the in-gel substrate (Monteiro and Mica l 1996). The protein kinase identified in this study displayed little activity when histones were used as the in-gel substrate, barely above endogenous autophosphorylation activity. Monteiro and Mica l employed a four hour kinase reaction while I have employed a one hour reaction. The four hour reaction is unnecessarily long and may lead the authors to incorrectly deduce histone specificity for this kinase. A 40 kDa kinase was also reported to be activated in mitotic HeLa cells (Heider et al. 1994). This kinase was hyperactivated by protein phosphatase treatment and was found to be distinct from p42 and p44 M A P kinases by immunoblotting. Both p42 and p44 M A P kinases are active early in G l and show little activity in nocodazole-arrested cells (Edelmann et al. 1996). This is the opposite of p45 kinase which is inactive in G l and active in nocodazole-arrested cells. It is not apparent i f the two unidentified 40 and 46 kDa mitotic HeLa cell kinases are identical or i f these kinases are the same as the p45 kinase. The pattern of kinase bands activated by phosphatase inhibitors in Cdc2 mutant FT210 cells is very similar to that reported by Heider et al. Unti l the p45 kinase is identified this issue wi l l remain unresolved. A purification strategy has been developed to identify the p45 kinase. The first two chromatographic steps, anion and cation exchange chromatography, have been effective in partially purifying the kinase. Protein kinase purification often requires an affinity chromatography step. I have used a substrate affinity column with the Ets-1(29-139) 64 substrate coupled to agarose beads. Unfortunately, p45 kinase d id not b ind reproducibly to the co lumn. It may be necessary to f i nd alternatives to this step i n order to completely puri fy the p45 kinase. One possibi l i ty is the use of a B lue Sepharose co lumn. The dye C ibac ron B l u e serves as an analog o f ADP-r ibose and binds strongly to the A T P b ind ing site o f kinases. Another alternative wou ld be to use M B P to create an aff inity co lumn. M B P is a much better substrate o f p45 kinase than Ets-1(29-139) and may b ind more effect ively to p45 kinase. The detection o f a novel 45 k D a mitotic protein kinase activated by protein phosphatase inhibitors in C d c 2 mutant cells indicates the existence o f Cdc2-independent pathways. These results also demonstrate the integral role of protein phosphatases in the control o f mitosis. B y ident i fy ing the 45 k D a protein kinase and investigating its role it w i l l be possible to differentiate between Cdc2-dependent and -independent mitotic events, furthering our knowledge of the mysteries of cel lular growth. 6.2 Identification of a Cdc2-independent mitotic histone kinase Chromosome condensation is one o f the most identif iable and dramatic events o f mitosis . The mechan ism whereby the dispersed interphase chromosomes condense into the mitot ic chromosomes is unresolved. Chromosome condensation at mitosis correlates w i th hyperphosphorylat ion o f histone H I and phosphorylat ion o f histone H 3 (Gur ley et al. 1978). H o w increased histone phosphorylat ion contributes to mitot ic chromosome condensation is complete ly unknown. The mitot ic histone protein kinases are largely unidentif ied. The activation o f C d c 2 kinase correlates w i th histone H I hyperphosphorylat ion. In C d c 2 mutant F T 2 1 0 cel ls induced to enter mitosis w i th protein phosphatase inhibitors, chromosome condensation occurs without increased histone H I phosphorylat ion (Guo et al. 1995). Th i s further strengthens the v iew that'histone H I is phosphorylated by C d c 2 kinase in vivo. 65 It is now apparent that there are Cdc2-independent pathways for chromosome condensation. Indeed, the Aspergillus nidulans kinase N I M A is necessary for entry into mitosis and aberrant expression of N I M A can lead to premature chromosome condensation (O'Connell et al. 1994). In HeLa cells, overexpression of NDVIA results in chromosome condensation in cells in the absence of Cdc2 activity (Lu and Hunter 1995). These observations indicate the existence of Cdc2-independent pathways for chromosome condensation. Chromosome condensation in the absence of Cdc2 kinase activity correlates with increased phosphorylation of histones H3 and H 2 A (Guo et al. 1995). Not surprisingly, the protein kinases responsible for these phosphorylation events in vivo are unknown. In vitro, both histones H 2 A and H3 can be phosphorylated by c-AMP-dependent protein kinase while histone H3 can also be phosphorylated by a chromatin-bound protein kinase and pp90rsk (Martinage et al. 1980; Shoemaker and Chalkley 1980; Taylor 1982; Chen et al. 1992). Other in vitro histone H 2 A kinases include a cyclic-GMP-dependent protein kinase and protein kinase C (Glass and Krebs 1979; Takeuchi et al. 1992). I have demonstrated the existence of a novel mitotic histone specific protein kinase of approximately 60 kDa. This kinase was determined to be specific to histones H3, H I and H 2 A in an in-gel kinase assay. A l l of these histones are phosphorylated in eukaryotic cells (Gurley et al. 1978). The histone-specific kinase was also activated by protein phosphatase inhibitors in cells synchronized to S and G2 phases of the cell cycle, which have little Cdc2 activity. This result agrees with the observation of premature chromosome condensation in FT210 cells following treatment with protein phosphatase inhibitors (Guo et al. 1995). It would be very interesting to purify this kinase to evaluate its regulation and role in histone phosphorylation and chromosome condensation. Furthermore, the activation of the 60 kDa histone kinase during S and G2 phases by protein phosphatase inhibitors suggests an essential role of protein phosphatases in the regulation of chromosome structure during interphase to maintain the chromosomes in a 66 decondensed state. These results indicate that at least one histone kinase is present and potentially activatable early in the cell cycle and that protein phosphatases are required to maintain its correct cell cycle activity. The role, i f any, of histone phosphorylation in chromosome condensation is not clear. One possibility is that the number of positive charges must be reduced to a threshold value for chromosome condensation to occur. The histones have a central globular domain with floppy, disordered and highly basic tails. These tails may lie along and bind the major groove of D N A thus making the linker D N A between nucleosomes rigid to maintain chromosome dispersion. Phosphorylating the tails would result in weaker interactions with the D N A due to charge repulsions and greater flexibility in the linker D N A . This model proposes that it is the overall number of phosphorylated residues in histone tails that is required for chromosome condensation, and not necessarily specific histones. Some results agree with this model, as condensed chromosomes in Cdc2 mutant cells are accompanied by increased hyperphosphorylation of histones H3 and H 2 A . This is in contrast to a normal mitosis where histone H3 is monophosphorylated and histone H I is hyperphosphorylated. Histone H3 in Cdc2 mutant cells is phosphorylated on at least two unidentified sites (personal communication from J. Th'ng). This increased phosphorylation of histone H3 may partially compensate for the loss of mitotic histone H I phosphorylation in these cells. Another model of chromosome condensation suggests that the histone tails may interact with scaffolding proteins in dispersed chromosomes. These scaffolding proteins may help maintain the distance between nucleosomes in dispersed chromosomes. Phosphorylation of the tails may disrupt these interactions allowing for chromosome condensation to occur. Histone tails have been demonstrated to be involved in protein-protein interactions. Indeed, in heterochromatin, which is probably analogous in structure to condensed chromosomes at mitosis, the silent information regulators SIR3 and SIR4 interact with the amino-terminal tails of histone H3 and H4 in maintaining transcriptional 67 repression by condensed heterochromatin (Hecht et al. 1995). Identification of other proteins involved in maintaining heterochromatin structure wi l l likely give important insights into the structure of condensed chromosomes at mitosis. Phosphorylated histone tails may also promote internucleosomal interaction through interactions occurring between the phosphorylated tails and histone globular domains of adjacent nucleosomes. Removal of the histone tails by trypsin digestion prevents salt-induced condensation of polynucleosomes in vitro (Garcia-Ramirez et al. 1992). This further indicates an essential role of the tails in chromosome condensation. 6.3 P lk l activity is dependent upon Cdc2 activity: implications for mitotic microtubule dynamics P l k l belongs to an emerging family of mitotic protein kinases which includes S. cerevisiae C D C 5 p , D. melanogaster Polo kinase and Xenopus P l x l . A l l have been demonstrated to have essential roles in mitosis. The recently identified P l x l may trigger mitosis by phosphorylating and activating Cdc25C (Kumagai and Dunphy 1996). Mutation of both Polo and C D C 5 p results in arrest late in mitosis with abnormal chromosomal segregation (Llamazares et al. 1991; Kitada et al. 1993). P l k l has been shown to be associated with the spindle apparatus and microtubules during mitosis as well as the putative microtubule motor, M K L P - 1 (Lee et al. 1995). This suggests a role for P l k l in chromosome segregation. P l k l is tightly cell cycle regulated. Both the m R N A transcript and the P l k l protein start to accumulate during G l phase reaching a maximal level at the G 2 / M transition (Lake et al. 1993; Golsteyn et al. 1994). The increased expression of P l k l in late G2 cannot fully account for the increased activity of P l k l at mitosis, which is 4-6 times higher than that of an equivalent amount of P l k l from interphase cells (Golsteyn et al. 1995). This suggests that the activity of P l k l is regulated by posttranslational modification, probably phosphorylation. M y results confirm that P l k l is posttranslationally modified in mitosis. 68 In Cdc2 mutant FT210 cells at 39°C, the activity of P l k l is very low, well below that of S phase cells. Conversely, immunoblot analysis reveals far more protein present at 39°C than in S phase. Data from in vivo phosphorylation experiments indicated that at 39°C P l k l was not phosphorylated whereas P l k l was phosphorylated at mitosis where it is maximally active. Furthermore, treatment of Cdc2 mutant FT210 cells at 39°C with protein phosphatase inhibitors resulted in no increase in P l k l activity. These results indicate that P l k l , unlike Cdc2, is activated rather than inhibited by phosphorylation and that protein phosphatases do not directly control the activity of P l k l in vivo. Control of P l k l activity by phosphorylation implies that P l k l is probably in a protein kinase signaling cascade. The protein kinase(s) responsible for the mitotic activation of P l k l are unknown. The kinetics of P l k l activation are very similar to that of Cdc2; reaching a maximum at the G 2 / M transition and being inactivated at the end of mitosis. This inactivation is presumably by degradation as P l k l protein levels are very low in G l and accumulate throughout the cell cycle. Although P l k l has no consensus Cdc2 sites, (S/T)PX(K/R) , it can be phosphorylated in vitro by Cdc2 but this has no effect on P l k l activity (Hamanaka et al. 1995). I have determined that P l k l activation is dependent upon Cdc2 activity. FT210 cells lacking Cdc2 at 39°C, have low P l k l activity even when induced into mitosis by treatment of the cells with protein phosphatase inhibitors. Cdc2 has been demonstrated to have a physiological role in mitotic spindle dynamics by directly phosphorylating Eg5 a kinesin-related motor protein which is necessary for bipolar spindle formation (Blangy et al. 1995). Indeed, Cdc2-dependent signaling pathways, including P l k l , appear to regulate the mitotic microtubule array and chromosome segregation. It wi l l be necessary to identify other members of the pathway to completely understand mitotic spindle dynamics. The inability of P l k l to be activated independently of Cdc2 in FT210 cells may be the reason why these cells cannot progress past metaphase. P l k l undergoes a dramatic 69 translocation from the spindle poles to the metaphase plate where it associates with microtubule midbodies and the kinesin-like protein M K L P - 1 (Golsteyn et al. 1995; Lee et al. 1995). The mechanism whereby P l k l translocates is currently unknown but may occur through interactions with a microtubule motor protein. Interestingly, the colocalization of P l k l and M K L P - 1 cannot be detected by immunofluorescent staining until anaphase, after P l k l has relocated from the spindle poles. M K L P - 1 may be involved in the segregation of chromosomes by causing microtubules to pass over each other. Mutant Cdc2 FT210 cells induced to enter mitosis cannot separate their chromosomes and proceed into anaphase. Therefore, I can speculate and propose a model for chromosome segregation (Fig. 25). P l k l is activated by a protein kinase(s) in a Cdc2-dependent pathway at the spindle poles, where Cdc2 is partly localized during mitosis (Bailley et al. 1989). Activated P l k l translocates from the spindle poles to the metaphase equatorial plate by an unknown mechanism. A t the metaphase plate, P l k l associates with and activates M K L P - 1 and possibly other unidentified proteins. Activated M K L P - 1 is then involved in the separation and segregation of the sister chromatids into the emerging daughter cells. The identification of P l k l as a mitotic kinase that is not activated by phosphatase inhibitors suggests some specificity of the effects of phosphatase inhibitors on induction of mitosis. Treatment with phosphatase inhibitors does not simply generally perturb the delicate balance of the opposing effects of protein kinase/phosphatase regulatory systems but rather only affects the activity of a subset of target protein kinases. These results also confirm the existence of Cdc2-independent mitotic pathways in higher eukaryotes. Indeed, Cdc2-independent pathways exist in the process of mitotic chromosome condensation as previously discussed. Interestingly, the chromosome condensation caused by the overexpression of N I M A in the absence of Cdc2 activity in H e L a cells occurs without the formation of mitotic spindles (Lu and Hunter 1995). This further strengthens the requirement and role of Cdc2 in mitotic microtubule dynamics in vivo. Investigating the members of these signaling pathways and determining their 70 A. Metaphase Figure 25. Model of the role of Plkl in chromosome segregation P l k l is phosphorylated in a Cdc2-dependent pathway by an unknown protein kinase(s) at the spindle poles at metaphase. At anaphase, P l k l translocates to the equatorial plane where it associates with and activates the microtubule motor protein, M K L P - 1 . Activated M K L P - 1 is involved in the separation and segregation of the sister chromatids into the daughter cells. 71 regulation and interactions with Cdc2-dependent pathways, such as the P l k l pathway, w i l l provide valuable insights into the control of cell division. 6.4 Conclusions Reversible protein phosphorylation at mitosis is an essential regulatory process ensuring that diverse mitotic events such as chromosome condensation and cytokinesis occur in the proper temporal sequence; resulting in each daughter cell receiving a complete copy of the genome. Protein kinases and phosphatases are responsible for these phosphorylation events and ultimately the fidelity of mitosis. Very few protein kinases and phosphatases involved in the events of mitosis have been identified. I was interested in identifying mitotic kinases activated independently of Cdc2 kinase. A n in-gel renaturation kinase assay was employed to investigate the activity of mitotic protein kinases activated by phosphatase inhibitors in temperature-sensitive FT210 cells lacking the major mitotic kinase Cdc2. This system allowed me to differentiate between Cdc2-dependent and -independent mitotic protein kinases. This work investigates and identifies two mitotic kinases, a histone-specific kinase and p45 kinase, which were activated by phosphatase inhibitors in FT210 cells lacking Cdc2. p45 kinase is a 45 kDa cell cycle-regulated mitotic protein kinase found in all cells. Furthermore, the p45 kinase was not the previously identified 46 kDa kinases: J N K 1 or M A P K A P - 2 . This work reports the partial purification of the p45 kinase and some preliminary characterization of the activity of the p45 kinase. p45 kinase activity was inhibited by the P K C specific inhibitor chelerythrine chloride. This may suggest a role of P K C in the activation of p45 kinase at mitosis. The general protein kinase inhibitor staurosporine did not affect the activity of p45 kinase in cells treated with phosphatase inhibitors but did inhibit the kinase in mitotic cells. A histone-specific protein kinase of approximately 60 kDa was identified. Induction of mitosis in FT210 cells at 39°C results in phenotypically normal condensed 72 chromosomes with hyperphosphorylation of histones H3 and H 2 A (Guo et al. 1995). The kinase displayed specificity for histones H3, H I and H 2 A . Finally, the mitotic protein kinase P l k l was not activated by phosphatase inhibitors in FT210 cells at 39°C. The activity of P l k l was determined to be dependent upon Cdc2 activity. This result demonstrated specificity in the activation of mitotic kinases by phosphatase inhibitors resulting in the induction of mitosis in FT210 cells lacking Cdc2. 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