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Regulation and function of protein Kinase CK2 in cancer cells Sayed, Mohamed Rabi 2002

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REGULATION A N D FUNCTION OF PROTEIN KINASE CK2 IN C A N C E R CELLS By M O H A M E D R. S A Y E D M . S c , University of British Columbia, 1998 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Faculty of Graduate Studies Experimental Medicine Program We accept this thesis as conforming To the required standard THE UNIVERSITY OF BRITISH C O L U M B I A October, 2001 © M O H A M E D R. S A Y E D , 2001 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 The University of British Columbia Vancouver, Canada Date Oct- Sty- O I DE-6 (2/88) ABSTRACT Protein kinase CK2 is a ubiquitous eukaryotic protein serine/threonine protein kinase present in both the nucleus and cytoplasm. In most cells, the holoenzyme form of CK2 is a constitutively active heterotetramer of two catalytic (aand a') and two non-catalytic P-subunits, but CK2(3-free pools of C K 2 a also exist. The activity of CK2 is increased in rapidly growing tissues, tumor cells and human cancers. Despite the identification of more than 160 substrates, many of which are important in cell division and differentiation, the signaling pathways that regulate this enzyme have been enigmatic. Here I provide evidence that protein kinase CK2 is regulated by the stress-activated p38 M A P kinase signaling pathway. I found that p38 M A P kinase, in a phosphorylation-dependent manner, can directly interact with and activate CK2 apparently through an allosteric mechanism. The tumor suppressor protein p53 is one of the key molecules involved in maintaining the stability of the genome, through both cell cycle arrest and apoptosis, especially under circumstances of genotoxic stress. p53 has previously been shown to be associated with protein kinase CK2, and undergo phosphorylation on Ser-392. However, the physiological regulation and the functional consequences of this event remain unknown. I found that this phosphorylation is dependent upon the stepwise activation of p38 M A P kinase and CK2 in response to cellular stress. Furthermore, this activation was demonstrated to be responsible for the biochemical regulation of the cyclin-dependent kinase-1 (CDK-1), as both 5,6-dichloro-l-((3-D-ribofuranosyl)-benzimidazole (DRB; a semi-specific inhibitor of CK2)) and antisense depletion of CK2, as well as SB203580 (a i i specific inhibitor of p38 M A P kinase) were each associated with an inhibition of its activation in response to nocodazole (microtubule inhibitor). I observed that depletion or inhibition of the catalytic subunits of CK2, in the presence of microtubule inhibitor (nocodazole), resulted in a compromise of the spindle assembly checkpoint. Furthermore, the CK2-depleted cells exposed to nocodazole underwent reduced apoptosis as well as abnormal duplication and alignment of centrosomes. These oncogenic properties were observed in both human cervical carcinoma cells (i.e. HeLa cells) and human colon tumor cells (HCT116). I also showed that this effect was dependent on the presence of functional wild-type p53, as this phenomenon was not apparent in HCT116 p53"/_ cells. My results support a novel role for CK2 in concert with p53, to maintain the stability of the genome. These findings may provide for an improved understanding of abnormalities of the spindle checkpoint in human cancers, which is a prerequisite for defining future therapies. i i i TABLE OF CONTENTS A B S T R A C T i i T A B L E OF CONTENTS iv LIST OF T A B L E S vi LIST OF FIGURES -v i i LIST OF ABBREVIATIONS ix A C K N O W L E D G E M E N T S . xii 1. INTRODUCTION 1 1.1. Protein Kinase CK2 1 1.1.1. Generalities 1 1.1.2. Molecular basis of CK2 specificity 8 1.1.3. Structural features 11 The catalytic subunits a/a' 11 The noncatalytic CK2 p-subunit 14 1.1.4. Regulation 16 The a and P subunit relationship 16 Activators and inhibitors of CK2 17 Regulation of CK2 activity 19 1.1.5. CK2 and cancer 21 1.2. p38 Mitogen -Activated Protein Kinase (p38 M A P kinase) 24 1.2.1. Overview 24 1.2.2. Regulation... , 25 1.3. The Tumor Suppressor p53 31 1.3.1. Overview 31 1.3.2. p53 structure and function 32 1.3.3. Regulation 34 N-terminal phosphorylation 35 • C-terminal phosphorylation 38 Responses to the activation of p53: Downstream events 42 p53 and cell cycle checkpoints 42 p53 and centrosome homeostasis 49 p53 and regulation of apoptosis 51 1.4. Current Problems and Objectives 56 2. M A T E R I A L A N D METHODS 57 2.1. Cell Culture and Lysates preparation 58 2.2. Cell Synchronization 59 2.4. CK2 Phosphotransferase activity assays 59 2.5. Immune-complex Kinase Assays 60 2.6. Immunoblotting 61 2.7. In vitro Binding Assay 61 2.8. Oligonucleotide Transfection 62 iv 2.9. Flow Cytometry 63 2.10. Mitotic Index 63 2.11. Apoptosis Assay 63 2.12. Immunofluorescence 64 3. STRESS INDUCED ACTIVATION OF PROTEIN KINASE CK2 B Y DIRECT INTERACTION WITH p38 M A P KINASE 65 3.1. Rationale and Hypothesis 65 3.2. Results 66 3.2.1. Stress factor- induced activation of CK2 and p38 M A P kinase 66 3.2.2. Phosphorylated and active p38 M A P kinase coelutes with CK2 from MonoQ...„ 69 3.2.3. Active p38 M A P kinase coimmunoprecipitates with CK2 72 3.2.4. p38a M A P kinase expressed as glutathione S-transferase fusion protein binds to and activates recombinant CK2 75 3.3. Discussion ...80 4. ACTIVATION A N D LOCALIZATION OF PROTEIN KINASE CK2 IN NOCODAZOLE-INDUCED MITOTIC ARREST 83 4.1. Rationale and Hypothesis 83 4.2. Results 84 4.2.1. Depletion or inhibition of CK2 compromises mitotic arrest 84 4.2.2. CK2 functions as a component of the spindle assembly checkpoint 88 4.2.3. Activation of CK2 and p38 M A P kinase in nocodazole-arrested cells 90 4.2.4. Subcellular immunolocalization of CK2 at different mitotic stages 93 4.3. Discussion.... 96 5. PROTEIN KINASE CK2 IS REQUIRED FOR p53 FUNCTION IN RESPONSE TO SPINDLE D A M A G E 99 5.1. Rationale and Hypothesis 99 5.2. Results 100 5.2.1. Nocodazole-induced p53 Ser-392 phosphorylation is mediated by CK2 and is SB203580 sensitive 100 5.2.2. Depletion of CK2 causes an override of mitotic arrest following spindle damage 104 5.2.3. Mitotic spindle checkpoint compromise requires p53 protein 109 5.2.4. Cells transfected with CK2 antisense escaped nocodazole-induced cell death 114 5.2.5. Abnormal centrosome amplification consequent upon CK2 depletion ..117 5.3. Discussion 120 6. CONCLUSION 125 7. FUTURE STUDIES 129 8. BIBLIOGRAPHY 131 v LIST OF TABLES CHAPTER 1. INTRODUCTION Table 1. Alphabetical list of proteins phosphorylated and interacting with protein kinase CK2 6 Table 2. Extracellular stimuli of p38 and functional outcome 28 vi LIST OF FIGURES CHAPTER 1. INTRODUCTION Figure 1. Functional elements of CK2 a and (3 subunits 10 CHAPTER 3. STRESS-INDUCED ACTIVATION OF PROTEIN KINASE CK2 B Y DIRECT INTERACTION WITH p3 8 M A P KINASE Figure 2. Stress factor induced activation of CK2 and p38 M A P kinase 68 Figure 3. Phosphorylated and active p38 M A P kinase coelutes with CK2 From Mono Q 71 Figure 4. Activation of CK2 in anisomycin-stimulated p38a M A P kinase 73 Figure 5. p38 M A P kinase coimmunoprecipitates with CK2 74 Figure 6A-C p38 a M A P kinase expressed as a glutathione S-transferase fusion...77-78 Figure 7. p38 M A P kinase does not phosphorylate CK2 in vitro 79 CHAPTER 4. ACTIVATION A N D LOCALIZATION OF PROTEIN KINASE CK2 IN NOCODAZOLE-INDUCED MITOTIC ARREST Figure 8. Depletion of CK2 compromises mitotic arrest 86 Figure 9. Failure of HeLa cells to undergo mitotic arrest in event of CK2 depletion or inhibition 87 Figure 10. Suppression of nocodazole-induced MPF activity by either CK2 Antisense or a specific inhibitor of p38 89 Figure 11. Activation of CK2 in nocodazole-arrested cells is dependent on p38 M A P kinase 91 Figure 12. Activation of p38 M A P kinase in nocodazole-arrested cells 92 Figure 13. Subcellular localization of CK2 in HeLa cells ..95 CHAPTER 5. PROTEIN KINASE CK2 IS REQUIRED FOR p53 FUNCTION IN RESPONSE TO SPINDLE D A M A G E Figure 14. Stress factor-induced phosphorylation of p53 at Ser-392 is Dependent on CK2 and p38 M A P kinase 102 Figure 15. CK2 is a major kinase phosphorylating p53 at Ser-392 in Nocodazole-treated cells 103 Figure 16. Mitotic arrest is compromised in the absence of CK2 106 Figure 17. Inhibition of cdc2 activity in CK2-depleted HeLa cells 107 Figure 18. Phosphorylation of cdc2 is independent of cyclin B degradation in CK2-depleted cells 108 Figure 19. p53 is required for CK2 to override the mitotic arrest I l l Figure 20. Mitotic indices of HCT 116 and HeLa cells 112 Figure 21. Cyclin B expression in HCT 116 p5 3 -/- cells 113 Figure 22. Suppression of apoptosis in the absence of CK2 in HeLa cells 115 vii Figure 23. Suppression of apoptosis in the absence of CK2 in HCT 116 cells 116 Figure 24. Abnormal amplification of centrosomes in the absence of CK2 119 CONCLUSION Figure 25. Schematic of events following nocodazole-induced spindle damage 128 viii LIST OF A B B R E V I A T O N S A P C AS A T M C A K CK2 C D K B A X Caspase D N A - P K DRB EGF EDTA Erk-1 Erk-2 FGF GM-CSF GSK-3 HSP27 IC IGF-1 IGF-BP3 IL Anaphase-promoting complex Antisense Ataxia telangiectasia mutated Cyclin-activating kinase Casein kinase II Cyclin-dependent kinase Bcl2 associated factor X Cysteine-aspartic acid specific protease DNA-dependent protein kinase 5,6-dichloro-1 -((3- D-ribofuranosyl)-benzimidazole Epidermal growth factor Ethylene diaminetetra-acetate Extracellular regulated kinase-1 Extracellular regulated kinase-2 Fibroblast growth factor Granulocyte macrophage colony-stimulating factor Glycogen synthase kinase-3 27-kDa heat shock protein Inhibitory concentration Insulin-like growth factor-1 Insulin growth factor-binding protein-3 Interleukin ix JNK c-jun N-terminal kinase kDa KiloDalton M A P Microtubule-associated protein M A P K Mitogen-activated protein kinase M A P K A P MAPK-activated protein Mr Relative molecular mass M W Molecular weight MEF Mouse embryonic fibroblast M K K M A P kinase kinase Mdm2 Murine double minute clone 2 MPF Maturation promoting factor NGF Nerve growth factor NLS Nuclear localization signal P A G E Polyacrylamide gel electrophoresis p38 M A P kinase 38 kDa mitogen-activated protein kinase p53 53 kDa tumor suppressor protein PBP Polybasic peptide P C N A Proliferating cell nuclear antigen PDGF Platelet-derived growth factor PIGs p53-induced genes P K A Protein kinase A P K C Protein kinase C PP2A Protein phosphatase 2A RB Retinoblastoma RGS Ribosomal gene cluster S Sense SB203580 SmithKline Beecham-203580; p38 M A P K inhibitor SDS Sodium dodecyl sulphate SH3 Src-homology domain-3 TAF T A T A box binding protein associated factor TBP TATA-binding protein TNF Tumor necrosis factor V E G F Vascular endothelial growth factor xi A C K N O W L E D G E M E N T S I dedicate this thesis to the people who supported me all the way through this. To parents and my friend Connie Wong. I would like also to thank my mentors and supervisors Bi l l Salh and Steven Pelech for their advice and guidance. xii 1. INTRODUCTION 1.1. Protein Kinase C K 2 1.1.1. Generalities Protein kinase CK2 (formerly termed casein kinase II) is a highly conserved protein-serine/threonine kinase that is ubiquitously expressed in eukaryotic organisms. CK2 from most sources is generally a heterotetramer with a molecular mass of approximately 130 kDa. This tetramer is composed of two subunits with a general stucture of a2 (32 or a a ' (32. The a and a ' are catalytically active by themselves and have molecular masses of 42-44 kDa (a) and 38 kDa (a'). The a and a ' subunits are structurally analogous but are known to be encoded by different genes (Litchfield et al., 1990). Although the a subunits are found in all species, the a ' subunits have not been found in Drosophila andXenopus (Allende and Allende, 1995). The (3 subunit exists in homodimers (usually 26 kDa in all animal cells) and is inactive, but confers both negative and positive regulation to the catalytic activity of a subunits (Pinna, 1990; Issinger, 1993). In addition, the [3 subunit affects the stabilization of a subunits against heat denaturation or proteolysis and, more importantly, it can change the specificity of its interaction with substrates and inhibitors (Allende and Allende, 1995; Chambaz, 1998). In Saccaromyces cerevisiae two forms of the (3 subunit exist, (3 and (3' (41 and 32 kDa), which are expressed by two separate genes (Bidwai et al., 1994), In Dictostelium discoideum and Zea mays there are no detectable (3 subunits, 1 despite the existence of intact CK2 catalytic activity (Allende and Allende, 1995; Guerra et al., 1999a). The a-subunit shares a high degree of homology with cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAP kinases) and glycogen synthase kinase-3 (GSK-3), whereas the non-catalytic (3-subunit is a relatively unique protein with no remarkable homology to other proteins (Pinna and Meggio, 1997; NCBI Genbank, 2001). The tetrameric holoenzyme is extremely stable. In vitro, the holoenzyme is formed upon mixing of the subunits by a self-assembly mechanism, and cannot be easily dissociated, except under denaturing conditions (Grankowski et al., 1991). Whether dissociation occurs in vivo once the holoenzyme has been formed is totally unknown. The determination of the crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays (recombinant maize CK2a) at 2.1 A resolution was fundamentally important, as it sheds light on some of the biochemical characteristics of this enzyme (Guerra et al., 1998; Niefind et al., 1998). CK2 is unusual among protein kinases in that it can use both ATP and GTP as phosphoryl donors with almost the same efficiency. Its heterotetrameric structure is rare among protein kinases and it has a predilection for phosphoacceptor residues having clusters of acidic amino acids on their C-terminal side. Such remarkable site specificity is dictated by a minimum consensus sequence, S/T-X-X-E/D/Sp/T (Tuazon and Traugh, 1991; Pinna and Meggio, 1997). Although CK2 was one of the first protein kinases discovered (Burnett and Kennedy, 1954; Pinna, 1990; Issinger, 1993; Allende and Allende, 1995; Pinna, 1997; Guerra and Issinger, 1999), its regulation by known second-messenger molecules and its physiological function have been elusive. Numerous reports indicate that CK2 is 2 involved in proliferation and tumorigenesis. CK2 phosphotransferase activity has been shown to be enhanced in transformed cell lines (Prowald et al., 1984, Chambaz, 1998), in solid tumors (Munstermann et al., 1990; Pistorius et al., 1991; Stalter et al., 1994; Landesman-Bollag et al., 2001), in rapidly proliferating tissue (Munstermann et al., 1990; Lebrin et al., 2001), and also during embryogenesis (Schneider et al., 1986; Mestres et al., 1994). In higher eukaryotic organisms, prominent CK2 activity has been found in embryonic brain, indicating an important role for CK2 in brain development (Mestres et al., 1994). It has been shown that embryonic tissues or organs contain higher concentrations of CK2 protein level and activity than in the adult counterparts over a wide range of species that include rat, chicken and the nematode C. elegans (Issinger, 1993; Allende and Allende, 1995). Studies using in situ hybridization for detection of mRNAs in mice and in zebrafish during embryonic development have demonstrated that mPvNAs for the a and (3 subunits of CK2 are especially abundant in the developing nervous system (Mestres et al., 1994). These findings correlate nicely with the observation that microinjection of antisense oligonucleotides directed against C K 2 a prevents neuritogenesis in neuroblastoma cells (Ulloa et al., 1993). In solid human tumors, e.g. mammary, colorectal and lung carcinomas, CK2 amount/activity is abnormally elevated (Issinger, 1993; Daya-Makin et al., 1994; Gapany et al., 1995; Faust et al., 1996; Guerra et al., 1999a; Landesman-Bollag et al., 2001; Romieu-Mourez et al., 2001). On the one hand, this elevated activity of CK2 has been suggested by Seldin and Leder (1995) to contribute to the pathogenesis of human cancer. On the other hand, CK2 can also negatively regulate cell proliferation (Allende and Allende, 1995; Pinna, 1997 Guerra et al., 1999a). It has been shown that CK2 may exert 3 its anti-proliferative activity by its interaction with the tumor suppressor protein p53 (Milne et al., 1992), or with protein phosphatase 2A (Heriche et al., 1997). Recent studies indicate that CK2 is not only active as a tetrameric holoenzyme but also as free subunits (Allende and Allende, 1995; X i n et al., 1999; Landsman-Bollag 2001). Stigare et al. (1993) have reported that the majority of the CK2 oc-subunit is tightly bound to intranuclear components other than the P-subunit. The regulatory P-subunit is produced far in excess of the a-subunit and is rapidly degraded (Guerra et al., 1999b). In cancer cells an abnormal distribution and ratio of a- and p- subunits was observed, and this dysregulation has been proposed to contribute to the malignant phenotype (Guerra et al., 1999b). Overexpression of CK2 has been studied in mice. It has been shown that transgenic mice, expressing elevated levels of CK2 a in lymphoid organs, display a stochastic propensity to develop lymphomas (Seldin and Leder, 1995). Go-expression of a c-myc transgene in addition to CK2 a results in neonatal leukemia, indicating that CK2 a can serve as an oncogene and that its dysregulated expression is capable of transforming lymphocytes (Seldin and Leder, 1995). Furthermore, it has been reported that CK2cc overexpression is involved in the loss of a p53 allele in thymic lymphomas (Ole-Moi et al., 1993). The role of CK2 in cancer is discussed below. Clues regarding the function of CK2 in vivo have also been obtained via identification of putative physiological substrates and by molecular genetic studies. Over 160 well characterized substrates of CK2 are currently known (Pinna, 1997; Pinna and Meggio, 1997). The alphabetical list of proteins phosphorylated by protein kinase CK2 is shown in Table 1. Among them are nuclear and cytoplasmic enzymes and structural proteins playing important roles in transcription, translation, signal transduction and cell-4 cycle regulation. Disruption of CK2 a-subunit genes in yeast revealed that the catalytic C K 2 a-subunit is essential for viability (Padmanabha et al., 1990), whereas overexpression did not lead to detectable phenotypic changes (Roussou and Draetta, 1994). In contrast, yeast strains lacking CK2 (3 are viable, but exhibit an altered phenotype, and overexpression of CK2 (3 also leads to an altered phenotype (Roussou and Draetta, 1994; Bidwai et al., 1995). Recent research indicates that CK2 subunits could be involved not only in forming the tetrameric CK2 holoenzyme, but also in binding to, and possibly regulating, other proteins such as transcription factors (e.g. p53 protein and TAL-1) and protein kinases, (e.g. A-Raf kinase and Mos) (Boldyreff and Issinger, 1997; Hagemann et al., 1997; Chen, et al., 1997; Xin et al., 1999). This seems to be especially interesting in light of the finding that CK2 (3/a-subunit distribution is unbalanced in clear cell renal carcinomas, with an excess of P-subunits in the carcinomas (Stalter et al., 1994). 5 Table 1. Alphabetical list of proteins phosphorylated and interacting with protein kinase CK2 (Pinna and Meggio, 1997) A l (human hnRNP) ABF-1 (yeast) Adenovirus Ela Androgen receptor c-jun c-Myb C-Myc C a 2 + channel blockers; receptor Caldesmon Calmodulin Calnexin Calreticulin (spinach) Calsequestrin Caveolin CDC37 CD45 tyrosine phosphatase Clathrin light P-chain Coactivatorpl5 (PC4) CREB C R E M CTP:phosphocol. cytidyltransferase DARPP-32 D N A ligase D N A topoisomerase I D N A topoisomerase II DSIP peptide Dynamin Dystrophin E47 EF-1 6 EF-1 P Egr-1 (early growth response) eIF-2a eIF-2p eIF-3 eIF-4B elF-5 eIF4e/p20 (S. cerevisiae) Engrailed protein (Drosophila) Epstein-Barr Nuclear Antigen 2 Epstein-Barr S M , P-protein Epstein-Barr virus Z E B R A protein Furin Cap43 (neuromodulin) GB glycoprotein (cytomegalovirus) GBF-1 transcription factor (Arabidopsis) Glycogen synthase Glycophorin gp96 (soybeans). . CRP94 of sarcoplasmic reticulum' Guanine nucleotide exchange factor HASPP28 (rat brain) HCP (hist-rich C a 2 + bind, protein) HDAgs (Hepatitis 8 virus antigens) HIV-1 Rev protein HrV-1 viral protein H M G - 1 4 H M G -1 HMG-like protein PI hnRNP HPV E7 Hsp 90 Human centrom. protein 8 (CENP-B) Human complement protease Human estrogen receptor IGF-11 receptor IGFBP-1 IkBa IkBp Inhibitor-2 o fPP l Insulin receptor IRS-1 L D lipoprotein receptor lipoprotein inhibitor Mannose-6-phosphate receptor M A P - I B Max protein M D M 2 Measles virus P-protein MEF2C (myocyte enhancer factor) MRFs (MRF4 and MyoD) mRNA particles M Y C N (human neuroblastoma cell) 6 Table 1. Continued NBP-60 (NLS binding protein) Neural cell adhesion molecule Nuclear DNA-binding protein p210 Nucleolar pi30 Nucleolar protein B23 Nucleolin Nucleoplasmin Ornithine decarboxylase Osteopontin PO, PI , and P2 ribosomal proteins pi20 cell prolif. nucleolar protein pi3 and p38 yeast ribosomal proteins p25 (Xenopus laevis) p34 (chloroplast ribonucleoprotein) p 3 4 « ic2 p38 RNA-binding protein p53 p65 synaptic vesicles p98 (sea urchin egg) Pea lamin like protein PHAS-1 P K A R-11 subunit F K C . Platelet Va and Vil la factors PP2C pp35 proliferation nuclear P-protein pp65 (human cytomegalovirus) Progesterone receptor Proteasome (30 kDa) Proteasome complex (C8 and C9) Prothymosin a Pseudorabies virus pp-62 PU-1 transcription factor RAS-17 Rat epididymal fat cell p22 Respiratory syncytial virus P-protein RIPP RIPP-1 and -11 phosphophoryns R N A polymerase I R N A polymerase 11 R N A polymerase III Sarcalumenin Serum response factor Stathmin SV40 large T antigen Synaptotagmin T-5ubstrate (wheat germ) Tal-1 Tau protein TFILA coactivator p 15 ' TRa2 (thyroid hormone receptor). Troponin-T Tryptophanyl-tRNA synthetase Tubulin VAMP/synaptobrevin Viral P-protein Vitamin D receptor VPI (polyoma virus capsid) V P U (HIV-1) V S V (ves. stomatitis virus) P-protein V Z V Waricella zoster virus) protein XFC-5-1 (Xenopus zinc finger) protein xNopp!80 (Xenopus laevis) Yeast SRP-40 (homologue to Noppl40), ZFPs (Xenopus laevis) 7 1.1.2. Molecular basis of CK2 specificity To gain information about the structural elements responsible for the unique ability of CK2 to recognize phospho acceptor sites specified by multiple acidic residues, a systematic mutational study has been performed in which a number of basic residues conserved in C K 2 a were replaced by alanine. By biochemical analysis of each of these mutants with the optimal substrate R R R A D D S D D D D D , a number of basic residues within the a subunit that have been implicated in substrate recognition, have been identified (Sarno et al., 1996; 1997a). By combining this information with modeling studies, a sequence of basic residues concentrated around the catalytic site, and responsible for the binding of individual acidic determinants in the phosphoacceptor substrate with a minimum consensus sequence, S/T-X-X-E/D/Sp/Tp has been highlighted, where X is often an acidic amino acid residue (Sarno et al., 1997a, 1998). These authors have shown that the main contacts are those between the aspartic acid residue at position n+1 and n+3 with Lys-198 and one or more of the lysines in the 7 4 K K K K 7 7 quartet located at the basic stretch between the conserved regions II and III (Sarno et al., 1997a, 1998). Additional contacts involve two basic residues that are inserted in the Gly-rich loop and possibly a lysyl residue in the subdomain V region (Lys-122) (Vaglio et al., 1996). Sarno et al. (1996; 1997b) have shown that by mutating basic residues in the 74-77 and in the 191-198 regions of the alpha-subunit, the negative regulation by the beta-subunit and by its N-terminal synthetic fragment CK2[3-(l-77) is drastically reduced. In contrast, the positive regulation by a C-terminal, CK2(3-(155-215)-peptide is unaffected or even increased. Moreover, the basal activity of the a subunit mutants K74-77A, 8 K79R80K83A, and R191R195K198A towards specific peptide substrates is stimulated by the (3 subunit many fold more than that of alpha wild type, while extra stimulation by P mutant D55L56E57A, observable with a wild type, is abolished with these mutants (Sarno et a l , 1996; 1997a). These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of the P subunit, is mediated by basic residues in the 74-83 and in the 191-198 sequences of the a subunit. These are also implicated in substrate recognition, consistent with the concept that the N-terminal acidic region of the p subunit operates as a pseudosubstrate (Sarno et al., 1997b). The structural analysis of CK2 is discussed below. A schematic illustration of CK2 residues implicated in substrate recognition and accounting for the markedly acidophilic nature of CK2 is shown in Figure 1. 9 a H e p a r i n i i l i ^ u b . ] r N L S ? H S P 9 0 1 9 1 1 9 8 R V A S K Y T K n + 1 H e p a r i n i S - s u b . i A u t o p h o s p h . sssp N i S t i m u L b y P B P DLEFOEELED . d o w n - r e g u l a t i o n DGAiTGTGH'HMLFMVHPEYOTKFJ'ANQrVPRLYGFKIH - a - b i n d i n g a n d p r o t e c t i o n - S t i m u l a t i o n o f b a s a l a c t i v i t y - S - 8 i n t e r a c t i o n Figure-1: Functional elements of CK2 a and (3 subunits. Regions and/or residues implicated in specific functions of a and (3 subunits are highlighted. The Roman numerals denote the sub-domains of the C K 2 a catalytic domains as defined by Hanks and Hunter (1995). The positions (relative to Ser/Thr) of the substrate specificity determinants recognized by basic residues of the a subunit are specified by the n+/n-notation. Arrows pointing up and down indicate stimulation and inhibition by heparin (or (3 subunit), respectively. NLS, nuclear localization signal; PBP, polybasic peptides (e.g. polylysine, histones); HSP 90, heat shock protein-90. Based on data from Pinna and Meggio (1997) and Pinna (1997). 10 1.1.3. Structural Features The catalytic subuits a/a' The primary structure of the CK2 catalytic subunits (either a or a') displays very highly conserved regions of the 12 subdomains that have become the hallmarks of the large superfamily of protein kinases (Litchfield et al., 1990). I wil l discuss the most important and well characterized domains of CK2a . Conserved region I (glycine loop) As in all kinases this region is involved in ATP binding and phosphotransfer. The first two glycines out of the three of the motif G - X - G - X - X - G are conserved (Pinna and Meggio, 1997). The third residue in CK2 a is replaced by a hydrophilic residue, serine, a feature that might confer ATPase activity by bridging water molecules in contact with ATP (Pinna and Meggio, 1997). Two features of this loop are notable: a lysine residue (K49) which has been shown by mutational analysis to contribute to the recognition of the acidic determinant at position n+2 in peptide substrates (Sarno et al., 1998); and a tyrosine (Tyr-50) which is homologous to the regulatory residue Tyr-15 of C D K , the phosphorylation of which (as well as that of the preceding Thr-14) suppresses the catalytic activity of C D K . The question of whether this tyrosine residue is ever phosphorylated in CK2 as a mechanism for its regulation remains unclear. 11 Basic stretch between conserved regions II and III This region comprises amino acids 74-80 which is conserved in all species and probably is the most striking hallmark of CK2 and is situated just upstream from the conserved glutamic acid that defines subdomain III. It is involved in substrate recognition (position n+3; Fig. 1) and interaction with the p-subunit. Mutational analyses have shown that the first part of this cluster (Lys-74 to Lys-77) (Hu and Rubin, 1990; Gatica et al., 1994; Vaglio et al., 1996), is implicated in CK2 inhibition by heparin. However, both the 74-77 and the 79-83 segments are implicated in substrate recognition at position n+3 (Guerra et al., 1999a). Although it appears that these basic residues are to some extent interchangeable as far as substrate recognition is concerned, Lys-77 seemingly plays a prominent role since individual replacement by alanine causes a dramatic increase of the phosphotransferase activity of the a subunits toward casein (Sarno et al., 1998). It has been shown that the 74-80 basic sequence of CK2cx interacts with the P-subunit (Sarno et al., 1998) and mediates the P subunit induced down regulation of the catalytic activity of CK2 (Pinna and Meggio, 1997). Competition experiments with heparin have revealed that the 74-77 basic quartet also mediates interactions with HSP90 (Miyata and Yahara, 1995). This basic region, in addition, has the hallmarks of a nuclear localization signal (NLS), which may be responsible for targeting CK2 to the cell nucleus (Rihs et al., 1991). These studies have been confirmed by the crystal structure of recombinant maize (rm) CK2 a (Niefind et al., 1998; Guerra et al., 1999a). The 12 structure of rm C K 2 a closely resembles the well-known structure of protein kinase A (PKA), with a P-rich N-terminal domain comprising the p strands 1-5 and the helix aC . The CK2 enzyme was crystallized in an active, open conformation where the N-terminal region, characterized by short a-helical motifs, forms extensive contacts with the lysine-rich cluster of the ocC helix and with the activation loop. The lysine-rich cluster is characterized by the K 7 4 K K K - I K R E I K 8 3 sequence. This cluster is involved in substrate recognition, inhibition by heparin, down-regulation exerted by the CK2P subunit, interaction with heat shock protein-90, and nuclear targeting mediated by a nuclear localization signal (Nigg et al., 1991, Niefind et al., 1998). The catalytic loop and PP2A binding motif This short segment contains the D X 1 K X 2 X 3 N sequence, spanning the highly conserved Asp and Asn residues that define subdomain VIB in CK2 (Figure 1) and is almost identical to P K A and CDKs. This region is important in peptide substrate recognition in P K A . Mutational studies in this region of CK2 were indicative of the acidophilic nature of this kinase (Dobrowolska et al., 1994). Just downstream from the catalytic loop is a sequence motif conserved in vertebrate and Drosophila C K 2 a (H-F-H/N-R-K-L) . This motif is also found in SV40 and is required for binding protein phosphatase 2 A in vitro (Heriche et al., 1997). 13 The subdomain VII-VIIIconnection (activation loop) The region between the DFG and APE triplets that defines subdomains VII and VIII in most protein kinases is altered in CK2 (Guerra et al., 1999a). This region contains the 'activation loop' (or 'T-loop') after the presence of a threonyl residue that is constitutively phosphorylated in active P K A (Pinna and Meggio, 1997). The activation loop includes residue(s) that when phosphorylated (either autocatalytic or promoted by other kinases) correlate directly with increases in the enzymatic activity of most kinases (Johnson et al., 1996). However, CK2tx does not contain either threonine or serine in its activation loop, nor has phosphorylation ever been reported in this region. These observations, in conjunction with the finding that the free catalytic subunit is spontaneously active, indicate that the activation loop of CK2 is displaced from the catalytic cleft and does not hamper enzymatic activity (Pinna and Meggio, 1997). The noncatalytic CK2 P-subunit The noncatalytic or the regulatory P-subunit of C K 2 is generally autophosphorylated in vivo and in vitro. Peptide mapping studies indicate that the autophosphorylated sites in the P subunit are Ser-2 and Ser-3 (Figure 1). CK2P is a relatively unique protein with no homology to regulatory subunits or domains of other protein kinases. It can be separated from the holoenzyme in vitro only under denaturing conditions. After expression of the CK2 subunits a and p separately in Escherichia coli and reconstitution of the holoenzyme, it was possible to study the role of the P-subunit by 14 comparing the features of the a-subunit alone with those of the holoenzyme (Grankowski et al., 1991). The P-subunit has three known functions: (1) it modulates the activity of the a-subunit in a substrate-dependent manner (Grankowski et al., 1991; Meggio et al., 1992b); (2) it contributes to the substrate specificity (Meggio et al., 1992b); and (3) it stabilizes the a-subunit (Issinger et al., 1992; Meggio et al., 1992b). The modulation of the C K 2 a activity with most substrates by the P subunit is a stimulatory effect. However, if calmodulin is used as the substrate, the P-subunit almost completely inhibits this activity. This inhibition can be overcome by the addition of polylysine (Meggio et al., 1992a). Mutagenesis studies on the P-subunit revealed an acidic stretch from amino acids 55-64, which is responsible for the inhibitory effect and for the stimulation by polylysine (Meggio et al., 1994a). Interestingly, mutations of acidic residues at positions 55, 57, and 59-64 of the P-subunit to alanine residue, led to a 4-fold more active holoenzyme than the wild-type enzyme after reconstitution with the a-subunit alone (Boldyreff et al., 1993). At the same time, these mutants are no longer responsive to the stimulator polylysine. This indicates that the acidic residues Asp-55 and Glu-57 have a general down-regulatory role, which can be overcome by polylysine, most likely by direct interaction with these residues. In the case of the substrate calmodulin, this down-regulation results in almost complete inhibition. Crosslinking and peptide studies have shown that the acidic amino acid stretch of CK2 P from residues 55-64 interacts with a corresponding basic stretch of the a-subunit (Krehan et al., 1996; Sarno et al., 1997b). However, this seems to be a low affinity electrostatic interaction (Guerra et al., 1999a; 1999b). 15 The structural features that are responsible for P subunit homodimerization, association with the a-subunit, protection against denaturation and proteolysis and up-regulation of the activity, are located in the C-terminal region between amino acids (155-215) (Kusk et al., 1995; Marin et al., 1997). The C-terminal region is also essential for interaction with protein kinases such as the tumor suppressor p53 (Xu et al., 1999), A -Raf (Boldyreff and Issinger, 1997; Hagemann et al., 1997), Mos (Chen et a l , 1997), p90 r s k (Kusk et al., 1999) and many 'cellular partners' of CK2 (table-1) (Pinna and Meggio, 1997; Kusk et al., 1999). 1.1.4. Regulation Despite the fact that this enzyme was discovered over 45 years ago (Allende and Allende, 1995; Pinna and Meggio, 1997), the mechanisms that regulate its activity under physiological conditions have been elusive. Here I will discuss some of aspects that have been reported to influence CK2 regulation. The a and P subunit relationship As mentioned above, two regions of the p-subunit have been shown to interact with the a subunit: the acidic stretch, composed of residues 55-64 in the N-terminal moiety, and the C-terminal region. The N-terminal acidic cluster of P makes electrostatic contacts with basic residues of the a-subunit that are otherwise implicated in substrate binding, and, therefore, is responsible for down-regulation of its activity (Sarno et al., 16 1997b). However, the tight physical interaction between the two subunits appears to be mediated by the C-terminal region of the p subunit. The precise location of the interacting domain within the a subunit is unknown. Furthermore, studies exploiting the two-hybrid system in yeast have shown that the entire sequence of the a-subunit, with the exception of only two short segments at the very N - and C-terminal ends, is required in order to ensure strong interaction with the P subunit (Kusk et al., 1995). This outcome is consistent with the view that the correct folding of the a-subunit is required in order to generate the structural elements that bind the p-subunit with high affinity. Activators and inhibitors of CK2 CK2 is unique amongst protein kinases in that it possesses basal activity, either in its canonical heterotetrameric form or as isolated catalytic subunits, being independent of any known second messenger and not susceptible to acute regulation by phosphorylation. This activity is especially intriguing i f it is borne in mind that CK2 is implicated in a variety of signal transduction pathways, as judged by the nature of many of its physiological targets. Despite its basal activity, CK2 can still be,affected by a number of compounds that have some physiological importance. The list includes compounds such as polyamines, specifically spermine (Allende and Allende, 1995). This compound can cause a two- to three-fold increase in enzyme activity. This stimulation is highly dependent on the concentration of M g 2 + and on the nature of the substrate. It has been reported that a number of polyanions, notably heparin, polyglutamic acid, and random polymers of glutamic acid and tyrosine, are powerful and relatively 17 selective inhibitors of CK2 (Pinna and Meggio, 1997). Their mode of action is generally competitive with respect to the phosphoacceptor substrate, although heparin does not exactly mimic the substrate conformation when it binds to C K 2 , as judged from mutational analysis of residues that are implicated in substrate conformation (Sarno et al., 1998). Pseudosubstrate peptides in which serine has been replaced by alanine are poor inhibitors due to a reduction in affinity (Pinna, 1990; Meggio et al., 1994b). In contrast, high affinity is maintained i f serine is replaced by tyrosine, consistent with the view that tyrosine can bind, although it is not phosphorylated, except under special circumstances. Consequently, the pseudosubstrate peptide E E E E E Y E E E E E E E is a selective and fairly efficient inhibitor of CK2 with an IC50 = 0.8 p M (Sarno et al., 1999). It has been shown that protein kinase inhibitors that are competitive with respect to ATP, such as the most potent broad-specific inhibitor of protein kinases, staurosporine, are poor inhibitors of CK2 (Meggio et al., 1995; Pinna and Meggio, 1997; Guerra et al., 1999a). This observation, in conjunction with the ability of CK2 to use GTP, as well as ATP, as a phosphodonor, supports the view that the ATP-binding site of CK2 has unique features that could be exploited to design highly specific inhibitors. This has been confirmed by the resolution of the crystal structure of C K 2 a (Niefind et al., 1998). Attempts to improve the selectivity of isoquinoline sulfonamide analog inhibitors of protein kinases have led to the development of a semi-specific inhibitor for CK2 that is derived from the halogenated benzimidazole/benzotriazole derivatives. This compound is 5,6- dichloro-l-(P-D-ribofuranosyl)-benzimidazole, commonly known and commercially available as D R B . DRB, first described as an inhibitor of transcription in eukaryotic systems (Sehgal and Tamm, 1978; Egyhazi et al., 1982) was later found to act through 18 the inhibition of CK2 activity (Zandomeni et a l , 1986). Its K i values, calculated with CK2 from various sources (Meggio et al., 1990; Szyszka et al., 1995; Guerra et al., 1999), range between 20 and 30 f iM. The selectivity of DRB-derived inhibitors is quite remarkable: neither P K A , protein kinase C, pseudorabies virus protein kinase, nor a number of protein-tyrosine kinases are appreciably affected by concentrations that are dramatically inhibitory toward CK2 (Guerra et al., 1999a). Regulation of CK2 activity Several years ago a series of reports presented evidence for an increase in the activity of CK2 after treatment of animal cells in culture with a number of mitogenic stimuli. These reports included experiments with serum (Carroll and Marshak, 1989), insulin and insulin-like growth factor-IGF-1 (Sommercorn et al., 1987; Klarlund and Czech, 1988;), epidermal growth factor (EGF), heat shock and U V radiation (Sommercorn et al., 1987; Ackerman et al., 1990; Gerber et al., 2000). In general, increases ranging from 30% to four-fold in the level of the activity of the CK2 enzyme were observed 10-30 min after treatment of cells with the stimulating factor. However, these results that appeared to indicate the occurrence of a rapid increase in C K 2 phosphotransferase activity after treatment of cells with proliferative stimuli, have been brought into question (Lin et al., 1992; Litchfield et al., 1992). In addition, it has been reported by one of the laboratories that originally described such an increase in the activity, that a systematic study of this phenomenon indicates that the levels of CK2 19 activity measured in vitro after mitogenic stimuli do not significantly change (Allende and Allende, 1995). Some other observations are also unclear. It has been reported that C K 2 phosphotransferase activity is enhanced many fold during progesterone-induced maturation of Rena temporaria oocytes (Kandror et al., 1989). By contrast, studying the same phenomenon with Xenopus laevis oocytes, Cicirelli et al., (1988) found only a modest 1.7-fold stimulation of CK2, whereas other protein kinases such as RSK, cdkl and ERK2, increased much more dramatically. Both these observations are in agreement with a report that indicated that in vitro the cdc2 kinase [also known as the maturation-promoting factor (MPF) and cyclin-dependent kinase-1 (CDK-1)] obtained from starfish oocytes, could phosphorylate and activate CK2 (Mulner-Lorillon et al., 1990; Smits and Mederna, 2001). This report also correlates this activation with the phosphorylation of a threonine residue in the (3 subunit. Litchfield et al. (1991, 1992) have reported that cdc2 kinase can phosphorylate CK2 in vivo and in vitro. These authors demonstrated that the phosphorylation occurred on Ser-209 of the P subunit of the mammalian protein. However, they were not able to demonstrate activation of the phosphorylated CK2 . Unfortunately, it has been shown that this serine (209) is absent from the (3-subunits of Drosophila, yeast and Arabidopis (Pinna and Meggio, 1997). Furthermore, human CK2|3 can be deprived of a C-terminal segment without any apparent change in the functional properties of CK2 (Meggio et al., 1993). Similarly, it has been shown that the in vitro phosphorylation by the cdc-2 of this site of the (3 subunit, does not seem to affect the in vitro capacity of [3 to activate the a subunit (Meggio et al., 1993). 20 Interestingly, it has been also shown that cdc-2 kinase phosphorylates CK2oc in cells arrested in mitosis (Litchfield et a l , 1992), and conversely that cdc-2 kinase can be phosphorylated at Ser-39 by CK2 during GI phase (Russo et al., 1992). However, there was no specific effect of this phosphorylation on CK2oc activity (Russo et al., 1992). The significance of the relationship between cdc-2 and CK2 remains to be elucidated (Allende and Allende, 1995; Pinna and Meggio, 1997). 1.1.5. C K 2 a n d c a n c e r The observation that C K 2 protein and activity are enhanced in rapidly proliferating tissues dates back 15 years. CK2 protein and enzyme activity appear to be elevated quantitatively in all tumors with proliferation potential (Issinger, 1993; Guerra and Issinger, 1999; Ahmed et al., 2000; Landesman-Bollag et al., 2001). Increases of CK2 phosphotransferase activity in cancer tissues were first shown in human colonic carcinomas (Seitz et al., 1989; Pistorius et al., 1991). These findings have been confirmed by many other authors, encompassing a large variety of solid tumors, e.g. breast (Munstermann et al., 1990), prostate (Ahmed, 1994), head and neck (Gapany et al., 1995), and lung tumors (Daya-Makin et al., 1994). Stalter et al. (1994) performed a statistical analysis of the activity and the expression of CK2 using renal clear cell carcinomas and the corresponding control tissues. The average protein kinase CK2 activity from 21 different kidney samples was shown to be 318 U/mg and that from the corresponding tumors, 610 U/mg. The subunit ratio of protein kinase C K 2 a in tumors/normal tissue (T/N) was 1.58 and that of the 21 protein kinase CK2[3 (T/N) was 2.65 (Stalter et al., 1994). The data indicated that the increases in protein kinase CK2 activity in tumor cells may correlate to some extent with a deregulation in subunit biosynthesis or degradation. This is at least partly due to the presence of an excess of enzymatically active protein kinase oc- subunit but also to a significantly higher presence of the noncatalytic [3-subunit (Stalter et al., 1994). Caveolin, an integral membrane component of caveolae, was first identified as the major phosphoprotein whose phosphorylation was elevated in v-Src transformed cells. As both v-Src-induced transformation and elevated caveolin phosphorylation were dependent on membrane attachment of v-Src, it has been suggested that caveolin is a critical target in v-Src transformation. CK2 can phosphorylate caveolin and thus appears to play a role in mitogenic signaling events, especially in the mechanism of v-Src oncogenesis (Sargiacomo et al., 1994). The observation of a relationship between elevated CK2 expression and hence increased activity as well as the proliferative state of the tumor is so far a phenomenological observation. It has been shown that overexpression of CK2cx leads to an increase in the frequency of lymphomas, although only to a modest extent, but it is significantly increased by coexpression with c-Myc (Landesman-Bollag et al., 1998). A similar mechanism is postulated by Kelliher et al., (1996) for Tal-1 action in Tal-l/CK2oc thymocytes. Expression of the Tal-1 transgene leads to lymphoid malignancy in mice that is accelerated by the presence of the C K 2 a transgene. The Tal-1 gene product is a serine phosphoprotein and bHLH (basic helix-loop-helix) transcription factor known to regulate embryonic hematogenesis (Kelliher et al., 1996). It has been suggested that there are two transcriptional regulatory systems that collaborate with CK2 dysregulation 22 and may serve as candidates to human lymphoid malignancies and pathogenicity (Guerra and Issinger, 1999). The first involves the b H L H transcription factor Myc. CK2 phosphorylates both Myc and its D N A binding partner Max (Bousset et al., 1993). Phosphorylation by CK2 inhibits D N A binding by Max/Max homodimers but does not affect D N A binding by Myc/Max heterodimers, thus favoring gene transcription (Berberich and Cole, 1992). The second candidate system comprises the T A L / L M O heterodimeric transcriptional regulators. LMO-2 is a zinc finger transcription factor with an essential function in erythropoiesis (Hwang and Baer, 1995). Tal-1 heterodimerizes with L M O proteins to form heterodimeric complexes that regulate transcription in early erythroid and myeloid cells and are essential for their development (Warren et al 1994). The Tal-1 and LMO-1 are two genes that are frequently rearranged or ectopically expressed in human T- cell acute lymphoblastic leukemia (Hwang and Baer, 1995). CK2 phosphorylates Tal-1 and overexpression of C K 2 a collaborates with Tal-1-induced T-cell acute leukemia (Kelliher et al., 1996). The tumor suppressor p53 can be regulated by CK2. Landesman-Bollag et al. (1998) have reported that the deficiency of the tumor suppressor p53 and misexpression of protein kinase C K 2 a collaborate in the development of thymic lymphoma in mice. In addition, it has been suggested that the dysregulation of CK2 might collaborate with the loss of a p53 allele to affect lymphocyte development or transformation (Xin et al., 1999). p53 is a target of CK2 phosphorylation, it has been shown that CK2 interacts and phosphorylates human p53 at Ser-392 (Ser-386 in murine p53) (Meek et al., 1990; Herrmann et al., 1991). The sequence surrounding this phosphorylation site is 23 X X X D 3 9 2 / 3 8 6 S D (NCBI, Genbank accession # BC003596). The interaction between p53 and CK2 is discussed in more detail below. 1.2. p3 8 Mitogen-Activated Protein kinase (p3 8 M A P kinase) 1.2.1. Overview The mammalian p38 M A P kinase family consists of at least four different homologous proteins, p38cx, p38p\ p38y and p388 (Widmann et a l , 1999). The different isoforms display distinct expression patterns (Tibbies and Woodgett, 1999; Wang et al., 1997) and data indicating isoform-specific activities have been reported (Wang et al., 1998) . However, the precise biochemical function that each isoform serves in vivo remains unclear. The p38a and p38(3 genes are ubiquitously expressed. However, p38y and 8 are differentially expressed in different tissues. p38y is predominantly expressed in skeletal muscle and p38S is enriched in lung, kidney, testis, pancreas and small intestine (Wang et al., 1997). p38y expression was reported to be induced during muscle differentiation and p388 expression was shown to be developmentally regulated (Ono and Han, 2000). p38a M A P kinase is activated by diverse cellular stress conditions (microtubule inhibitors, ultraviolet and y irradiation, osmotic shock, heat shock, oxidative stress, lipopolysaccharide, protein synthesis inhibitors) (Takenaka et al., 1998; Tibbies and Woodgett, 1999), certain cytokines (interleukin-1, -2, -3, -7, -8; [IL-1, IL-2, IL-3, IL-7, IL8], tumor necrosis factor; TNFcx), growth factors like granulocyte macrophage colony-stimulating factor (GM-CSF), fibroblast growth factor (FGF), nerve growth factor 24 (NGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and G protein-coupled receptors (Widmann, 1999; Ono and Han, 2000; Mainiero et al., 2000). Table 2 lists some stimuli reported in recent years that lead to the activation of p38a. Although the other three p38 group members, p38p\ p38y and p385 were cloned several years ago, there is little information concerning their activation in different cells under diverse conditions (Ono and Han, 2000). 1.2.2. Regulation p38 M A P kinase is activated by dual phosphorylation of threonine and tyrosine residues in the Thr-Gly-Tyr activation motif between kinase subdomains VII and VIII (Widmann et al., 1999). Stimulation of p38 involves the activation of several dual protein kinases, the M A P kinase kinases (MKKs), including M K K 3 , M K K 4 and M K K 6 , which have been reported to phosphorylate p38 in vitro. There are reports indicating that distinct upstream kinases selectively activate p38 isoforms. M K K 3 cannot effectively activate p38P like M K K 6 does; p388 can be activated by M K K 3 and M K K 6 , and also by the c-Jun N-terminal kinase (JNK) kinases, M K K 4 and M K K 7 (Derijard et al., 1995; Han et al., 1996). These data indicate that the activation of p38 isoforms can be both specifically regulated through different regulators and coactivated by the same upstream regulators. p38 M A P kinase can phosphorylate and activate the M A P kinase-activated protein (MAPKAP) kinase, 2 and 3, which in turn phosphorylate small heat-shock proteins such 25 as the 27-kDa heat shock protein (HSP27) (Paul et al., 1997). Other substrates for p38 have been identified including HSP70 and transcription factors such as, ATF2, Elk, CHOP (also known as G A D D 153), MEF2C and CREB (Hung et a l , 1998; Widmann et al., 1999). Because p38 M A P kinase can phosphorylate many different substrates, it is reasonable to suggest that p38 affects many different biological functions. To investigate the biological function of p38 M A P kinase, a kinase inhibitor of p38 [pyridinyl-imidazole compound SmithKline Beecham (SB) 203580] is often used. It has been shown that SB 203580 is extremely specific for p38oc and p38|3 isoforms in vitro and in vivo (Cuenda et al., 1995; Beyart et al., 1996; Cuenda et al., 1997; Engelman et al., 1998) and has no inhibitory activity on all kinases tested including in vitro, including the other M A P kinases (Engelman et al., 1998). The crystal structure of p38 in complex with SB 203580 revealed that the inhibitor binds within the ATP pocket of the kinase and this binding interferes with the p38-substrate interaction (Tong et al., 1997; Young et al., 1997). It has been shown that a single amino acid substitution within the ATP binding pocket of p38 M A P kinase alters the sensitivity of p38 to the inhibitors and offers an explanation for the specificity of this class of inhibitors (Gum et al., 1998). It was found that the administration of SB 203580 blocks the production of cytokines (IL-1 and TNFcc) in stimulated monocytes (Lee et al., 1994). On the other hand, inhibition of p38 M A P kinase by SB 203580 prevents IL-2 and IL-7 -driven T-cell proliferation, indicating a role for p38 in cytokine-stimulated cellular proliferation (Crawley et al., 1997). On the other hand, p38 M A P kinase has been shown to be implicated in apoptosis (Widmann et al., 1999). Several cellular stresses such as osmotic shock and ultraviolet irradiation that cause apoptosis, also activate p38 M A P kinase 26 activity (Whitmarsh et al., 1996). Blockage of p38 M A P kinase activity by SB 203580 impaired p38-mediated apoptosis in T cells (Ichijo et al., 1997). Recently, p38 M A P kinase, but not E rk l , Erk2 or INK, was shown to be activated in mammalian cultured cells when the cells were arrested in M phase by disruption of the spindle with nocodazole (Takenaka et al., 1998). Addition of activated recombinant p38 to Xenopus cell-free extracts caused arrest of the extracts in M phase, and injection of activated p38 into cleaving embryos induced mitotic arrest. Treatment of NIH 3T3 cells with a specific inhibitor of p38 suppressed activation of the checkpoint by nocodazole. Thus, p38 functions as a component of the spindle assembly checkpoint in somatic cell cycles (Takenaka et al., 1998). In addition, p38 has been shown to interact with the tumor suppressor p53 and phosphorylates the murine C-terminal region of murine p53 at Ser-389 (Ser-392 in human) and the N-terminal region at Ser-33 in response to genotoxic stress such as U V and y-radiation (Bulavin et al., 1999; Keller et al., 1999; Sanchez-Prieto et al., 2000). This phosphorylation has been shown to affect p53-DNA binding and transcriptional activity (Keller et al., 1999; Sanchez-Prieto et al., 2000). How p38 M A P kinase participates in so many different biological processes remains unknown. It has been suggested that the multiple functions and different responses of p38 may be due to the availability of downstream substrates (Paul et al., 1997). Nebreda and Porras (2000) have stated that the critical determinant of understanding the molecular mechanisms that allow p38 M A P kinase to produce different cellular behaviors, is the identification of the physiologically relevant p38 target or targets. 27 Table 2. 2000). Extracellular stimuli of p38 and functional outcome (Ono and Han Stimuli Functional outcome Pathogens LPS Soluble staphylococcal peptidoglycan (pPGN) Plasma-opsonized Staphylococcus aureus (S-SA) Superantigen staphylococcal enterotoxin B (SEB) Macrophage-activating lipopeptide (MALP)-2 Mycoplasma fermentas lipid-associated membrane Echovirusl (EVI) Murine hepatitis virus strain 3 (MHV-3) prothrombinase H2O2 production TNF-a production IL-1, TNF production IL-8 production Jun B expression Macrophage production Simian immunodeficiency virus Herpes simplex virus lype-I (HSV-1) Clostridium botulinum C, toxin c-Jun expression Cytokines TNF-a IL-1 IL-2. IL-7 IL-17 (COX)-2 IL-18 Proliferation Cyclooxygenase And IL-8 production HIV type I and IL-8 production T N F - a and GM-CSF Respiratory burst response Growth factors TGF(3 Chemotaxis, actin organization 28 Table 2. Continued Stimuli Functional outcome NGF NGF withdrawal PDGF V E G F Differentiation Apoptosis Proliferation Actin organization, Stress environment Heat shock Hyperosmolarity Hypoosmolarity Ultraviolet Oxygen radical metabolites (H2O2, menadione) Stretch cardiac myocyte Cardiac hypertrophy Cyclic stretch Proliferation Hypoxia Ischemia/reperfusion As, V , Zn and Cd Arsenite Heart Repair E R K activation Others CD40 cross-linking N F - K B activation, B cell proliferation B cell antigen receptor cross-linking Okadaic acid (MMP-1) Collagenase-I expression Phenylephrine Norepinephrine Carbachol Differentiation HSP phosphorylation Vascular smooth Angiotensin 11 muscle cell Proliferation, N a + / H + exchange 29 Table 2. Continued Stimuli Functional outcome PAF Synthetic oligonucleotides (CpG DNA) Adenosine Ceramide (C2, C6 and SMase) S-nitrosoglutathione CD473 (retinoid) Calphostin-C Anisomycin Sodium salicylate Taxol Glutamate Gonadotropin-releasing hormone (GnRH) Cholecystokinin (CCK) GM-CSF cells Steel locus factor (SLF) cells FGF expression IGF Endothelin-I Hypertrophy Thrombin Three dimensional collagen Ischemic heart Preconditioning COX-2 production Apoptosis Apoptosis Actin organization Development of hemopoietic Development of hemopoietic Regulation of gene Anti-apoptosis, gene Expression Cardiac myocyte Collagenase-3 (MMP-13) expression 30 1.3. The Tumor Suppressor p53 1.3.1. Overview A large body of evidence places the transcription factor p53 at a critical point of converging pathways from diverse cellular insults, which can elicit coordinated cellular responses that result in adaptation to these insults (Hall et al., 1996a). Much important information have been derived from the analysis of clinical biopsy material, and this indicates that abnormalities of p53 are among the most prevalent molecular abnormalities in human (and other animal) cancer, with missense point mutations in the sequence-specific D N A binding domain of p53 and allelic loss at the p53 locus on chromosome 17 being particularly prevalent. In addition, p53 is a frequent target for inactivation by virally encoded proteins such as SV40 large T antigen, the HPV E6 ORF, hepatitis B X antigen, and adenovirus E l a (Hall et al., 1996a; Prives and Hall, 1999). A wealth of information has been well documented concerning p53 and its role in normal cells in maintaining the integrity of the genome and in neoplasia (Ko and Prives, 1996; Prives and Hall, 1999). p53 suppresses tumor growth by two distinct mechanisms. First, p53 induces apoptosis, a tightly regulated pathway of programmed cell death involving changes in mitochondrial physiology, cysteine protease activation and D N A degradation (Miyashita and Reed, 1995; Moll and Zaik, 2001; Shen and White, 2001). p53 also prevents growth by halting the cell cycle (Prives and Hall, 1999). p53 inhibits G l / S transition in cells exposed to D N A damage by causing accumulation of p21WAFl (El-Deiry et al., 1995). p53 also regulates a mitotic spindle checkpoint that prevents D N A synthesis before chromosome segregation (Cross et al., 1995). In addition, p53 31 controls a G2 checkpoint and expression of the wild-type protein can prevent G2 /M transition in mouse and human cell lines (Prives and Hall, 1999; Taylor and Stark, 2001). Loss of these checkpoints in p53 deficient cells can lead to polyploidy, chromosomal breaks and centrosome amplification, indicating that regulation of p53 contributes to the maintenance of D N A integrity (Cross et al., 1995; Fukasawa et al., 1996). The biology of p53 and its role to maintain the genomic stability of the cell through both cell cycle arrest and apoptosis is discussed below. 1.3.2. p53 structure and function The transcription factor p53 is a modular protein with several regions with distinct, but interdependent, functions (Arrowsmith and Morine 1996; Soussi and May, 1996). These functions are coordinated such that the p53 protein within a cell acts to integrate signals emanating from a wide range of cellular stresses, and allows the cell to respond to these insults by activating a set of genes whose products facilitate adaptive and protective activities, including apoptosis and growth arrest (Arrowsmith and Morine, 1996; Ko and Prives, 1996; Soussi, 1996). To function in such a complex manner, p53 protein is subject to a diverse array of regulatory mechanisms that keep this tumor suppressor protein in check until needed (Ko and Prives, 1996; Prives and Hall, 1999; Taylor and Stark, 2001). The biochemical properties that underpin the complex biology are intimately correlated with the structure of the p53 molecule. The core of the p53 protein is a region (within residues 100-300) which folds in such a way so as to form a domain which can interact with D N A in a sequence-specific 32 manner (El-Deiry et a l , 1992; Bourdon et al., 1997; El-Deiry et al., 1998a). The majority of missense mutations seen in human (and animal) tumor occur in regions of the gene encoding this domain such that either critical residues involved in D N A contact are altered or that the whole conformation of the core domain of the protein is disrupted. The consequence of either of these events is the loss of the ability of p53 to specifically bind to D N A in a sequence-specific manner. The binding to D N A is optimal when the protein is in a tetrameric state as a consequence of interactions of four separate p53 molecules via the tetramerization domain (Jeffrey et al., 1995). The C terminal region is composed of predominantly basic residues and forms a region that has key regulatory properties. This region undergoes modification by acetylation, phosphorylation, O-glycosylation, and R N A binding has been also reported, but the physiological significance of these posttranslational modifications remains uncertain (Prives and Hall, 1999). The D N A binding domain is separated from the transcriptional activation domain by a region containing a series of repeated proline residues typical of a polypeptide that can interact with signal transduction molecules that contain a SH3 (src-homology domain-3) binding domain (Goria et al., 1996). Through this domain p53 is influenced by diverse signaling molecules including the c-abl oncogene (Yuan et al., 1996). The acidic N-terminal transcriptional activation domain allows p53 protein, in the context of its specific binding to a target D N A sequence, to recruit the basal transcriptional machinery required for transcribing new mRNA and by so doing, activate the expression of target genes. This region is also critically involved in regulating the stability and activity of p53 protein via interactions with proteins such as Mdm2 (murine double minute clone 2), which allows targeting of p53 to the ubiquitin-mediated proteolytic machinery (Ko and Prives, 1996). 33 Mdm2 binding also blocks the ability of p53 to interact with the transcriptional apparatus (Prives and Hall, 1999). Modification of p53 by phosphorylation may alter many of these properties and, in particular, the interaction of p53 with other proteins such as Mdm2. By this means, much of the regulation of the p53 pathway occurs. 1.3.3. Regulation p53 is a nuclear phosphoprotein, and several protein kinases have been demonstrated to be able to phosphorylate p53 at sites believed to be of physiological relevance. However, it is not clear how cellular stresses communicate and regulate p53 function. There are a number of phosphorylation sites on p53 that are altered after D N A damage and, in some cases, new protein kinases have been shown to be able to phosphorylate p53 at these sites. Furthermore, such phosphorylation events have been shown to result in alterations in the protein which make it more stable and more active. Recently, it has been shown that DNA-damage induced phosphorylation of p53 may achieve optimal p53 activation and stability by inhibiting M D M 2 binding to p53 protein (Zhang and Xiong, 2001). Phosphorylation, however, is not the only way in which p53 is regulated. Interestingly, another form of p53 modification, acetylation, has also been demonstrated and shown to be D N A damage-inducible (Gu et al., 1997) and the two may be temporally and mechanistically linked (Hupp, 1999). 34 N-terminal phosphorylation The N-terminus region of p53 consists of two transcription activation subdomains extending between residues 1 and 63, and a region spanning residues 60-97, containing several copies of the sequence P X X P , which plays an important role in the induction of apoptosis by p53 (Ko and Prives, 1996). Several phosphorylation sites have been mapped within the N-terminus of human and/or murine p53 including, casein kinase I (CK I) sites on murine p53: Ser-4, Ser-6, and Ser-9; and DNA-activated protein kinase (DNA-PK) sites: murine and human Ser-15 and human Ser-37 (Meek, 1994; Jayaraman and Prives, 1999). Ser-33 in human p53 is phosphorylated in vivo, and at least one protein kinase, the TFIIH-associated trimeric cyclin-activating kinase complex (CAK) , consisting of cyclin H , CDK7, and p36/MATl , has been shown to phosphorylate p53 at that site in vitro (Prives and Hall, 1999). By contrast, murine p53 is phosphorylated at residues 33 and 34 in vivo and can be phosphorylated in vitro by p38 M A P kinase JNK, respectively (Keller et a l , 1999; Milne et al., 1995). JNK can also phosphorylate human p53 at other N-terminal sites, although the exact site is unknown (Adler et al., 1997). In addition, the amino terminal region of p53 can be phosphorylated in vitro by Erkl and Erk2 at residues Ser-73 and Ser-83 (Adler et al., 1997). It should be noted that there are a number of other potential threonine and serine phosphate acceptors within the N-terminal region of p53. Although two-dimensional phosphotryptic mapping has provided strong support for the identification of the known sites, and perhaps can be tentatively used to rule out others, it remains possible that in some cells or under some conditions, novel sites may be utilized and such sites may indeed turn out to be very interesting. Although 35 demonstration of the existence of sites and relevant kinases is important, showing that a given site is phosphorylated in vivo in response to a given stimulus takes us considerably further in understanding the biological relevance of such modifications. A barrier to determining stress-induced changes in phosphorylation is that it is difficult to compare accurately the phosphorylation status of p53 from normal cells, where it is often present in nearly undetectable amounts, with that in stressed cells, where its levels have risen dramatically, often by one or two orders of magnitude (Prives and Hall, 1999). One important technical advance has been the use of phosphorylation site-specific antibodies. These reagents have made possible the rapid identification of specific phosphorylation events, either constitutive or inducible, and a number of groups have now reported advances with such antibodies. The first site to be shown to be inducibly phosphorylated after D N A damage was Ser-15 (Siliciano et al., 1997), as mentioned above, previously demonstrated to be a substrate for the DNA-dependent protein kinase (DNA-PK) (Lees-Miller et al., 1992). Recently, another kinase, the A T M (ataxia telangiectasia mutated) protein, the product of A-T gene (the human genetic disorder ataxia telangiectasia) has also been shown to be able to phosphorylate this site (Banin et al., 1998; Canman et al., 1998) and it is likely that additional kinases wil l prove capable of. carrying out this modification as well. Importantly the activity of the A T M protein kinase is significantly increased when cells are subjected to D N A damage. However, the observations that several protein kinases can phosphorylate a D N A damage-responsive site leave critical questions to be solved. It is still unknown which is the primary kinase that actually phosphorylates p53 at this (or any) site in vivo. To make matters even more complicated, it is now known that Ser-15 is not the only D N A damage-inducible site on p53. It was 36 recently shown that both Ser-33 and Ser-37 are also D N A damage-inducible (Jayaraman and Prives, 1999; Keller et al., 1999). Furthermore, the Meek laboratory has identified additional protein kinases for which p53 is a substrate whose activities are D N A damage-inducible (Milne et al., 1995). Moreover, Silicano et al. (1997) reported, in addition to Ser-15, another site within the first 20 amino acids of p53 that was increased in irradiated cells. What is the significance of phosphorylation of N-terminal sites? First, it is often the case that modification of a given site leads to conformational changes that are propagated to other regions of the phosphorylated protein. Second, p53 makes specific contact with a number of proteins (see below) and with DNA, and phosphorylation can affect these interactions. The N-terminus of p53 associates with both components of the transcriptional machinery, with TAFs (TATA box binding protein associated factor), and with its negative regulator, Mdm2 (Jayaraman and Prives, 1999; Prives and Hall, 1999). Shieh et al. (1997) demonstrated that phosphorylation of p53 with D N A - P K in vitro both markedly reduces the ability of p53 to bind to Mdm2 and causes a significant change in conformation of the N-terminus. Mdm2 both destabilizes p53 and represses transactivation by p53, and so this result provides a plausible explanation for how p53 is induced after D N A damage. It is likely that additional N-terminal D N A damage-inducible modifications will also be shown to affect the interactions of p53 with Mdm2, and it was recently reported that phosphorylation of human p53 by JNK inhibits its interaction with Mdm2 (Fuchs et al., 1998). It is also possible that phosphorylation at N -terminal sites will be shown to affect its interaction with transcription factors, as it was 37 reported that phosphorylation at Ser-15 and Ser-37 decreases the ability of p53 to bind to TFIID (Jayaraman and Prives, 1999). C-terminal phosphorylation The C-terminus of p53 has been described both as a region whose function is to control sequence-specific D N A binding and as a D N A damage recognition domain (Ko and Prives, 1996). It possesses autonomous D N A binding and strand reassociation abilities but to date, there has been no evidence of sequence specificity in these interactions (Ko and Prives, 1996). The p53 protein exists in solution predominantly as a tetramer and tetramers are the most efficient D N A binding form of the protein (Hupp, 1999). The structure of the oligomerization domain of human p53 reveals a (3-sheet-turn-cx-helix motif"which forms a pair of dimers (Hupp, 1999). Mutation of the p53 tetramerization region substituting residues with alanine or hydrophobic amino acids leads to altered oligomerization properties (Hupp, 1999). Substitution of this region with a heterologous tetramerization domain, however, produces a protein with many of the activities of wild-type p53 (Hupp, 1999; Jayaraman and Prives, 1999). It is interesting that disruption or loss of oligomerization function is associated with loss of cell cycle arrest but not growth suppression as measured by colony formation assays (Ishioka et al., 1995; Moll and Pellegata, 1995). Recently mutations have been identified in this domain (Lomaxetal, 1997). The effects of covalent modification of the C-terminal region of p53 on its D N A binding properties continue to spark much interest. Hupp et al. (1992) were the first to 38 discover that phosphorylation, antibody interaction or deletion of the C-terminus stimulates D N A binding of inert bacterially expressed p53, and this pivotal observation has led to a plethora of related findings. It is now known that phosphorylation at CK2 sites (Ser-392) or P K C (protein kinase C) sites (Ser- 371, 376, and 378) within this region result in stimulation of binding (Ko and Prives, 1996). The mechanism by which the C-terminus regulates D N A binding is not yet fully understood. Two models have been proposed: in the first, it has been hypothesized that the C-terminus allosterically regulates conversion of p53 from an inert 'latent' form to one which is active for D N A binding (Hupp et al., 1994). Evidence for this model is based on the observation that p53 can be isolated from baculovirus-infected insect cells or synthesized in vitro in a form that binds D N A only weakly but which, upon treatments as outlined above, becomes activated. The second model postulates that binding by the central domain is hindered by the interactions of the C-terminus with longer D N A molecules (Bayle et al., 1995; Anderson et al., 1997). It is not clear at this point which of these two models (or both) is correct. Validation of the allosteric model will require physical evidence for a conformational alteration in p53 protein upon one or more of the modifications noted previously. Indeed, it is entirely possible that both explanations contribute to the way in which p53 is regulated by its C-terminus. There have been a number of current developments in understanding how phosphorylation regulates p53 D N A binding. We must preface this optimistic statement by noting that attempts to discern phenotypes of phosphorylation site mutants in p53 have been confusing at best and disappointing at worst. Mutation of murine phosphorylation sites at N - and C-termini have frequently yielded p53 proteins which are roughly 39 equivalent to wild-type protein in standard transient transfection assays of transcriptional activation of p53- responsive reporters (Ko and Prives, 1996). Hao et al. (1996), however, reported that wild-type p53 in Gl-arrested murine cells is inactive, while mutation of the murine CK2 site to a charged residue (Glu) rescued p53 function. Additionally, mutation of the murine CK2 site to alanine had little effect on transactivation of the cyclin-dependent kinase inhibitor, p21, and RGC (ribosomal gene cluster) reporters, while it markedly inhibited the ability of p53 to repress the SV40 promoter (Hall et al., 1996a). Although it is not yet well understood, we might speculate that an interaction of the C-terminus with a protein component of the general transcription machinery such as TBP or p300, both of which have been implicated in the repression function of p53, either might require p53 to be phosphorylated at that site or might be affected by mutating Ser-392 (Prives and Hall, 1999). Further insight into the mechanism by which p53 is stimulated by CK2 is derived from the observation that p53 is bound stably by the beta subunit of CK2 (Appel et al., 1995; Hall et al., 1996b) and after phosphorylation, p53 can no longer reanneal D N A (Filhol et al., 1996). This interaction, as well as phosphorylation, may contribute to the effect of the protein kinase on p53 D N A binding. Inhibition of p53 annealing of D N A by CK2 phosphorylation supports the proposal that the C-terminus, when bound to D N A , interferes with specific binding by the central region. Note, however, that Sakaguchi et al. (1998) have demonstrated that peptides spanning the C-terminal protein (residues 303-393) display a ten-fold greater association constant for reversible tetramer formation after phosphorylation by CK2. Whether this striking observation is also true for full length p53 awaits validation by experimentation. Importantly, Kapoor and Lozano (1998) and 40 Lu et al. (1998), using antibodies specific for p53 phosphorylated at the CK2 site, showed that U V irradiation, but not gamma irradiation (IR), induces phosphorylation at this site. Another binding site at the C-terminal of human p53 is for C D K . It has been identified within residues 330- 339 (Wagner et al., 1998). Interestingly, substitution of Ser-392, but not Ser-315, with the acidic residue Asp led to reduced interaction of C D K with p53 (Sakaguchi et al., 1998). Reciprocally, whereas phosphorylation of Ser-392 increases tetramerization, this effect is reversed when p53 is phosphorylated at Ser-315 (Sakaguchi et al., 1998). Taken together, these data indicate an inter-dependent relationship between the CK2 and C D K sites on p53. 41 Responses to the activation of p53: Downstream events The downstream events mediated by p53 culminate in either cell cycle arrest or maintenance of chromosomal diploidy or mitotic fidelity when cells are exposed to genotoxic stresses. I will discuss briefly those events that pertain to the objective of this thesis. p53 and cell cycle checkpoints Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions and ensure that critical events such as D N A replication and chromosome segregation are completed with high fidelity. In addition, checkpoints respond to damage by arresting the cell cycle to provide time for repair and by inducing transcription of genes that facilitate repair. Checkpoint loss results in genomic instability and has been implicated in the transformation of normal cells into cancer cells. Recent advances have revealed signal transduction pathways that transmit checkpoint signals in response to D N A damage, replication blocks, and spindle damage. Checkpoint pathways have components shared among all eukaryotes, underscoring the conservation of cell cycle regulatory machinery. Checkpoints are regulated by several cell cycle transitions that are dependent upon the activity of cyclin-dependent kinases (Cdks), and inhibition of these kinases is a common mechanism by which cell cycle arrest. These enzymes are composed of a kinase subunit, Cdk, and an activating subunit, cyclin, in a complex that is subject to many levels of regulation (Villard et al., 2001). 42 Although, the most well-understood component of p53 function is its ability to induce a GI arrest as a result of D N A damage, several recent reports have implicated a role for p53 in G2/M and the spindle assembly checkpoints (Taylor and Stark, 2001). In response to D N A damage, the DNA-damage checkpoint detects damaged D N A and generates a signal that arrests cells in the GI (Gapl) phase of the cell cycle, slows down S phase (DNA synthesis), arrests cells in the G2/M (Gap2/Mitosis) phase, and induces the transcription of repair genes. The most well-understood component of D N A damage checkpoint is p53 function for its ability to induce a GI arrest via p21 (WAF1) pathway. In response to D N A damage, p53 has been shown to induce the expression of the p21(WAFl) C D K inhibitor, which appeared to be directly controlled by p53 transcription activation through two specific p53 DNA-binding response elements located within the p21 promotor (El-Deiry, 1995; El-Deiry et al., 1998b; Liu and Kulesz-Martin, 2001). Recent studies have revealed the possible existence of distinct signaling pathways that may preferentially utilize one or more of the other p53-binding elements within the p21 promotor (Resnick-Silveman, 1998). Once activated, p21 protein binds to cyclin-C D K complexes and inhibits their kinase activity (El-Deiry et al., 1998a; Liu and Kulesz-Martin, 2001). Because C D K kinase activity is required for various cell cycle transitions, p21 is a potent cell cycle inhibitor. p21 binds with its greatest affinity to GI cyclin-CDK complexes, and binds poorly to cyclin B/cdc2 during mitosis (El-Deiry et al., 1998a). p21 is a member of the p27 and p57 KIP/CIP family of universal cell cycle inhibitors which can inhibit cyclin D/CDK4/6 complexes during Gl /S and G2 cell cycle transition (Pines, 1999). p21 also binds to the proliferating cell nuclear antigen (PCNA) and can inhibit the processivity of D N A replication (El-Deiry et al., 1998b). 43 In mitosis, the proper segregation of chromosomes requires the execution of a number of processes: a bipolar spindle must be assembled; chromosomes must attach to the spindles through the kinetochore, a protein structure that forms on the centromeres of chromosomes. The kinetochores of sister chromatids must bind to spindle fibers attached to opposite poles and the properly attached chromosomes must arrive at the metaphase plate. The spindle assembly checkpoint prevents the onset of anaphase, the actual segregation of chromosomes, until these processes have been properly accomplished. Once these events take place, cells can execute anaphase and daughter cells separate (cytokinesis) and then progress into the next cell cycle (Pines, 1999; Gardner and Burke, 2000; Villard et al., 2001). Disruption of the spindle with microtubule-depolymerizing drugs such as nocodazole and colchicine arrest cells in mitosis (Runder and Murray, 1996), and this arrest depends on the integrity of the spindle assembly checkpoint. The induction of mitotic arrest by the effect of the microtubule-depolymerizing drugs has been shown to be related to the elevated level of activation of the cyclin-dependent kinase-1 (CDK-1); cdc2/cyclin B complex. Adaptation to prolonged checkpoint arrest can occur by inhibitory phosphorylation of cdc2 pr degradation of cyclin B (this effect is discussed below). The cell cycle G2/M transition and progression through mitosis is driven by the kinase activity of a complex referred to as maturation- or M-phase-promoting factor (MPF) or CDK-1 . This complex consists of a catalytic subunit (34-kDa cyclin-dependent kinase, cdc2), a regulatory subunit (cyclin B proteins), and associated proteins (Norbury and Nurse, 1992). Entry into mitosis requires cdc2 activation, a process that depends 44 upon an increase in cyclin B expression and the dephosphorylation of cdc2. Progression through mitosis and cytokinesis requires the subsequent inactivation of cdc2, which depends in part on cyclin B degradation. Experiments with yeast indicate that the mitotic spindle cell cycle checkpoint feeds into the cell cycle regulatory machinery at mitosis by a pathway that delays the degradation of cyclin B and maintains cdc2 kinase activity (Elledge, 1996; Villard et al., 2001). Thus, cyclin B is degraded and cdc2 is inactivated only after certain aspects of mitosis related to spindle assembly and disassembly are properly completed. Human CDK-1 was identified through complementation of a cdc2 mutant in yeast and as a protein that cross-reacted with antibodies to yeast cdc2 (Mowal and Stewart, 1998). Cyclin B has been described as a protein with no enzymatic activity and whose level increases during the cell cycle and then is destroyed late in mitosis; it is essential for cdc2 kinase activation and specificity (Norbury and Nurse, 1992; Ewen, 2000). Cdc2 activation is a multi-step process that begins when it binds to its regulatory subunit cyclin B , whose levels rise during G2 and peak in mitosis. Activation of cdc2 requires both cyclin binding and phosphorylation at position Thr-161 by cyclin-activating kinase (CAK) (Norbury and Nurse, 1992; Mowal and Stewart, 1998). C A K kinase was first found in Xenopus pM015 kinase and has sequence similarity to cdc2, now called CDK7 (Solomon et al., 1993; Mowal and Stewart, 1998). The phosphorylation of cdc2 at Thr-161 has been suggested to enhance binding of cyclin B (Mowal and Stewart, 1998). This is based on the structural analysis of the CDK2/cyclin A complex which shows that in the absence of binding to cyclin A , the regulatory T loop that is located at the ATP-binding site, blocks protein substrate binding 45 (Russo et al., 1996). Cyclin binding reorientates the ATP-binding site and allows movement of the T-loop to allow substrate binding and make it more accessible for phosphorylation by C A K (Russo et al., 1996). Phosphorylation of Thr-160 (equivalent to Thr-161 in cdc2) in the T-loop moves the loop further and stabilizes CDK2/cyclin A by increasing the number of contacts (Russo et al., 1996; Bartek and Lukas, 2001). CDK1 kinase phosphorylates critical proteins involved in S phase and M phase transitions resulting in D N A synthesis, chromosome condensation, cytoskeletal reorganization, nuclear lamin disassembly, and cell shape changes (Norbury and Nurse, 1992; Ekholm and Reed, 2000). Some targets include histone H I , nuclear lamins, vimentin, a microtubule-associated protein 115, p53 protein and others (Mowal and Stewart, 1998). Before mitosis, cyclin-B-cdc2 complexes are held in an inactive state by phosphorylation of cdc2 at Thr-14 and Tyr-15, which is catalyzed by the protein kinases Weel (which phosphorylates Tyr-15 only) and Myt l (which phosphorylates both Thr-14 and Tyr-15) (Pines, 1999; Ewen, 2000; Smits and Medema, 2001). Dephosphorylation is carried out by the dual-specificity protein phosphatase Cdc25C. Cdc2 activation at the onset of mitosis results from the concurrent inhibition of Weel and M y t l and stimulation of the phosphatase Cdc25C (Pines, 1999; Ewen, 2000; Smits and Medema, 2001). Activation of Cdc25C requires phosphorylation of several sites in the Cdc25C amino-terminal domain; this is catalyzed by at least two kinases. One of these kinases appears to be the Polo-related kinase (Plk); the Xenopus Plk homolog, Plx, phosphorylates and activates Cdc25C in vitro and is required for Cdc25C activation in frog egg extracts (Kumagai and Dunphy, 1996). Activating phosphorylation of Cdc25C is also carried out 46 by the Cyclin-Bl-cdc2 complex itself (Hoffman et al., 1993; Smits and Medema, 2001). The ability of cdc2 to activate its own activator provides the potential for a positive feedback loop in Cdkl activation, whereby partial activation of Cdc25C (perhaps by Plk) could lead to complete and overwhelming cdc2 activation. This positive feedback loop may be enhanced by the ability of cdc2 to phosphorylate and inactivate Weel (Patra et al., 1999). There is also evidence that cdc2 stimulates Plk activity in frog egg extracts, providing another potential mechanism for positive feedback (Abrieu et al., 1998). There are two different cyclin B proteins in mammalian cells. Cyclin B2 is a non-essential protein that associates with the Golgi apparatus and probably plays a role in Golgi remodelling during mitosis ( Jackman et al., 1995; Brande et al., 1998). Cyclin BI is an essential protein that is thought to be responsible for most of the other actions of cdc2 in the cytoplasm and nucleus (Brande et al., 1998; Villard et al., 2001). Recent data have indicated a role for the p53 tumor suppressor in regulating G2 and the mitotic spindle cell cycle checkpoints to maintain D N A integrity. It has been shown that p53 may interfere with the control of cyclin B metabolism by the mitotic checkpoint pathway and facilitate the accumulation of cells with a polyploid D N A content (Hixon et al., 1998). In addition, studies by Taylor et al. (1999) have shown that overexpression of p53 causes G2 arrest, attributable in part to the loss of cdc2 activity. Transcription of cdc2 and cyclin B I , determined using reporter constructs driven by the two promoters, was suppressed in response to the induction of p53. Suppression requires the regions -287 to -123 of the cyclin BI promoter and -104 to -74 of the cdc2 promoter. p53 did not affect the inhibitory phosphorylations of cdc2 at Thr-14 or Tyr-15 or the activity of the cyclin-dependent kinase that activates cdc2 by phosphorylating it at Thr-47 161. Overexpression of p53 was shown also to interfere with the accumulation of cdc2/cyclin B l in the nucleus, required for cells to enter mitosis (Taylor et al., 1999). Along a similar theme, Park et al. (1999), have shown that over expression of p53 in human bladder carcinoma cells released from Gl /S arrest, causes them cells to become arrested at G2/M transition due to repression of cyclin B l level. It has been shown that p53 can inhibit cyclin B l promotor activity and it is likely that the p53 mediates a decrease in cyclin B l mRNA that result from a reduction in the rate of cyclin B l transcription (Innocente et al., 1999). Several laboratories have reported that the loss or mutation of p53 result in cellular polyploidy and loss of mitotic fidelity, implicating an important role of p53 in the mitotic spindle cell cycle checkpoint (Cross et al., 1995; Fukasawa et al., 1996). It has been shown that fibroblasts isolated from individuals with Li-Fraumeni syndrome have a marked tendency to become heteroploid (or accumulation of more than one round of D N A without proper chromosomal segregation cell division, a process also called D N A endoreduplication) in culture (Bischoff et al., 1990). These individuals are born with heterozygous mutations in the p53 tumor suppressor gene (Livingstone et al., 1992) . Heteroploidy is also commonly found in p53 null cells in culture (Harper et al., 1993) and in vivo (Cross et al., 1995) in p53 knockout mice. Intriguingly, overexpression of a mutant p53 protein on a p53 null background accelerated the appearance of polyploidy in a myelomonocytic cell line (Peled et al., 1996). Also, the expression of mutant p53 proteins in human colon carcinoma cells and murine cell lines causes karyotypic abnormalities, including an increase in ploidy levels during growth in culture (Agapova et al., 1996). It has also been shown that Li-Fraumeni syndrome fibroblasts 48 that carry heterozygous structural p53 mutant proteins progress to polyploidy when incubated in the presence of mitotic spindle inhibitors (Gualberto et al., 1998). However, normal human fibroblasts, p53 null Li-Fraumeni fibroblasts, and normal human fibroblasts infected with a retrovirus that expresses the human papillomavirus 16 E6, which binds to and promotes the degradation of p53, exhibit growth arrest when incubated in the presence of mitotic inhibitors (Gualberto et al., 1998). Although, Lanni and Jacks (1998) have recently reported that p53 null mouse fibroblasts have a normal mitotic spindle checkpoint, these fibroblasts may progress to polyploidy due to the inactivation of a p53^dependent postmitotic checkpoint (Lanni and Jacks, 1998). In addition, inactivation of wild-type p53 by the overexpression of a truncated (C-terminus) p53 protein in a murine prolymphocyte cell line leads to polyploidy (Minn et al., 1996). p53 and centrosome homeostasis Recently, it has been proposed that p53 actively participates in maintaining the stability of the genome through regulation of centrosome duplication or as a monitor that limits centrosome overproduction. Fukasawa et al. (1996), have reported that the regulation of the centrosome duplication cycle is abrogated in the absence of p53, resulting in hyperamplification of centrosomes. In animal cells, the centrosome is composed of a pair of centrioles (core components of centrosome) and surrounding amorphous pericentriolar materials (Bornens, 1992). The centrosome, as a major microtubule organizing center, participates in various cellular events throughout the cell cycle. Functions of the centrosome are 49 perhaps most prominently featured during mitosis, where, two centrosomes become spindle poles and establish bipolarity, direct spindle microtubule assembly, and determine the cleavage furrow plane, all of which are required for the balanced segregation of chromosomes. In addition, during interphase the centrosome nucleates and spatially organizes the cytoplasmic microtubules, thus controlling redistribution of cellular organelles and establishment of cellular integrity (Bornens, 1992). Because each daughter cell receives only one centrosome after mitotic decision, the centrosome must duplicate during the mitotic cycle of the daughter cell. In addition, the centrosome must duplicate only once before the subsequent cell division. The duplication cycle encompasses all phases of the cell cycle. Duplication begins near the Gl /S boundary and is completed in G2 (Bornens, 1992). There are two major regulatory mechanisms imposed on centrosome duplication. One is the correct timing of initiation. The second is the prevention of reduplication of centrosomes once they have duplicated. Another important aspect of the centrosome duplication is that it always occurs in precise coordination with D N A replication. Loss of centrosome duplication cycle regulation or uncoupling of the centrosome duplication cycle from D N A replication cycle leads to abnormal amplification of centrosomes, which in turn profoundly affects mitotic fidelity (Bornens, 1992; Villard et al., 2001). Control of both initiation of centrosome duplication and suppression of reduplication appears to be abrogated in p53 (-/-) mouse embryonic fibroblast cells (MEF) (Fukasa'wa et a l , 1996). In these cells, Fukasawa et al. (1996) were able to show that centrosomes have undergone multiple cycles of duplication in a single cell cycle. In many cases, the abnormally amplified multiple copies of centrosomes co-migrated to the 50 poles, establishing pseudobipolarity. A small proportion of these cells with such pseudobipolar spindle underwent normal chromosome segregation and cytokinesis. However, a large proportion of cells with multiple spindle poles have been shown to undergo cell death in both in vitro culture of p53 (-/-) MEFs and in vivo p53 (-/-) mice (Fukasawa et al., 1996; Fukasawa, 1997; Tapore and Fukasawa, 2000). p53 and regulation of apoptosis Although the mechanisms of p53-dependent apoptosis are not clear, p53 plays a role in triggering apoptosis under several different physiological conditions. Normal thymocytes wil l undergo apoptosis in response to D N A damage, whereas thymocytes from p53 -/- mice do not undergo apoptosis in response to the same stimulus (Lowe et al., 1993b; Mol l and Zaika, 2001). Similarly, in mice that are irradiated, the stem cells of the small and large intestines undergo apoptosis, which does not occur in p53-/- mice. However, it is equally clear that not all apoptotic events are p53-mediated. For example, immature thymocytes from p53 -/- mice die of apoptosis when exposed to glucocorticoids or compounds that trigger T cell receptor pathways (Lowe et al., 1993; Shen and White, 2001). Physiological signals or activation of a specific signal transduction pathway can block p53-mediated apoptosis. Myeloid leukemic cells or murine erythroleukemic cells will undergo apoptosis when p53 is overexpressed and activated (Yonish-Rouach et al., 1991). Treatment of the myeloid cells with interleukin-6 or the erythroid cells with erythopoeitin blocks the p53-mediated apoptic pathway (Yonish-Rouach et a l , 1991). In 51 this case, communication between p53 and a signal transduction pathway results in the reversal of p53-mediated apoptosis. p53 can also initiate apoptosis in response to the expression of a viral or cellular oncogene or the absence of a critical tumor suppressor gene product retinoblastoma protein (Rb) (El-Deiry et a l , 1998a). Rb is the major target of cyclin D/CDK4 for cell cycle regulation (Levine, 1997). The Rb protein regulates E2F-DP transcription factor complexes (E2F-1, -2, and - 3 and DP-1, -2, and -3) which in turn regulate a number of genes including those encoding cyclin E, cyclin A and proliferating cell nuclear antigen (PCNA) required to initiate and propagate the S phase of the cell cycle (Levine, 1997). The expression of the adenovirus E l A protein in rat embryo fibroblasts has been shown to stabilize and activate p53. The resultant cells die of apoptosis (Debbas and White, 1993). The adenoviruses normally express the E1B-55 kDa protein, which binds to p53 and blocks its transcription factor activity, acts like bcl-2 to block apoptosis downstream of p53 activation (El-Deiry et al., 1998a). The human papilloma virus E7 protein acts like E l A and induces p5 3-mediated apoptosis. In response, the human papilloma virus genome encodes the E6 oncogene product to bind to p53 and promotes its degradation. Transgenic mice expressing E7 in the retina photoreceptor cells show extensive apoptosis (Howes et al., 1994). The expression of E7 in these same cells but in a p53-/- mouse results in a reduced frequency of apoptosis and an increased frequency of development of retinal tumors (Howes et al., 1994). The E l A and E7 proteins bind to the Rb protein and inactivate its ability to regulate the transcription factor complex, E2F-DP-1 activity (Levine, 1997). Such an unregulated E2F-1 activity in concert with activated p53 results in apoptosis (Levine, 1997). Cells overexpressing E2F-1 and a temperature-52 sensitive p53 undergo apoptosis at 32°C but not 37-39°C, where p53 is inactive (Wu and Levine, 1994). Other oncogenes also trigger p53-mediated apoptosis. Cells overexpressing myc and a temperature-sensitive p53 have a temperature-sensitive apoptotic response (Wagner et al., 1994). Thus, a number of factors affect the decision of a cell to enter a p53 -mediated cell cycle arrest or apoptotic pathway. Under conditions in which the D N A or cell cycle spindle machinery is damaged, survival factors for the cells are limiting, or an activated oncogene is forcing the cell into a replicative cycle (E1A, E7, E2F-1, or myc), p53-mediated apoptosis prevails. In this way, cells with unstable genomes (due to D N A or spindle damage) or cells in an abnormal environment (i.e., located in a place with limiting survival factors) with activated oncogenes that commit them to enter the cell cycle are eliminated in a p53-dependent apoptotic event (Levine, 1997). This is most likely the reason why so many cancerous cells select against wild-type p53 function (Levine, 1997). These observations bring up a number of questions. Clearly, the p53-mediated cell cycle arrest requires p53-dependent transcription and one or more of its downstream genes. Does p53-mediated apoptosis require the transcriptional activity of p53? Furthermore, how does p53 sense the presence of an activated oncogene in a cell (or a pathway activated by an oncogene?). A number of experiments addressing the first question, concerning the need for p53-mediated transcription to induce apoptosis, have concluded that both a p53-mediated transcriptional activity and a p53 activity not requiring transcription can play a role in apoptosis, and the choice depends on the cell type or experimental situation (Levine, 1997; El-Deiry et al., 1998a). In several cell 53 types, p53-mediated apoptosis initiated by D N A damage occurs in the presence of actinomycin D or cycloheximide, which block R N A or protein synthesis (Caelles et al., 1994). Similarly, a temperature-sensitive p53 in a cell with an activated myc induces apoptosis normally at 32°C in the presence of cycloheximide (Wagner et a l , 1994). It has been shown clearly that the transcriptional activation or the translation of p53- regulated gene products are not required in these cases for apoptosis (Caelles et al., 1994; Wagner et al., 1994). Similarly, the introduction of a p53 cDNA fragment that encodes amino acid residues 1-214 into HeLa cells induces apoptosis (Haupt et al., 1995). This fragment fails to bind to D N A or act as a transcription factor. Similarly, p53 with mutations in the transactivating domain (residues 22 and 23) that is unable to activate transcription still induces apoptosis in HeLa cells (Haupt et al., 1995). In contrast, the expression of E l A in baby rat kidney cells or mouse cells failed to induce apoptosis when p53 contained mutations that alter p53-specific D N A binding or transcriptional activation (Sabbatini et al., 1995). Indeed, the mutant p53 that produces amino acid residues 1-214 induced apoptosis in HeLa cells, but failed to induce apoptosis in another cell type, a human lung carcinoma, cell line H1299 (Haupt et al., 1995). Thus, it has been suggested that p53 may use transcriptional activation or direct protein signaling (protein-protein interactions or some other activity) or both to initiate apoptosis (Levine, 1997). There are a number of genes that are regulated by p53 (in at least some cell types). The earliest mediators of p53-dependent apoptosis to be identified were pro-apoptotic bax gene, p53 induced genes (PIGs), insulin growth factor-binding protein-3 (IGF-BP3) and others (Polyak, et a l , 1997; El-Deiry et al., 1998b; Shen and White, 2001). It is clear that no one target of p53 will be sufficient to recapitulate the p53-54 dependent apoptosis phenotype under all physiological conditions. However, it is established that Bax and IGF-BP3 can be directly regulated by p53 and can influence the decision to commit to apoptosis (Miyashita and Reed, 1995; Buckbinder et al., 1995). Overexpression of the anti-apoptotic protein Bcl-x (or the adenovirus E1B-19 K D a protein) can block p53-mediated apoptosis (El-Deiry et al., 1998b; Miyashita and Reed, 1995). Bax binds to Bcl-x and antagonizes its ability to block apoptosis, so a p53-dependent Bax synthesis could tip the scales toward apoptosis (Levine, 1997). The mechanism of action of the Bcl2-Bax family of gene products remains unclear. Another p5 3-regulated gene product that could affect growth regulation is the insulin-like growth factor-binding protein-3 (IGF-BP3) (Buckbinder et al., 1995). IGF-BP3 blocks the IGF mitotic signaling pathway by binding to IGF and preventing its interaction with its receptor. Thus, the blocking of IGF activity could enhance apoptosis or lower the mitogenic response of cells (Buckbinder et al., 1995). It has been reported that the Fas/APO-1 gene may also be regulated by p53 (Owen-Scaub et al., 1995). The Fas receptor (also known as APO-1 is a potent inducer of apoptosis in hematopoietic or liver cells exposed to Fas ligand (Nagata, 1997). p53 responsive elements have not yet been demonstrated in the Fas/APO-1 gene and so proof that it is truly a p53-regulated gene remains to be provided (Levine, 1997; Wang et al., 1999; Shen and White, 2001). 55 1.4. Current problems and objectives As outlined in the above discussion, the signaling pathways that regulate protein kinase C K 2 have been elusive since the discovery of this highly conserved and ubiquitous protein-serine/threonine kinase over 45 years ago. Furthermore, the tumor suppressor p53 is one of the key molecules involved in maintaining stability of the genome, through cell cycle arrest and apoptosis in response to genotoxic stress. p53 has previously been shown to be associated with protein kinase C K 2 , and undergo phosphorylation on Ser-392. However, the mechanism by which Ser-392 is regulated, under circumstances of cellular stress remain to be elucidated. The goals of this project were: 1. To investigate whether stress-activated signaling regulate protein kinase CK2, and uncover the mechanisms that underlie such regulation. 2. To characterize the physiological regulation and functional consequence of p53 phosphorylation at Ser-392 by CK2 in response to cellular stress especially in the event of mitotic crises in cancer cell cycle. 56 2. MATERIAL AND METHODS 2.1. Cell Culture and Lysates Preparation. Human cervical carcinoma HeLa cells were obtained from A T C C and human colon tumor HCT116 (p53 +/+ and p53 -/-, kindly donated by Dr. B. Vogelstein (Bunz et al., 1998). Cells were grown for 3 days until near confluency at 37°C and 5% C 0 2 on 175 cm 2 flasks in 10% fetal calf serum (FCS) supplemented D M E M and McCoy's medium (GIBCO), respectively. Cells were split at 5x 105 cells AO cm 2 dish 24 hour prior to treatment. For cell stimulation, HeLa cells were starved with 0.5% FCS for 15-20 h before exposure to stimuli. Starved cells were incubated with 10 p M SB203580 (Calbiochem) for 30 min prior to addition of 10 pg/ml anisomycin (Sigma) for 30 min, 50 p M arsenite (Sigma) for 15 min, or 20 ng/ml TNF ex for 15 min. Adherent cells were washed twice with ice-cold, phosphate-buffered saline (PBS), scraped, then lysed in 50 m M Hepes-NaOH (pH 7.2), 100 m M NaCl, 1 m M EGTA, and 20 m M NaF in Buffer A (1 m M sodium orthovanadate, 1% aprotinin, 1 mM phenylmethylsulfonylfluoride, 1 p M pepstatin, 0.5 pg/ml leupeptin, 10 pg/ml soybean trypsin inhibitor and 1% Non-idet P-40). Lysates were subjected to brief sonication for 2 x 10 s, centrifuged at 15,000 rpm for 5 min at 4°C, and the supernatants were collected and stored frozen prior to use. The protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as a standard. In some experiments, anisomycin-treated cells were lysed with ice-cold 100 m M Tris-HCl (pH 8), 100 m M NaCl, 50 m M MgCl2, 0.3 m M EDTA, 0.5 m M ZnCl2 and 1% 57 Nonidet P-40. After sonication for 2 x 10 s, the lysates were centrifuged at 14,000 rpm for 5 min at 4°C and the supernatant was incubated with alkaline phosphatase (Calbiochem) (0.5 U/jig) for 2 h at 37°C. Ten volumes of ice-cold Buffer B (10 m M MOPS (pH 7.2), 25 m M p-glycerophosphate, 2 m M EDTA, 5 m M E G T A and 2 m M sodium orthovanadate) were added and the sample was subjected to MonoQ anion-exchange chromatography. 2.2. Cell Synchronization For cell synchronization at G l / S phase, HeLa cells were treated with double thymidine block (two 16 h periods of exposure to 2 m M thymidine separated by a 10 h exposure without thymidine). The cells were then rinsed twice with phosphate buffer saline (PBS) and cultured in complete growth medium. Nocodazole (100-400 ng/ml) was added to either exponentially growing cells for the indicated times or to G l / S synchronized cells for 10 h. In some experiments HeLa cells were cultured in D M E M containing FCS (0.5%) for 30 h to synchronize cells in GO phase, fresh D M E M supplemented with 10% FCS was then added to induce the cells to re-enter the cell cycle. After 18 h, nocodazole (400 ng/ml) was added before cells entered M phase, as described (Takenaka et al., 1998). Mitotic cells were collected by mechanical shake off, 9 h after release from thymidine block (Gl/S) or 22-26 h from GO release (Takenaka et al., 1998). In some experiments, the potent and specific CK2 or p38 M A P K inhibitors; 5,6-dichloro-1-p-D-ribofuranosylbenzimidazole (DRB) (20 L I M ) and SB203580 (10 aM) (Calbiochem) respectively, were used 2 h prior to the addition of nocodazole. 58 2.3. MonoQ Chromatography and Immunoprecipitation. Cell lysates (2 mg protein) were diluted 1:10 in Buffer B and loaded onto a MonoQ (1-ml; Pharmacia) column at 4°C. The anion-exchange column was eluted with a 20 ml linear gradient of 0-0.8 M NaCl in Buffer B and 0.5 ml fractions were collected at flow rate of lml/min. Immunoprecipitations were performed with a rabbit polyclonal antibody for p38oc (Santa Cruz Biotechnology Cat. # SC-535-G), and a mouse monoclonal antibody for CK2(3 (kindly donated by O.-G. Issinger, Biokemisk Institut, Odense, Denmark). Cell extracts (0.5 mg) were incubated with 4 |ig of the appropriate antibody and 20 ul of protein A-Sepharose in the presence of 0.1% SDS for 3 h at 4°C. The beads were washed four times with 20 m M Tris-HCl (pH 7.4) and 150 m M NaCl in Buffer A . 2.4. CK2 Phosphotransferase Activity Assays CK2 phosphotransferase activity following Mono Q column chromatography was assayed when 5 ul of MonoQ fractions were incubated in a final volume of 25 ul with 5 ug of partially phosphorylated casein or the specific CK2 substrate R R A D D S D D D D D and 100 u M [y- 3 2 P]ATP (2.5 uCi/assay in Buffer C (12 m M MOPS (pH 7.2), and 15 m M MgCl 2 ) for 10 or 15 min at 30 °C. These and other CK2 assays were performed in the absence and presence of either 20 ug/ml heparin (Sigma), 20 uM DRB (Sigma) or 10 u M SB203580 (CalBiochem). The phosphorylation of casein was quantitated by spotting 20 ul on to a 1.5 cm 2 piece of Whatman P-81 phosphocellulose paper. The papers were 59 washed extensively in 1% (w/v) phosphoric acid, transferred into 6-ml plastic vials containing 0.5 ml of Ecolume (ICN) scintillation fluid, and the incorporated radioactivity was measured in a Wallac (LKB) scintillation counter. Recombinant CK2 holoenzyme (Calbiochem) (1 pg) was incubated with 250 ng of the wild-type glutathione-S-transferase (GST)-p38oc (Kinetek Pharmaceuticals, Inc.) or wild-type GST-Erkl (Kinetek Pharmaceuticals, Inc.) in a final volume of 25 pi with 10 pCi of 100 p M [y32-P]ATP in Buffer C for 15 min at 30°C. Reactions were terminated by the addition of SDS-PAGE sample buffer (Laemmli, 1970). After SDS-PAGE, radioactivity incorporated into CK2 was detected by autoradiography. 2.5. Immune-Complex Kinase Assays Immunoprecipitations were performed by incubating 500 pg of whole lysate with 4 pg of CK2cx monoclonal (kindly donated by Dr. Olaf Issinger), -cdc2 polyclonal (donated by Kinetek Pharmaceuticals, Inc) or -p38oc (Santa Cruz Biotechnology) polyclonal antibody and 20 pi of protein A-Sepharose (preblocked with 1% BSA) for 3-4 h at 4°C. The beads were washed four times with 20 m M Tris-HCl (pH 7.4), 150 m M NaCl , 1% NP-40 and the aforementioned protease and phosphatase inhibitors. The immunoprecipitated beads were washed one additional time with buffer A (12 m M MOPS (pH 7.2) and 15 m M MgC^) , and incubated with either 5 pg of casein of the specific CK2 substrate RRADDSDDDDD, the p38 M A P kinase substrate ATF2, or with the cdc2 substrate HI-histone (Calbiochem) in a final volume of 25 pi with 5 pCi or 10 pCi of 100 p M [y-3 2P] ATP in Buffer A , for 10 or 15 min at 30°C. Reactions were 60 stopped by either spotting 20 pi onto 1.5 cm 2 piece of Whatman P-81 phosphocellulose paper and the incorporated radioactivity was measured in a Wallec (LKB) scintillation counter, or by the addition of SDS-PAGE sample buffer. After boiling, the samples were subjected to SDS-PAGE for autoradiography with X-ray film or by immunoblotting analysis. 2.6. Immunoblotting Western blotting was carried out with 0.1 to 1 pg/ml of trie rabbit polyclonal antibodies: phospho-p38a (New England Biolabs, Cat. # 921 IS), p38cx, (StressGen Biotechnologies, Cat. # KAP-MA0.09), CK2tx-III (Hagemann et al., 1997), and CK2(3 (Kinetek Pharmaceuticals, Inc.), mouse monoclonal cyclin BI (Pharmingen, Cat. # 14551 A) , p53 (StressGen, Cat. # KAM-CC002), phospho-p53 Ser-392, phospho-cdc2 (New England Biolabs, Cat. # 9281S, 9110S, respectively), mouse monoclonal CK2oc (kindly donated by Dr. Olaf-Georg Issinger), rabbit polyclonal anti-p34 cdc2 (Kinetek Pharmaceuticals, Inc), or p38cx (Santa-Cruz Biotechnology) antibodies, were also used. 2.7. In vitro Binding assay Approximately 2 pg of recombinant CK2 holoenzyme (Calbiochem) were mixed with 400 ng of either GST-p38, GST or GST-Erk l . The recombinant proteins were incubated for 1 h at 4°C in Buffer D [12.5 m M MOPS (pH 7.2), 12.5 m M (3-glycerophosphate, 0.5 m M EGTA, 7.5 m M MgCl2, and 0.5 m M NaF], and the GST was 61 captured by addition of 25 pi of a 1:1 slurry of glutathione-Sepharose 4B beads for another hour at 4°C. In some experiments, 20 p M SB203580, 20 pg/ml heparin or 20 p M DRB was incubated with the GST-p38a or the recombinant CK2 for 15 min at 30°C prior to the addition of the recombinant CK2 or GST-p38, respectively. The beads were washed 3 times with Buffer D containing 0.1% NP-40 and the bound proteins were eluted by addition of SDS-PAGE sample buffer. After boiling, the samples were subjected to SDS-PAGE on 12% acrylamide gels and immunoblotted with either CK2a-I I I or CK2(3 antibodies. 2.8. Oligonucleotide Transfection Cells were grown in medium containing FCS (0.5%) for 24 h prior to transfection, washed twice with PBS and supplemented with fresh medium containing 10% FCS. CK2oc-a' antisense or sense oligonucleotide (2 pg/ml) was transfected into cells using Effectene (Qiagen) according to the manufacturer's protocol. CK2a-a ' antisense and sense were designed as previously described (Ulloa et al., 1993), with a modification in which the entire oligonucleotides were phosphorothioated; antisense (5' to 3'): G T A A T C A T C TTG A T T A C C C C A or sense (5' to 3'): TGG GGT A A T C A A G A T G A T T A C . Cells were incubated with the oligonucleotides for 18-24 h prior to any either treatments. 62 2.9. Flow Cytometry Cells were trypsinized, washed with PBS and fixed with 70% ethanol at the indicated time points then stored at -20°C. The fixed cells were then stained with propidium iodide (50 Ug/ml) with RNase (10 ug/ml). The stained cells were analyzed for D N A content by fluorescence-activated cell sorting (FACS) in a FACScan (Becton Dickinson Instruments). The forward and side scatter gates were set to exclude any dead cells from the analysis; 10,000 events within this gate were acquired per sample. 2.10. Mitotic Index Cells were collected by incubation with trypsin containing EDTA, centrifuged, counted for normalization and then fixed in a solution containing 3.7% formaldehyde, 0.5% NP-40 and Hoescht 33258 (10 |Xg/mL) in PBS. Nuclei were visualized by fluorescent microscopy. Nuclei with condensed, evenly stained chromosomes were scored as mitotic (Bunz, et al., 1998). At least 600 cells were counted for each determination. 2.11. Apoptosis Assay Cells were collected at the indicated time points by incubation with trypsin-E D T A , centrifuged and washed twice with ice-cold PBS, then stained with Annexin-V FITC and propidium iodide (PI) according to the manufacturer's protocol (Pharmingen). 63 Cells were analyzed using the CellQuest software on a Becton Dickinson FACScan and 10,000 events were acquired per sample. 2.12. Immunofluorescence Cells were grown on coverslips (-70% confluent) and fixed at -20°C with 100% methanol overnight. After being blocked in PBS containing 4% bovine serum albumin, cells were doubly stained with mouse monoclonal anti-CK2a and rabbit polyclonal anti-y-tubulin (Sigma) or mouse monoclonal anti-p38a (Transduction Laboratories) and anti-y-tubulin for 3 h. Cells were washed six times with PBS containing 0.1 % Triton X-100 and incubated for 1 h with goat anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC) and goat anti-rabbit IgG conjugated to rhodamine (both at 1:100 dilution, Jackson Laboratory). After six washes in PBS, cells were counterstained with 4', 6-diamidino-2-phenylindole (DAPI) (0.1 (ig/mL in PBS, Molecular Probes). Coverslips were mounted on glass slides with anti-fade (Molecular Probes) and analyzed via fluorescent microscopy. 64 3. STRESS INDUCED ACTIVATION OF PROTEIN KINASE CK2 BY DIRECT INTERACTION WITH p38 MAP KINASE 3.1. Rationale and Hypothesis The mechanisms that regulate CK2 phosphotransferase have been elusive since it has not been shown to be regulated by any of the well-known second messenger molecules. However, its activity can be affected by a number of compounds that may also be involved in p38 M A P kinase activation such as tumor necrosis factor (TNF), IGF-1, and insulin (Pinna and Meggio, 1997; Widmann, 1999). p38 M A P kinase has been also shown to play multiple roles a role in cell growth and division through unknown targets (Nebreda and Porras, 2000). Furthermore, preliminary data from our laboratory showed constitutive elevation of p38a and CK2 expression levels in many primary breast, colon and lung tumors. Using gel-based protein renaturation method for protein kinase activity in crude extracts from the former human solid tumors, the major autophosphorylated protein kinase was. identified as CK2 (B. Salh and S. Pelech, unpublished observations). I therefore tested whether stress-activated p38 M A P kinase might participate in CK2 regulation. 65 3.2. RESULTS 3.2.1. Stress factor-induced activation of CK2 and p38 M A P kinase To explore the role of stress stimuli in the regulation of CK2, we investigated the. effect of various stress stimuli, the protein synthesis inhibitor; anisomycin, the mitochondrial oxidation inhibitor; arsenite and the inflammatory cytokine; TNF-a , that have been shown to induce p38-stress signaling pathway (Hazzalin et al., 1998; Bernstam and Nriagu, 2000; L iu et al., 2000; Ono and Han, 2000; Namgung and Xia , 2001). Serum-deprived HeLa cells were treated with these agents and the casein phosphotransferase activity of CK2 was assessed following MonoQ fast protein liquid chromatography of the cell lysates. Anisomycin treatment caused an 8-fold activation of CK2, which eluted with 0.6 M NaCl in primarily fraction numbers 18 and 19 (Fig. 2A). This was confirmed by the sensitivity of the casein phosphotransferase activity peak to the CK2 inhibitor heparin (Fig. 2A), and the elution of the a, a' and (3 subunits of CK2 in these MonoQ fractions as revealed by immunoblotting analysis (Fig. 3 A , and D). Exposure of the serum-starved cells to the mitochondrial oxidation inhibitor arsenite or TNF-a for 15 min was also observed to stimulate CK2 activity by 3- to 4-fold (Fig. 2B). Furthermore, using a highly specific CK2 substrate peptide (RRADDSDDDDD) , cytosolic extracts from HeLa cells treated with arsenite or anisomysin, respectively, contained 6.33 ± 0.06 and 7.44 ± 0.03 (mean ± S.E., n = 3) fold more phospho-transferase activity than was detectable with cytosol from non-treated cells (data not shown). 66 Under the same conditions that activated CK2, anisomycin, arsenite and TNF-a also produced marked increases in the phosphorylation of the p38 M A P kinase as determined by immunoblotting with an antibody specific for the activating phosphorylation site of p38 isoforms (Fig. 2C). To assess whether p38 M A P kinase mediated the stress factors-induced activation of CK2, we took advantage of the specific p38 inhibitor SB203580. Treatment of the serum-deprived HeLa cells with SB203580 for 30 min prior to their exposure to anisomycin, arsenite or T N F a abrogated the ability of each of these agents to stimulate CK2 (Fig. 2 A and B). 67 A 15000 H Figure 2: Stress factor induced activation of C K 2 and p38 M A P kinase. A , B, HeLa cells were serum deprived (0.5%) for 18 hours and then treated with either anisomycin 10 pg/ml (30 min), arsenite 50 pg/ml (15 min) or TNFoc 20 ng/ml (15 min) resulted in the activation of CK2, which elutes in fractions 18 and 19 following MonoQ chromatography of the cell lysates. These activations could be blocked by pretreatment of the cells with the p38 M A P kinase inhibitor SB203580 (10 pM) or during the CK2 assay by heparin (20 pg/ml). C, Immunoblotting of cell lysates with an antibody specific for the phosphorylated form of p38 M A P kinase indicated that anisomycin, arsenite or T N F a activated p38 in HeLa cells. Results are representative of at least three experiments. 68 3.2.2. Phosphorylated and active p38 M A P kinase coelutes with CK2 from Mono Q As the anisomycin treatment consistently elicited the most striking activation of CK2, we exploited this agent to further explore the mechanism for the CK2 stimulation. There were no differences in the intensity of immunostaining of the a, a' and [3 subunits of CK2 in serum-deprived, anisomycin and SB203580-treated cells (Fig. 3A to F). Consequently, the anisomycin appeared to increase the specific enzyme activity of CK2 without alteration of the a, a'/(3 subunit ratio or the total amount of CK2 protein. There was also no apparent reduction in the mobilities of the CK2 subunits following SDS-polyacrylamide gel electrophoresis of the MonoQ fractionated lysates from cells exposed to anisomycin. Such retardation of CK2 subunits usually reflects their increased phosphorylation. CK2 subunits have been reported to interact with many other proteins, including the protein kinases Mos, Raf A and Rsk, which bind to CK2(3 (Guerra et al., 1999). Therefore, we examined whether a p38 M A P kinase might directly associate with CK2. The peak of immunoreactivity of the cx-isoform of p38 was found to coelute with CK2 following MonoQ fractionation in fractions 18-20, and this was most pronounced in lysates from anisomycin-treated cells (Fig. 3G, and H). The densitometric analysis showed a 1.6 fold increse of p38 protein eluted in fractions 18-20 in anisomycin-trated cells (results not shown). In contrast, the amount of p38cx coeluting with CK2 was substantially reduced in extracts from cells treated with both anisomycin and SB203580, and a large portion of the M A P kinase failed to bind to the MonoQ column and eluted in wash through fractions 4 to 7 (Fig. 31). A n even more dramatic redistribution of p38cx 69 away from CK2 was produced when the lysates from cells exposed to anisomycin were incubated with alkaline phosphatase to dephosphorylate the p38oc prior to MonoQ chromatography (Fig. 3J). CK2 subunits were not detectable by immunoblotting in MonoQ fractions 4 to 7 neither before nor after alkaline phosphatase treatment and it eluted in fractions 18 to 20 without any change in protein levels (results not shown). Furthermore, the alkaline phosphatase treatment abolished the anisomycin-induced activation of CK2 (data not shown). The implication of these experiments was that p38cc M A P kinase must be in an active conformation in order to maximally bind and activate CK2. 70 MonoQ fraction number 16 17 18 19 2 0 21 16 17 18 19 21 24 Figure 3: Phosphorylated and active p38 M A P kinase coelutes with C K 2 from MonoQ. Following MonoQ fractionation, lysates from HeLa cells were immunoblotted with anti-CK2-III (recognizes oc/a' subunits), C K 2 p or p38a antibody. Figure shows that lysates from untreated (A,D, and G) or those exposed to anisomycin in the absence (B,E,H,J) and presence of SB203580 (C,F, and I), there were no detectable changes in the distribution or amount of immunoreactive CK2 a- (A to C) and P- subunits (D to F). p38 eluted principally in the wash through and CK2-containing fractions. Following anisomycin treatment of cells, the amount of p38 associated with CK2 was increased (H) as compared to untreated cells (G) or SB203580-pretreated cells subsequently exposed to anisomycin (I). J, following incubation of lysates from ansiomycin-treated cells with alkaline phosphatase (0.5 units/ug), most p38 eluted in the wash through fractions from MonoQ. 71 3.2.3. Active p38 M A P kinase coimmunoprecipitates with CK2 While the preceding data for CK2 and p38a residing in a common complex was only correlative, direct evidence was obtained through coimmunoprecipitation studies. The p38a antibody was used to immunoprecipitate equivalent amounts of p38oc from lysates of serum-deprived, anisomycin-, and SB203580 + anisomycin-treated cells (Fig. 4). The casein phosphotransferase activity was approximately 4-fold higher in the immunocomplexes from anisomycin-treated as compared to untreated cells, and this was reduced to less than a 2-fold stimulation by the SB203580 (Fig. 4). These immunocomplexes with p38cx antibody were shown to contain both the a and (3 subunits of CK2 by immunoblotting, and their presence was increased in the immunocomplexes from anisomycin-treated cells (Fig. 5B and C). Reciprocally, in immunocomplexes obtained with an antibody specific for CK2p\ p38awas largely immunodetected in samples from anisomycin-treated cells, with little i f any evident in immunocomplexes from serum-starved or SB203580 + anisomycin -treated cells (Fig. 5 D). 72 0 S ta rved S B 2 0 3 5 8 0 An i somyc i n + An i somyc i n Figure 4: Activation of CK2 in anisomycin-stimulated p38a MAP kinase immunoprecipitates. Lysates of anisomycin-treated HeLa cells (10 ug/ml; 30 min.) were immunoprecipitated with a p38oc polyclonal antibody. CK2 kinase assay was determined using casein or RRADDSDDDDD substrate substrate (5 ug). Averages and standard error of the mean of three separate experiments are shown. 73 S ta rved S B 2 0 3 5 8 0 An i s omyc i n I.P. I.B. + An i somyc i n S ta rved S B 2 0 3 5 8 0 An i s omyc i n + An i somyc i n Figure 5: p38a M A P kinase coimmunoprecipitates with C K 2 . Lysates from anisomycin-treated cells were immunoprecipitated with anti-p38a or CK2(3 antibody and then immunoblotted with anti-p38cc, CK2tx or CK2p antibody. Immunoprecipitates (A to C) obtained with a p38a polyclonal antibody from lysates of anisomycin-treated cells featured higher levels of immunoreactive CK2cx and CK2P subunits than from untreated or anisomycin and SB203580-treated cells. D, Immunoprecipitates obtained with the CK2P antibody from lysates of anisomycin-treated cells displayed higher levels of immunoreactive p38oc than from untreated or anisomycin and SB203580-treated cells. 74 3.2.4. p38a M A P kinase expressed as glutathione S-transferase fusion protein binds to and activates recombinant CK2 Further support for the direct interaction of CK2 and p38awas obtained through the use of recombinant CK2 holoenzyme (CK2a/(3) and recombinant p38oc expressed as a glutathione S-transferase (GST) fusion protein. A basal activity of the GST-p38 and CK2 were shown when assayed ATF2 or casein substrate, respectively, in vitro (results not shown). GST-p38 displayed binding for recombinant CK2 protein (Fig. 6C). The interaction of CK2 for GST-p38a was specific, since no binding of CK2 a was detected when either GST or GST fused to the M A P kinase Erkl was substituted for GST-p38 a (Fig. 6C). GST-p38 stimulated the casein phosphotransferase activity of recombinant CK2 by 2-fold, and this was sensitive to SB203580 and the CK2 inhibitors heparin and 5,6-dichloro-ribifuranosylbenzimidazole (DRB) (Fig. 6A). When the CK2/GST-p38cc complex immobilized on glutathione-Sepharose was incubated with [y P]ATP, there was strong phosphorylation of the f3- subunit of CK2 (Fig. 6B), but not the a-subunit of CK2 (data not shown). This phosphorylation was abolished when the incubations with [y- 3 2P]ATP were carried out with SB203580, DRB or heparin. When the presence of C K 2 a was ascertained by immunoblotting, the DRB and heparin did not dissociate the CK2/GST-p38a complex, whereas SB203580 did (Fig. 6C). These findings indicated that the radiolabelling of C K 2 p in the presence of GST-p38 was largely due to autophosphorylation, apparently from the stimulation of CK2 catalytic activity. This was further supported by experiments in which the CK2 was preincubated with unlabeled ATP for 15 min to saturate the autophosphorylation sites, and then with [y- 3 2P]ATP in 75 the absence and presence of GST-p38oc for an additional 10 min. Under these circumstances, there was no enhanced activity of the recombinant CK2 toward casein substrate with the addition of GST-p38a. However, approximately two-fold increases in the casein phosphotransferase activity of CK2 were observed when GST-p38a added in the presence of [y 3 2p] ATP alone (Fig. 7). The latter result indicates that p38 M A P kinase is unlikely to be able to phosphorylate CK2 directly. 76 A CK2 + + + + + GST-p38 + + + + + SB203580 - - + DRB + Heparin + Figure 6 A-C : p38 a MAP kinase expressed as a glutathione S-transferase fusion protein binds to and activates recombinant CK2. A, The GST-p38a-stimulated casein phosphotransferase activity of CK2 is inhibited by SB203580, DRB and heparin in vitro. Two ug of the recombinant CK2 were incubated with 400 ng of GST-p38 in the presence and absence of SB203580 (20 u M for 15 min), DRB (20 u M for 15 min) or heparin (20 Ug/ml for 15 min) for 1 h at 4°C. CK2 kinase activity was measured according to the material and methods using casein as the substrate. Averages and standard error of the mean of three separate experiments are shown. 77 < CK2 + + + - + + GST-p38 + + + + - + GST-Erk1 - - - _ + -SB203580 + DRB + Heparin CK2 + + + + + GST-p38 + + + + -GST-Erk1 GST + SB203580 + DRB + Heparin + | p * -CK2oc Figure 6 B&C: B, The GST-p38 a-stimulated autophosphorylation of CK2P subunit is inhibited by SB203580, DRB and heparin in vitro. Two | lg of recombinant CK2 were mixed with GST-p38, GST, or GST-ERK1 in the presence and absence of SB203580, D R B or heparin and immobilized on glutathione beads as described in material and methods. Five (Xg of the CK2 specific substrate RRDDSDDDDD were incubated with the immunoprecipitated beads for 15 min at 30 °C in the presence of [y-32p] ATP and the reaction was stopped by adding SDS sample buffer. The radioactivity incorporated into CK2 was measured by autoradiography. No detectable C K 2 a autophosphorylation was observed. C, CK2 binds to GST-p38oc, but not the Erkl M A P kinase, and the association is inhibited by SB203580, but not by DRB or heparin. The immunoprecipitates from the above treatments were probed with CK2oc antibody. The right most lane shows the migration position of recombinant CK2oc. 78 2000 Unlabeled ATP Labeled ATP CK2 CK2+ CK2+ GST p38 GST p38 GST p38 Figure 7: p38 M A P kinase does hot phosphorylate C K 2 in vitro. C K 2 was preincubated with unlabeled ATP for 15 min to saturate the autophosphorylation sites, and then with [y- 3 2P]ATP in the absence and presence of GST-p38a for an additional 10 min, lanes 1 and 3. The activity of CK2 was measured using 5 ug of casein as the substrate. Lanes 2 and 4 show the casein phosphotransferase activity of the recombinant CK2 preincubated with GST-p38 or p38 alone, respectively, in the presence of [y-3 2 P]ATP alone. Averages and standard error of the mean of three separate experiments are shown. .79 3.3. Discussion Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we were able to demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-a stimulate the specific enzyme activity of CK2 in the human cervical carcinoma HeLa cells by up to 8-fold, and this was blocked by the p38cx M A P kinase inhibitor, SB203580. Our results clearly demonstrated that p38 phosphorylation is a prerequisite for interaction with CK2 and the pyridinyl imidazole compound SB203580, p38 M A P kinase specific inhibitor, prevented complex formation. Further characterization of the CK2-p38 interaction was carried out by immunoprecipitation and immunoblotting experiments. We observed increase amounts of the C K 2 a protein in stress-stimulated cells which was decreased upon SB203580 treatment. However, in examining the immunoblots with anti-CK2(3-antibody, it was interesting to observe that CK2(3 was more abundant in cells stimulated by various stress stimuli and to a smaller extent in starved cells. Similar results were observed in anti-CK2 (3 immunoprecipitates. Pretreatment with SB203580, samples from anisomycin-treated cells, completely abolished the presence of CK2(3 protein (Fig. 5). These results indicate that p38 M A P kinase may engage in a complex formation with CK2 , perhaps via a protein-protein interaction. This was dependent on the activation and phosphorylation of p38 (Fig. 2-3), since pretreatment with SB203580 and alkaline phosphatase prevented the complex formation. Furthermore, we demonstrated that the p38 M A P kinase was not able to directly phosphorylate CK2 (Fig. 7). Further support for the direct interaction 80 between CK2 and p38 was obtained through the use of recombinant CK2 and p38. Our results clearly showed that p38 had a great affinity for CK2 in vitro that led to increase in C K 2 activation by 2 fold and the p38 M A P kinase specific inhibitor, SB203580, disrupted this interaction (Fig. 6). SB203580 was shown to have no effect on the activity of recombinant CK2 alone in vitro (data not shown). SB203580 has been reported to affect p38 M A P kinase activity by binding within the ATP pocket at the hinge region that is involved in protein-protein interaction (Gum and Young, 1999). Thus, the p38 inhibitor SB203580 attenuates CK2 activation, perhaps by interfering in the protein-protein interaction between CK2 and p38. Our data indicate for the first time that following activation of p38 M A P kinase, CK2 is activated by a protein-protein interaction that leads to autophosphorylation of the CK2p subunit (Fig 6B). CK2Phas been shown to undergo autophosphorylation when it is assembled with and activated by CK2cx (Pinna, 1997; Guerra and Issinger, 1999). Furthermore, CK2(3 is required for formation of the tetrameric CK2 complex, both negative and positive regulation, specificity and stability of CK2cc (Pinna, 1997; Guerra and Issinger, 1999). It is conceivable that the activation of CK2cx by the activated p38 may attract or induce the formation of cx-P dimerization. This scenario is very attractive since the map of a modeled tertiary structure of the region of oc-P link revealed that the CK2P binding site in CK2a is located in the region linking two lobes but on the other side of the activation loop of the kinase domain (Guerra et al., 1998; Niefind et al., 1998)D. Thus it does not hinder the access of substrates to the kinase and is proximal to the ATP binding region. Based on the finding that the dimeric form of the C-terminal P fragment is mainly responsible for cx-P interaction, and it binds more efficiently than its 81 monomeric form, this indicates that either (3 dimerization is a prerequisite for tight binding of a, or each of the catalytic subunit needs to make contact for high affinity association (Guerra et a l , 1998; Guerra et a l , 1999). It is possible that p38 binding with P may promote a conformational change that facilitates subsequent binding of the catalytic subunit of CK2 that is already bound to p38. Collectively, our results support a model in which phosphorylated p38a is able to engage CK2 in a complex through direct protein-protein interaction. It would appear that an active conformation of p38a is needed for binding, since the SB203580 prevented complex formation. Perhaps, through an allosteric effect exerted by active p38oc, CK2 may become stimulated to phosphorylate other targets. It is unclear i f the increased autophosphorylation of CK2 may be sufficient to preserve the state of activation of CK2 after p38a is dissociated. 82 4. ACTIVATION AND L O C A L I Z A T I O N OF PROTEIN KINASE C K 2 IN NOCODAZOLE-INDUCED MITOTIC ARREST 4.1. Rationale and Hypothesis The above results have implicated protein kinase CK2 in mediation of stress signaling downstream of p38 M A P kinase. Takenaka et al. (1998) have shown that the p38 M A P kinase, but not p42/p44 M A P K or JNK was activated in the mitotic arrest by disruption of the spindle with nocodazole. This study indicated that p38 M A P kinase functions as a component of the spindle assembly checkpoint in the somatic cell cycle and contributes in mitotic stress possibly modulating CDK-1 function. In view of the association of p38 with CK2 in the previous chapter, I therefore explored whether CK2 participated in the regulation of spindle assembly. 83 4.2. Results 4.2.1. Depletion or inhibition of CK2 compromises mitotic arrest To investigate whether CK2 is required for mitotic checkpoint function, we employed antisense to the alpha subunit to deplete the protein kinase. We transfected HeLa cells with either C K 2 (a/a' subunits) antisense (AS) or sense (S) oligodeoxyribonucleotide. The expected depletion of CK2 upon AS oligonucleotide treatment was confirmed by assaying the intracellular protein level of CK2 a/a ' subunits by immunoblotting (Fig. 8B). The densitometric analysis showed 80% reduction of CK2 protein from Fig. 8B in As-treated cells (results not shown). Furthermore, we showed negligible activity of CK2 in cells transfected with CK2 antisense (data not shown). Previous studies, using the same antisense oligonucleotide, have demonstrated not only complete depletion of the catalytic CK2 a/a ' subunits (Ulloa et al., 1993), but also inhibition of sorting of the regulatory P subunit, preventing its translocation to the nuclei and dimerization with a subunits (Ulloa et al., 1994). Cells were then synchronized at G0/G1 phase and released into the cell cycle, by the addition of serum-containing medium (10%) as previously described (Takenaka et al., 1998). Nocodazole, a drug that disrupts the mitotic spindle by inhibiting microtubule polymerization, was added before cells entered the mitotic (M) phase. After 9-10 hours, microscopic examination of transfected cells with either S oligonucleotide or buffer alone revealed that many of them became rounded, indicative of cells arrested in mitosis (Fig. 8A). In contrast, cells transfected with AS showed fewer rounded cells. We then measured the percentage of 84 cells in mitosis (mitotic index, MI) after nocodazole treatment. The average MI of cells with either S-treated or buffer was 63% (1008 of 1600 cells), whereas the MI of cells transfected with AS was 11% (176 of 1600) (Fig. 9). These results indicated that CK2 depletion was responsible for a reduction in the mitotic cell fractions induced by nocodazole. Similar results were observed using the CK2 specific inhibitor DRB that was added two hours prior to nocodazole exposure (Fig. 9). 85 A Control CK2a Sense + Nocodazole B Blot: a - CK2a ^ ^ ^ ^ Blot: a - GAPDH 2 § I = * s O CD .2 o a c o | o Figure 8: Depletion of CK2 compromises mitotic arrest. A , HeLa cells, treated with CK2 sense, antisense oligonucleotides or left untreated (control), were synchronized at G0/G1 phase in serum deprived medium (0.5%) for 30 hours. Cells were then released into the cell cycle by addition of serum (10%). After 18 hours, cells exposed to nocodazole (400 ng/ml) for 12 hours before being photographed. B, Depletion of the endogenous CK2oc in HeLa cells transfected with CK2cx-a' antisense. Protein extracts were subjected to protein immunoblot analysis with monoclonal anti-CK2cx antibody. Lower Panel shows equal amount of proteins loaded by immunoblotting with polyclonal a -GAPDH antibody. 86 80 "I Figure 9: Failure of HeLa cells to undergo mitotic arrest in the event of CK2 depletion or inhibition. HeLa cells, treated with CK2 sense, antisense or left untreated, were deprived of serum (0.5%) for 30 hours and released into the cell cycle by addition of serum. After 18 hours, cells exposed to nocodazole (400 ng/ml) for 12 hours. The percentage of cells in mitosis (mitotic index) were measured. Nuclei with condensed, evenly stained chromosomes with Hoescht 33258 were scored as mitotic cells. At least 600 cells were visualized in three independent experiments. Averages and standard error of the mean of three separate experiments are shown. 87 4.2.2. CK2 functions as a component of the spindle assembly checkpoint The spindle assembly checkpoint is the mechanism that prevents the onset of anaphase in cells that have defects in spindle assembly or chromosomal alignment on the spindle (Li and Nicklas, 1995). As I indicated above, it has been demonstrated that inhibition of p38 M A P kinase activity, in response to spindle disruption, compromises the spindle assembly checkpoint (Takenaka et al., 1998). Based upon our earlier observations that p38 M A P kinase and CK2 interact directly (Chapter 3), we therefore tested whether CK2 might also participate in the spindle assembly checkpoint of the somatic cell cycle. Transfected HeLa cells with S, A S , or left untreated were synchronized at G l / S phase boundary by a double thymidine block and released into the cell cycle. Cells were then either treated with nocodazole for 10 hours or released without treatment for 9 hours during which the mitotic cells were collected by mechanical shake off. Microscopic examination (results not shown) and lysates from shake off and nocodazole-treated cells contained kinase activity toward histone HI , a measure of CDK-1 consistent with the released cells from Gl /S block being in M phase (Fig. 10A). A dramatic increase in CDK-1 activity was observed in nocodazole-treated cells, indicative of mitotic arrest (Fig. 10A and B). These activities were substantially suppressed in AS-transfected cells after nocodazole exposure. Thus, the activation of mitotic spindle assembly checkpoint by the microtubule inhibitor, nocodazole, was compromised by depletion of CK2. In agreement with Takenaka et al. (1998), our results indicate that the activation of CDK-1 by nocodazole was also suppressed in cells pre-treated with the p38 M A P kinase specific inhibitor SB203580(Fig. 9A and B). 88 A H1 kinase B O + Figure 10: A , Suppression of nocodazole-induced M P F activity by either C K 2 antisense or a specific inhibitor of p38. HeLa cells were synchronized at G l - S by double thymidine block and either collected (control) or released into cell cycle. After release, the cells were incubated with or without nocodazole (400 ng/ml). In some experiments SB203580 (10 uM) or DRB (20 | l M ) was added two hours prior to nocodazole treatment. Cell lysates were then prepared and histone HI phosphotransferase activities were measured, as equal amount of proteins were loaded and subjected to SDS-PAGE for autoradiography. B, cdc2 activity is downstream to C K 2 and p38 M A P kinase. Protein extracts from the above treatments were immunoprecipitated with anti-cdc2 antibody, phosphotransferase activity was assayed toward histone H I . Average and standard error of the mean of three separate experiments are shown. 89 4.2.3. Activation of CK2 and p38 M A P kinase in nocodazole-arrested cells To determine whether nocodazole-induced activation of p38 and CK2 occurred specifically in M phase cells as a result of spindle assembly defect, or nonspecifically irrespective of the cell cycle, we treated cells with double thymidine block for G l / S synchronization and either collected or released into cell cycle and then subjected to nocodazole (400 ng/ml) for 10 hours. In agreement with previous findings (Takenaka et al., 1998), we also observed that p38 activity in the immune-complex kinase assays (using anti-p38cx M A P kinase antibody) in nocodazole-arrested mitotic cells was three-four times higher than that from the metaphase cells (Fig. 11). Furthermore, we have shown that neither the activation nor the phosphorylation of p38 M A P kinase was affected in cells treated with CK2 antisense and exposed to nocodazole (Fig. 11 and 15). We next examined whether CK2 was activated in nocodazole-treated cells or in normal M phase. Immune-complex kinase assays for CK2 revealed that the activity of CK2 from nocodazole-arrested mitotic cell lysates was four times greater than that from interphase cells (control) (Fig. 12). There was negligible stimulation of CK2 activity compared with the control in mitotic cells (shake-off), and the nocodazole-induced activation of C K 2 was attenuated by inhibition of p38 M A P kinase using SB203580 (Fig. 12). Collectively, with our studies (Chapter 3) and previous studies (Takenaka et al., 1998), these results raised the possibility that p38 function in the spindle assembly checkpoint is mediated by its down stream target, CK2. Thes result led us to propose that CK2 may function downstream of p38 to maintain the activation of CDK-1 in response to spindle damage. 90 Figure 11: Activation of p38 M A P kinase in nocodazole-arrested cells. p38 protein in cell lysates, prepared from the above treatment (Fig. 10), was immunoprecipitated with anti-p38a antibody and assayed for phosphotransferase activity toward p38 M A P kinase substrate, activating transcription factor 2 (ATF2). The lower panel shows that equal amounts of p38 protein were immunoprecipitated. Average and standard error of the mean of three separate experiments are shown. 91 I.P.: a-CK2oc Blot: a - CK2a CO z Figure 12: Activation of C K 2 in nocodazole-arrested cells is dependent on p38 M A P kinase. CK2 protein in cell lysates, prepared from the above treatments (Fig. 10), was immunoprecipitated with a monoclonal anti-CK2cc antibody and assayed for phosphotransferase activity towards the CK2 specific substrate R R A D D S D D D D D . Average and standard error of the mean of three separate experiments are shown. The lower panel shows that equal amounts of CK2 protein were immunoprecipitated. 92 4.2.4. Subcellular immunolocalization of CK2 at different mitotic stages The centrosome is the major microtubule organizing center in mammalian cells. Functions of the centrosomes are prominently featured during mitosis, where they are required for establishment of spindle bipolarity, spindle microtubule assembly and for determination of the cleavage furrow plane, all of which required for balanced segregation of chromosomes (Winey et al., 1996). Recently, McKendrick et al. (1999) reported increases in p53 and CK2 immunofluorescence within the centrosomes, in C57MG cells treated with the topoisomerase I inhibitor, camptothecin, indicating important functions of these molecules in the microtubule organization. We therefore examined the subcellular localization of CK2 throughout the cell cycle in the presence and absence of the microtubule inhibitor, nocodazole. We identified centrosomes with a monoclonal antibody to y-tubulin (anti-y-tubulin) (Oakley et a l , 1990; Joshi, 1994) and the CK2 catalytic subunit by monoclonal antibody to C K 2 a (anti-CK2a). HeLa cells were synchronized at G0/G1 phase as described in material and methods (Takenaka, et al., 1998) and then released into the cell cycle in the absence and presence of nocodazole. Fig. 13 shows the results of triple label staining with anti-y-tubulin (fluorescein staining), anti-CK2oc (rhodamine) and D N A staining (DAPI). Control experiments were performed by using CK2 a or y antibody-depleted mouse serum (data not shown). There was no obvious concentration of CK2 in the interphase states (prophase through prometaphase) (Fig. 13), much of CK2 staining remained perinuclear, and diffusely cytoplasmic. During metaphase and anaphase, some CK2 staining was detectable in the spindle (Fig. 13). However, a remarkable increase in 93 CK2 staining was observed on the spindle body in cells arrested in metaphase by nocodazole treatment. In cells pretreated with SB203580 and then exposed to nocodazole, the staining of CK2 was perinuclear and no detectable staining was observed within the spindle. This result indicates that CK2 may colocalize with the spindle body in response to nocodazole treatment. In addition, it strongly implicates p38 M A P kinase in CK2 translocation to the spindle body in response to spindle damage. 94 CK2cc y-tubulin DAPI prometaphase Metaphase Nocodazole SB203580 + Nocodazole Anaphase Figure 13: Subcellular localization of C K 2 in HeLa cells. Exponentially growing HeLa cells were exposed to nocodazole (200ng/ml) for 18 hours in the presence and absence of SB203580, then were fixed and triple-stained with monoclonal antibody to CK2cx, antibody to centrosome (y-tubulin) and DAPI. Arrows indicate the centrosome localization. Cells observed with a x 100 oil immersion objective lens. 95 4.3. Discuss ion As I discussed in the Introduction, the cell cycle G2/M transition and progression are driven by the activation of CDK-1 (cdc2-cyclin B) complex. This process is a critical event to ensure that chromosomes attach to and become aligned on the mitotic spindle before the inactivation of the C D K - 1 . Thus exit from mitosis and progression to anaphase requires the subsequent inactivation of CDK-1. This process of cell division is highly monitored by a checkpoint termed the spindle assembly checkpoint. Inhibition of spindle assembly with depolymerizing drugs such as nocodazole, activates the spindle assembly checkpoint and prevents the onset of anaphase. This event involves mitotic arrest by elevation of CDK-1 and inhibition of its degradation. Previous work by Takenaka et al. (1998) has shown that p38 M A P kinase is a component of the spindle assembly checkpoint. It has been shown that activation of the spindle assembly checkpoint by disruption of the spindle with nocodazole results in activation of p38 M A P kinase and mitotic arrest (Takenaka et al., 1998). During mitotic arrest by nocodazole, p38 M A P kinase was shown to maintain activation of CDK-1 in response to spindle damage, this is to ensure that cells cannot override the mitotic arrest, induced by the microtubule inhibitor (Takenaka et al., 1998). Here we showed that the function of p38 M A P kinase in the spindle assembly checkpoint activation might due to the activation of its down stream target CK2. Our results have shown that depletion of CK2 by CK2 antisense resulted in failure of the mitotic arrest by nocodazole (Fig. 8 to 9). This is consistent with the results obtained by using dominant negative p38 M A P kinase and p38 specific inhibitor, SB203580 96 (Takenaka et al., 1998). We have shown that the number of mitotic cells induced by nocodazole were significantly reduced by CK2 depletion. To investigate whether CK2 depletion causes the loss of the checkpoint function, we synchronized CK2-depleted HeLa cells at G l / S phase by double thymidine block and then released them into cell cycle. Before mitosis, we treated the cells with nocodazole. Our results show that the failure of the mitotic arrest correlated with inhibition of cdc2 activity (Fig. 10). Similar results were obtained using the CK2 inhibitor DRB (Fig. 10). Our findings revealed that CK2 was activated only in response to nocodazole and was not activated during normal mitosis (Fig. 12). Therefore, this result clearly shows that CK2 is not a component of the spindle assembly but it is a component of the spindle checkpoint. Since CK2 depletion or inhibition compromised the spindle checkpoint activation by nocodazole, we determined whether p38 M A P kinase is part of the same pathway by using the specific inhibitor of p38, SB203580. Similar results were observed. The activation of CDK-1 by nocodazole was suppressed in cells treated with SB203580 (Fig. 10). The activation of CK2, in response to nocodazole, was suppressed by the p38 inhibitor (Fig. 12). However, the activation of p38 M A P kinase was not affected by CK2 antisense (Fig. 11). Therefore we conclude from these results that CK2 functions as a component of the spindle assembly checkpoint in the somatic cell cycle in response to spindle damage, perhaps by sustaining or maintaining the activity of CDK-1 and mitotic arrest. This function is likely mediated by the CK2 upstream target, p38 M A P kinase. Finally, we observed a striking immunofluorescent staining of the C K 2 a within the spindle body in response to nocodazole (Fig. 13). This significant staining of CK2 in nocodazole-treated cells was dramatically reduced and became perinuclear in cells 97 pretreated with SB203580. Interestingly, our results also show that the CK2 staining within the spindle body during interphase and metaphase was negligible (Fig. 13). Our data strongly indicate a translocation of CK2 to the spindle body in response to spindle damage. The translocation of CK2 implicates p38 M A P kinase signaling pathway, indicating an cooperation between these two molecules following spindle damage. 98 5. PROTEIN KINASE CK2 IS REQUIRED FOR p53 FUNCTION IN RESPONSE TO SPINDLE DAMAGE 5.1. Rationale and Hypothesis Protein kinase CK2 phosphorylates the human tumor suppressor p53 at Ser-392 (Ser-386 in murine p53) (Meek et al., 1990; Herrmann et al., 1991). Phosphorylation by CK2 has been shown to enhance p53 sequence-specific D N A binding in vitro (Hupp et al., 1992), which in turn contributes to its transactivation function in vitro (Mundt et al., 1997). In vivo, the phosphorylation of p53 protein at the CK2 site has been reported to regulate p53-specific transcriptional repression (Hall et al., 1996). Recently, the tumor suppressor p53 has been proposed to be a component of the spindle assembly checkpoint, indicating its essential role to ensure maintenance of diploidy and D N A integrity (Cross et al., 1995; Lanni and Jacks, 1998). Our latter results indicated a role for CK2 in response to spindle damage. We further investigated whether CK2 function in spindle assembly checkpoint involves the p53 molecule. 99 5.2. Results 5.2.1. Nocodazole-induced p53 Ser- 392 phosphorylation is mediated by CK2 and is SB203580 sensitive To test the hypothesis that the p53 is a downstream target of the stress stimuli anisomycin, arsenite, and TNFoc, we performed immunoblotting studies of lysates from HeLa cells, that had been treated with these factors, using a p53 Ser-392 phosphorylation site-specific antibody. As shown in Fig. 14 the treatment of serum-deprived HeLa cells with any of these agents enhanced the phosphorylation of p53 at this site. The phosphorylation of p53 in response to anisomycin and TNFoc, but not arsenite, was markedly attenuated by either DRB or SB203580. This result indicates that the phosphorylation of p53 at Ser-392 site in response to anisomycin or TNFoc was mediated by p38 M A P kinase and CK2. In the case of arsenite, we have observed no change in p53 phosphorylation in cells pretreated with the p38 M A P kinase inhibitor, SB203580 (Fig. 14). Thus, we suggest that there may be an alternative pathway, in addition to the p38 M A P kinase-CK2 route, by which p53 can be phosphorylated at the Ser-392 site. Interestingly, it has been shown that arsenite induces p53 accumulation and phosphorylation through an ATM-dependent pathway (Yih and Lee, 2000). We reexamined the above finding in the context of mitotic arrest induced by nocodazole. We synchronized HeLa cells transfected with CK2 sense, antisense oligonucleotides, or buffer at G l / S phase by double thymidine block and then released them into cell cycle in the presence and absence of nocodazole. Figure 15 shows that the 100 hyperphosphorylation of p53 at Ser-392 due to nocodazole, was completely suppressed in cells either transfected with AS or treated with the p38 specific inhibitor-,SB203580. This effect was independent of changes in p53 protein expression (lower panel). The depletion of CK2 did not affect the phosphorylation or the activation of p38 upon nocodazole treatment (Eig. 11 and 15), confirming its role downstream of p38 M A P kinase activation. Our results also show that p53 phosphorylation at Ser-392 in normal mitotic cells was negligible. Thus, these results indicate that CK2 phosphorylates p53 at Ser-392, due to the nocodazole-induced activation of the spindle assembly checkpoint, and p38 M A P kinase is an upstream regulator. The phosphorylation of p53 at Ser-392 in response to the mitotic arrest by nocodazole correlates nicely with the activation of CK2 and p38 M A P kinase in the event of mitotic arrest by nocodazole (Fig. 11 and 12). It should be noted that HeLa cells are known to harbor human papilloma virus (HPV), which has been shown to target p53 for degradation (Scheffner, 1993). However, it has been reported that cervical carcinoma cells, (including HeLa), express functional wild-type p53, which is sufficient to activate the transcription of cellular target genes (Butz et al., 1995). We were able to detect p53 protein in these cells and p21 was induced (data not shown). Fig. 15 shows the level of p53 expression observed in our studies. 101 Anisomysin - + - - - + + -Arsenite - . + - + - - - -TNFoc - - + + . . + SB203580 - . . . . . DRB - . . . + + + . : Figure 14: Stress factor-induced phosphorylation of p53 at Ser-392 is dependent on CK2 and p38 MAP kinase. H e L a cells were serum starved (0.5%) for 18 hours and then treated with anisomycin (10 mg/ml for 30 min), arsenite (50 p M for 15 min), or TNFoc (20 ng/ml for 15 min). Cel l Lysates were immunoblotted with anti- p53 Ser-392. Figure shows enhanced phosphorylation of p53 as detected with a p53 Ser-392 phospho-site antibody. The increased phosphorylation in response to anisomycin and TNFoc could be reduced by pretreatment of the cells with the C K 2 inhibitor D R B (20 p M ) and by the p38 M A P kinase inhibitor SB203580 (10 p M ) . Results are representative o f three experiments. 102 phospho-p53-(serine 392) phospho-p38• p53 co + 0 ) 2 p « 5^ N 00 N C ™ o O X " D CO " D O 5 O CN 8 C N O -5 o mo ^ z Sc. z CO Z C J + Blot: a-p53 Figure 15: CK2 is the major kinase phosphorylating p53 at Ser-392. HeLa cells were synchronized at G l - S by double thymidine block and then released into cell cycle. Mitotic cells were either collected (shake off) after 9 hours or were incubated with or without nocodazole (400 ng/ml) for 10 hours. In some experiments SB203580 (10 uM) or DRB (20 |J,M) was added two hours prior to nocodazole treatment. Cell lysates from the above treatment were immunoblotted with phospho site-specific antibodies to p53 (Ser-392) and p38oc, or polyclonal antibody to p53 protein. 103 5.2.2. Depletion of CK2 causes an override of mitotic arrest following spindle damage Having demonstrated an effect of CK2 depletion on mitotic arrest (Fig. 8 to 9), CDK-1 activity and p53 phosphorylation (Fig. 10 and Fig 15), we wanted to determine the cell cycle characteristics of these cells in more detail. Therefore, exponentially growing HeLa cells were transfected with CK2 AS, S or buffer alone. Cells were then synchronized in G0/G1 phase by serum starvation (0.5%) for 30 hours and then released into the cell cycle by addition of 10% serum containing medium, as previously described (Takenaka et al., 1998). Nocodazole (200 ng/ml) was added before the cells entered mitosis for the indicated times shown. Fig. 16 shows the F A C S analysis data of synchronized HeLa cells, in the presence and absence of nocodazole. Fig. 16 shows that cells, after 6 hours following release into the cell cycle, progressed through S phase and entered G2 and mitosis with similar kinetics (Fig. 16). However, from 12 hours, the behavior of these populations was dramatically different. Sense-treated cells exposed to nocodazole continued to accumulate in mitosis for the next 12 hours with D N A content of 4N (Fig. 16). The time-dependent increase of cdc2 activity (Fig. 17), as well as corresponding cyclin B expression (Fig. 18A), and mitotic index (Fig. 20) by 18 hours, all confirm that cells remained blocked in mitosis upon exposure to nocodazole. Significantly, cells depleted of CK2 appeared to progress into the cell cycle, compromising the mitotic block (Fig. 16), with a large population of cells progressing into a Gl-l ike state with D N A content of 2N populations by 24 hours. The mitotic index (Fig. 20) of the cycling cells indicates that >30% of cells entered mitosis by 24 hours, despite disruption of mitotic assembly. Thus, CK2-depleted cells treated with 104 nocodazole appeared to exhibit an override of the mitotic assembly checkpoint and an apparent reentry into S phase. This was also manifested by an alteration of cyclin B expression (Fig. 18A) and cdc2 inactivation (Fig. 17). To further investigate i f the expression of cyclin B correlated with phosphorylation-dependent inactivation of cdc2, the electrophoretic mobility of cdc2 was used to assess whether it was phosphorylated on Thr-14, Tyr-15 or both. Figure 18B shows the phosphorylation stages of cdc2 upon nocodazole stimulation in the presence and absence of CK2 antisense. The most gel-retarded form of cdc2, species 'a ' , is phosphorylated on both Thr-14 and Tyr-15 whereas the intermediate form, species 'b', is phosphorylated on Thr-14 or Tyr-15, but not both (Atherton-Fessler et al., 1994; L iu et al., 1997). The fastest migrating form of cdc2 (species 'c') is not phosphorylated on either Thr-14 or Tyr-15, and represents either the active form of the kinase (phosphorylated on Thr-161 and bound to cyclin B), or cdc2 that is not bound to cyclin (monomeric, inactive cdc2) (Atherton-Fessler et al., 1994; Liu et al., 1997). Figure 18B demonstrates that the phosphorylation state of cdc2 remains unchanged in AS-transfected cells, whereas cyclin B turnover is altered (Fig. 18A). Collectively, these results indicate that the inhibition of cdc2 activation in CK2-depleted cells upon nocodazole exposure, was not correlated with changes in the phosphorylation of cdc2, but was likely due to the altered cyclin B kinetics. Similar results were also obtained by using the CK2 inhibitor, DRB (data not shown). 105 6h 12h 18h 24h c o o 0 1000 FL2-G 0 1000 FLZ-0 o IOOO FL2-A G1/G0=52| G2/M=30| 0 1000 FL 2- ft 0 1000 FL2-A 0 1000 FL2-A FL2-A 0 1000 FL2-A 0 1000 0 1000 0 1000 0 1000 FL2-A FL2-ft FL2-A FL 2 - ft Figure 16: Mitotic arrest is compromised in the absence of CK2. Synchronized HeLa cells at G0/G1, by serum starvation (0.5%) for 30 hours were treated with CK2 Sense (control) or CK2 Antisense and then released into cell cycle by addition of 10% serum. Cells were collected for flow cytometric analysis in the presence and absence of nocodazole (200 ng/ml) at the indicated time. The percentage of the D N A contents is shown within the figure. 106 Figure 17: Inhibition of cdc2 activity in CK2-depleted HeLa cells. Transfected cells with CK2 Sense and CK2 Antisense oligonucleotides were exposed to nocodazole (200 ng/ml) for the indicated times or left untreated. Control is a representative of cells transfected with CK2 Sense or left untreated. Cell lysates were immunoprecipitated with anti-cdc2 antibody and assayed for cdc2 phosphotransferase activity toward histone HI . Average and standard error of the mean of three separate experiments are shown. 107 o o 2 8 o z o o CM o * o o z u o CM o * O o z o o CM o * O o z Blot: a-cyclin-B — b —c Blot: a-cdc2 Figure 18: Phosphorylation of cdc2 is independent of cyclin B degradation in C K 2 -depleted cells. Transfected cells with CK2 Sense and CK2 Antisense oligonucleotides were synchronized at G0/G1 phase and then exposed to nocodazole (200 ng/ml) or collected at G0/G1 phase. Lysates from above treatment were either subjected to immunoblotting with anti-cyclin B (A) or cdc2 (B) protein, (a), (b) and (c) indicate the different stages of the electrophoretic mobility of cdc2 protein. 108 5.2.3. Mitotic spindle checkpoint compromise requires p53 protein p53 has been shown to be involved in G2/M phase to maintain cell diploidy (Ko, 1996). Several studies have shown that p53"/_ cells accumulate 4N, 8N and 16N populations upon exposure to microtubule inhibitors for >48 hours (Cross et al., 1995; Lanni and Jacks, 1998). From our findings thus far, we were unable to directly implicate p53 in the altered spindle checkpoint arrest observed following CK2 depletion. Therefore, we investigated this hypothesis in the colorectal M I N (microsatellite instability) HCT116 human colon cancer cell line (Bunz et al., 1998), because it has an intact spindle-dependent checkpoint (Bunz et al., 1998; Cahill et al., 1998). As expected, HCT116 cells with either wild type (wt) p53 + / + or with disrupted p53_/" alleles (Bunz et al., 1998) [we have confirmed that there was no detectable p53 protein in p53"A cell line, (data not shown)], were shown to be arrested in M phase upon nocodazole exposure (Fig. 19). However, p53 + / + cells transfected with CK2 AS , in common with HeLa cells similarly treated, also appeared to show a dramatically compromised mitotic spindle checkpoint with 32% of the cells in G0/G1 compared with only 3% for p53"/_ cells, after 24 hours (Flow cytometric statistical analysis of Fig. 19). Furthermore approximately 30%o of the cells had reached mitosis by 24 hours. In striking contrast, p53_/" cells transfected with CK2 AS maintained a high mitotic index comparable with nocodazole treated p53_/" cells (Fig. 20), accumulating 4N with diminished 2N populations (Fig. 19). Thus, it appeared that the normal checkpoint status of MIN cells, HCT116 with wild type p53, following CK2 depletion and spindle damage, is converted to that characteristic of CIN (chromosomal instability) cells (Cahill et al., 1998). 109 We evaluated whether cyclin B expression was affected in p53"'" and p53 + ' + cells. Figure 21 shows that cyclin B was not affected by depletion of CK2 in p53_/", while the p53 + / + exhibited a down-regulation of cyclin B expression in response to antisense CK2 depletion (Fig. 21) in keeping with the HeLa cell data. Another important finding to emerge from investigation of the HCT116 (p53"/_ or p53 + / +) cells was that antisense depletion of CK2 did not influence the ability of the cells to reach the mitotic checkpoint. This is exemplified by the fact that both at 18 hours and at 24 hours, there are equivalent fractions of cells at the G2 /M stage for both nocodazole, and nocodazole + antisense treated p53"A cells (60% and 61% at 18 h, and 67% and 65% at 24 hours, respectively). In addition, the FACS analysis profile of CK2 AS-treated HCT116 p53 + / + cells alone (Fig. 19) shows that these cells exhibit the same kinetics as cells transfected with CK2 sense (Fig. 19) or left untreated (data not shown). These results reduce the possibility that AS treated HeLa cells underwent an S phase delay. Furthermore, our results showed that neither the activity nor the protein levels of CK2 were affected in HCT116 p53-/" cells (results not shown). Therefore, these studies seem to indicate that abrogation of the mitotic assembly checkpoint and cyclin B degradation are dependent on p53 protein, and in addition, are regulated by CK2. 110 Figure 19: p53 is required for CK2 to override the mitotic arrest. Synchronized HCT116 (p53+ / +) and HCT116 (p53_/") cell line at G0/G1, by serum starvation (0.5%) for 30 hours were treated with CK2 Sense (control) or CK2 Antisense and then released into cell cycle by addition of 10%) serum. Cells were collected for flow cytometric analysis in the presence and absence of nocodazole (200 ng/ml) at the indicated time. I l l 6 12 18 24 Time (hours) u - HCT116(p53+/+) HCT116(p53-/-) •a- HeLa CK2 AS (HeLa) » - CK2 AS (HCT116-p53-/-) CK2AS (HCT116-p53+/+) Figure 20: Mitotic indices of HCT116 and HeLa cells. Cells were treated with nocodazole (200 ng/ml) at the indicated times. Nuclei with condensed, evenly stained chromosomes with Hoescht 33258 were scored as mitotic cells. At least 600 cells were visualized in three independent experiments. 112 o Em c o o N co "O o o o Z + o 00 co < "8 CM o * o o z 0 o N CD •g o o o + o 00 co <E CM o * O o z Blot: tx-Cyclin B I8h 24h B Blot: a-Cyclin B Blot: a-GAPDH o N co o o o 2 + o 00 co <E CM u * o o z 0 o N CO •g o o o j2 + o 00 S < E CM o * O o z 18h 24h Figure 21: Cyclin B expression in HCT116 p53 and p53" cells. CK2-depleted (AS-treated) and Sense-treated p53'A (A) and p53 + / + (B) cells were exposed to nocodazole (200 ng/ml) at the indicated times, or left untreated (control). Lysates were immunoblotted with anti-cyclin B . Equal amount of proteins is shown in the lower panel by immunoblotting with ant-GAPDH polyclonal antibody (B). 113 5.2.4. Cells transfected with CK2 antisense escaped nocodazole-induced cell death The tumor suppressor p53 has been shown to mediate apoptosis in several cell types (Moll and Zaika, 2001). Loss of p53 prevents thymocytes from undergoing apoptosis after gamma irradiation, and decreases the efficacy of chemotherapy in vitro, and in vivo (Lowe et al., 1993; Ko and Prives, 1996; Moll and Zaika, 2001). To examine cell death in cells transfected with CK2 AS, we analyzed and quantified the phenotypic changes characteristic of apoptotic cells by double staining HeLa cells with annexin V and propidium iodide (PI). Exponentially growing control or AS-transfected cells were exposed to nocodazole, and then harvested every 6 hours. Figure 22 shows that 22% of control cells had undergone apoptosis after 24 hours of exposure with nocodazole. These observations are consistent with previous studies in certain cell lines, which undergo apoptosis following persistent exposure to nocodazole (Minn et al., 1996; Taylor and McKeon, 1997). In contrast, cells transfected with CK2 A S showed a significant, reproducible 50% reduction in apoptosis. A n even greater reduction in apoptosis (72%) consequent upon CK2 depletion was observed using HCT116 p53 + / + cells (Fig. 23 A). As expected, p53_/" cells exhibited a much lower frequency of apoptosis in response to nocodazole in comparison to p53 + / + cells (10% vs. 32.2%). Furthermore, our results show that there is no change in the percentage of apoptotic cell fractions in HCT116 p53" A cells transfected with CK2 A S and exposed to nocodazole (Fig. 23B). In further support the co-operativity between CK2 and p53, it is apparent from this data that p53-/" cells do not exhibit a reduction of apoptotic cell fraction upon CK2 depletion. 114 Control Nocodazole <U y--TJ O • 6h tfj,. 3 r— •J Annexin-YFITC A: L = D 10 10' NT 10J 10H A . Annexin-YFITC L . 3.3% 10" 10' 10' 10 91.0% Annexin-VFITC : 4.5% 18h g^o. 10' 10' lP"l<rl A : Annexm-YFITC L . D: 3.6% 10u 101 vr 10J 90.9% : 4.0% CK2 Antisense + Nocodazole = 3.1% 10" 10' 10' 10* ;91.8% Annexin-VFITC L = = 3.6% D = 8.0% : 82.5% = 6.9% 10' 10' 10J 10"* A = 7.0% Annexin-VFITC L = 83.6% D = 6.3% 104 A : L = 10' 10' 10J 10 Annexin-VFITC A L D 10u 10' 10' 10" = 3.6% Annexin-VFITC = 90.5% = 4.8% : 8.0% 81.0% : 9.3% 10' 10' 10J 10 Annexin-VFITC 4 A : L = : 6.0% 82.1% : 7.1% 10' 10' 10J 10n Annexin-YFITC : 7.9% 77.9% : 9.0% = 22.0% = 59.9% = 15.1% 10' 10' 10J 1 0 \ Annexin-VFITC A : L = D: : 10.3% 78.3% : 8.6% Figure 22: Suppression of apoptosis in the absence of CK2 in HeLa cells. Quantitative analysis of the apoptotic cells using annexin-V FITC in exponentially growing HeLa cells-transfected with CK2 antisense or Sense-treated (control) in the presence and absence of nocodazole (100 ng/ml) for 24 h. The flow cytometric analysis was performed on 100,000 cells and the percentage of apoptotic (A, the right lower quadrant), live (L, the left lower quadrant) and dead (D, the top right quadrant) cells was measured in three separate experiments. 115 B Control Sense iir r w Annexin-YFITC A = 3.2% L = 91.9% D = 4.0% Nocodazole + Sense iou w1 «r «r ion Annexin-YFITC A = 32.2% L = 53.2% D=15.0% CK2 Antisense + Nocodazole ' 1 " i iou io1 ir ioJ ton Annexin-VFITC A = 8.9% L = 78.9% D = 7.0% ' I " 5 iou io' iir ioJ ton Annexin-YFITC A = 3.5% L = 90.7% D = 4.1% 10u io1 io2 to3 101 Annexin-VFITC A = 10.0% L = 78.9% D = 10.9% r io' IQl ioJ ion Annexirr-V FITC A = 10.3% L = 78.3% D = 11.1% Figure 23: Suppression of apoptosis in the absence of CK2 in HCT116 cells. Quantitative analysis of the apoptotic cells using annexin-V FITC in exponentially growing HCT116 p53 + / + (A) and HCT116 p53"/_ (B), transfected with CK2 Antisense or Sense-treated (control) in the presence and absence of nocodazole (100 ng/ml) for 24 h. The flow cytometric analysis was performed on 100,000 cells and the percentage, of apoptotic (A), live (L) and dead (D) cells was measured in three separate experiments. 116 5.2.5. Abnormal centrosome amplification consequent upon CK2 depletion Loss of regulation of centrosome duplication cycle, or uncoupling of the centrosome duplication cycle from the D N A replication cycle leads to abnormal amplification of centrosomes, which in turn profoundly affects mitotic fidelity. Centrosome defects have been implicated in mitotic abnormalities and tumor progression in human cancer (Carroll et al., 1999; Lingle et al., 1999). Both initiation and centrosome duplication and suppression of reduplication appears to be controlled by p53 gene (Fukasawa et al., 1996). To investigate whether the loss of CK2 has a similar effect on centrosome behavior, we synchronized HeLa cells transfected with CK2 AS or Sense at G0/G1 phase, then released them into the cell cycle. Nocodazole was then added before the entry into M phase and cells were harvested and fixed every one hour. Cells were stained with anti-y tubulin for centrosome visualization and DAPI for nuclei. Figure 24 shows that CK2-depleted cells had initiated anaphase despite spindle damage, dividing into two daughter cells with abnormal positioning of the two centrosomes (A). Some cells had formed multiple copies of centrosomes (B) which indicated that they had undergone reduplication. This result shows that the acquisition of an abnormal number of centrosomes was due to direct consequence of CK2 depletion. Surprisingly, we also found that these cells had undergone cytokinesis despite the abnormal centrosome amplification, producing cells with two copies or more of centrosomes (Fig. 24C). Figure 24D shows that the Sense-transfected HeLa cells, exposed to nocodazole, arrest in mitosis. Fig. 24E-F shows normal mitotic spindle assembly and anaphase in CK2-depleted cells, in the absence of nocodazole. These cells 117 show normal spindle with a typical bipolar array of anti-parallel microtubules organized by two centrosomes at the poles (mitosis) (E), and anaphase with typical alignment of centrosomes in the two daughter cells (F). Thus, these observations may implicate CK2 in the regulation of centrosome duplication. 118 y-tubulin DAPI A B C D E F Figure 24: Abnormal amplification of centrosomes following AS- mediated C K 2 depletion. Synchronized HeLa cells transfected with CK2 antisense and sense oligonucleotides were immunostained with anti-y tubulin, DAPI and CK2cx antibodies. A to C, CK2-depleted cells were exposed to nocodazole (200ng/ml),undergoes anaphase (Ato B) and Cytokinesis (C). Arrows indicate the centrosome localization. D, nocodazole-arrested CK2 sense-transfected cells; E , CK2-depleted cells in early anaphase and F, control cells in late anaphase (in the absence of nocodazole). Cells observed with a 100 x oil immersion objective lens. 119 5.3. Discussion In this study we have demonstrated for the first time, a significant functional co-operation between protein kinase C K 2 and p53, in modulation of cdc2 phosphotransferase activity and the mitotic checkpoint arrest following nocodazole-induced spindle damage. This effect was influenced by the p38 M A P kinase, as both the biochemical events and the cell-cycle changes could be reversed using SB203580. Nocodazole-induced CK2 activation correlated with increased levels of cyclin B expression and cell cycle arrest, whereas the compromised checkpoint was associated with a reduction in levels of cyclin B and consequently a reduction in cdc2 activation. The latter event was shown to be dependent upon p53. The reduction in apoptosis exhibited by CK2 depleted cells, accompanied by the significantly reduced mitotic index provides the strongest support for the notion that the mitotic checkpoint arrest was breached. The identical cell cycle distribution pattern observed for both nocodazole-treated and AS CK2-depleted p53_/" cells (Figure 23) provides the most compelling evidence against an alternative cell cycle modulating role for depletion of CK2 in mediating the above responses. The C-terminal domain of p53 is a target for CK2 phosphorylation. CK2 and p53 associate in vivo via binding of the regulatory (3 subunit of CK2 to the C-terminal domain (Appel et al., 1995; Gotz et al., 1999). The effect of this phosphorylation upon the growth suppressing and transactivating activities of p53 is controversial (Hupp et al., 1992; Rolley and Milner, 1994; Mundt et al., 1997). We have shown that protein kinase CK2 is required for p53 to maintain genomic stability in response to spindle damage. We 120 have found that p53's function during M phase in response to spindle damage, which has previously been demonstrated to prohibit re-duplication of D N A (Notterman et al., 1998) and centrosomes (Fukasawa et al., 1996) without completion of mitosis, was dependent on the direct effect of CK2 on p53. This effect correlated with phosphorylation of the p53 protein at Ser- 392 (Fig. 15). CK2 was found to function as a component of the mitotic spindle checkpoint as demonstrated in Figures 10, 17 and 18. This was demonstrated by showing that the sustained activation of p34 c d c 2 /cyclin B , due to activation of the spindle assembly checkpoint by nocodazole was completely suppressed by either depletion of CK2 catalytic subunits or inhibition of its activity using antisense or DRB, respectively. We have also shown that depletion or inhibition of CK2 did not affect the inhibitory phosphorylation of cdc2 at Thr-14 or Tyr-15, nor the activity of cdc2 by phosphorylating Thr-161 (Fig. 17-18), but did affect the expression of the regulatory cyclin B (Fig. 18). The question remained as to whether the override of the mitotic arrest, observed in the absence of CK2, might be due to p53-mediated abrogated expression of cyclin B. It has been shown that HeLa cells with a mitotic inhibitor such as nocodazole for 12 to 48 h results in elevated cyclin B levels (Kung et al., 1990; Pines, 1999). These data led to the conclusion that prolonged inhibition of the segregation of chromosomes in human cells is associated with sustained cyclin B levels and mitotic arrest. Hixon et al. (1998) have reported that cells expressing p53 mutants show an unscheduled degradation of cyclin B protein in response to mitotic spindle-depolymerizing drugs. It has been also shown that activation or overexpression of p53 can directly regulate cyclin B expression by lowering the intracellular levels of cyclin B mRNA (Innocente et al., 1999; Taylor et 121 al., 1999). These data indicated that inactivation of cdc2 kinase through cyclin B repression is an essential step in p53-mediated mitotic arrest. Therefore, we determined the expression of cyclin B protein. Our results showed that the cyclin B expression level (Fig. 21 A) was not affected in the HCT116 p53"/_ cell line, in the absence or inhibition of CK2, during persistent activation of the mitotic spindle checkpoint by microtubule-destabilizing drugs (in contrast to p53 + / + cells Fig. 2IB). Furthermore, our cell cycle analysis data showed that cells lacking functional p53 underwent mitotic delay for up to 24 hours (Fig. 19). Although it has been shown that p53 does not affect the initiation, duration or magnitude of the mitotic delay following disruption of the mitotic spindle, it is an essential element of the mitotic checkpoint which prohibits re-duplication of D N A (Notterman et al., 1998). Previous results have shown that mutation of p53 protein at the C K 2 phosphorylation site (Ser-392) inhibited the ability of p53 to cause growth repression but had no effect on its transactivation activity (Hall et al., 1996b). p53 transrepression has been suggested to be required for induction of p53-dependent apoptosis (Sionov and Haupt, 1999). Via regulation through the carboxyl terminus of p53, it has been suggested that p53 can exist in two conformations with different properties, one configuration might be inhibited for D N A binding but might remain active for other activities (Ko and Prives, 1996). We thus propose that the depletion of CK2, which causes suppression of p53 protein phosphorylation at Ser-392, may confer upon p53 a gain of oncogenic properties phenotype that results in increased cell survival and post-transcriptional degradation of 122 cyclin B , compromising the mitotic checkpoint arrest that leads to genomic instability. In other words, p53 may lose the ability to maintain mitotic integrity, in the absence or dysfunction of CK2, by a loss of its ability to appropriately regulate cyclin B levels, as well as an inability to excute apoptosis. This was clearly demonstrated not only in HeLa cells but also in the colorectal M I N cancer cell line, which has an intact spindle checkpoint. Chromosome instability reflects the occurrence of defective mitosis, including unequal distribution of chromosomes to daughter cells and failure to undergo cytokinesis, which leads to generation of aneuploid cells. Both in vivo and in vitro, chromosome instability has been shown to correlate with loss or mutation of p53 protein (Fukasawa et al., 1996; Fukasawa, 1997). It has been shown that loss of p53 protein in mouse embryonic fibroblasts results in the acquisition of multiple copies of centrosomes upon nocodazole treatment (Fukasawa et al., 1996; Tarapore and Fukasawa, 2000). These cells have shown to have abnormally amplified multiple copies of centrosomes that co-migrate to the poles, establishing pseudobipolarity (Fukasawa et al., 1996; Tarapore and Fukasawa, 2000). Cells with such pseudobipolar spindles have been shown to undergo normal chromosome segregation and cytokinesis (Tarapore and Fukasawa, 2000). Similarly, our results showed that the loss of CK2 in HeLa cells, exposed to nocodazole, resulted in an abnormal number of centrosomes that failed to position along the bipolar axis (Fig. 24). These cells were also shown to undergo cytokinesis, despite leaving mitosis with a disrupted spindle. Although this result is yet to be verified by genetic manipulation that is ongoing in our laboratory, our results indicate that CK2 may be 123 involved in a p53-dependent regulation of centrosome duplication in response to spindle damage. 124 6. CONCLUSION In this thesis I have shown that protein kinase CK2 may be regulated by the stress-stimulated p38 M A P kinase signaling pathway. This is the first placement of CK2 within a specific signaling pathway. I have showed that upon phosphorylation and activation of p38a by different stress stimuli, it is able to engage CK2 in a complex through direct interaction with CK2 enzyme. Through an allosteric effect exerted by active p38oc, CK2 becomes stimulated to phosphorylate other targets such as the tumor suppressor p53 protein. We have shown that stress-activated CK2 mediated by the direct protein-protein interaction with p38 M A P kinase regulates the phosphorylation of the C-terminus of p53 protein at Ser-392. The implication of this phosphorylation is that it may have a potential function in coordination of cellular processes in response to genotoxic stresses such as protein synthesis, mitochondrial function and cell cycle progression. Protein kinase CK2 may have an essential role in maintaining the integrity of cellular function by collaborating with tumor suppressors such as p53. In this report, we have shown that phosphorylation of p53 at Ser-392 was correlated with the regulation of p53 function in response to spindle damage. Gur results have demonstrated that the p38-CK2 complex function in concert with p53 to ensure mitotic fidelity. Abrogation of CK2 activity in response to mitotic crisis may confer upon p53 oncogenic properties manifested in cell survival and chromosomal instability. Therefore we hypothesize that under cellular stress, CK2 functions as a tumor suppressor in concert with p53. Thus I propose a signaling pathway that regulates p53 in the event of mitotic crisis such as spindle damage. Upon microtubule disruption, we have shown that p38 125 M A P kinase is activated and associates with CK2, regulating CK2's activity and its translocation to the spindle body or centrosomes. This, in turn, exerted a direct effect on the binding and phosphorylation of p53, modifying p53's function to ensure maintenance of genomic fidelity (Fig. 25). Protein kinase CK2 activity is elevated in a multitude of human cancer cells including leukemias and solid tumors (Pinna and Meggio, 1997; X u et al., 1999). Seldin and Leder (1995) have demonstrated that CK2 upregulation was critical to the pathogenesis human cancer by overexpressing the catalytic a subunit in transgenic mice. C K 2 a transgenic mice develop lymphoma beginning at about six months of age with an incidence of 15-20% per year (Seldin and Leder, 1995; X u et al., 1999). Similarly, overexpression of C K 2 a in the mammary gland of transgenic mice causes hyperplasia and dysplasia of the female mammary gland (Landsman-Bollag et al., 2001). In addition Landesman-Bollag et al. (1998) have demonstrated that overexpression of CK2cx contributed to the loss of p53 alleles. Thus, it has been proposed that CK2 might be a 'proto-oncogene', a gene whose dysregulated or ectopic expression can cause cancer (Seldin and Leder, 1995; Guerra et al., 1999; Xin et al, 1999). In contrast to the above reports, we hypothesize that protein kinase CK2 may function as a tumor suppressor in cooperation with p53 protein. However, in the event of misexpression of CK2cx or CK2J3 subunit, which is common in most tumors, CK2 may have a negative effect on p53 function that alter its transrepression activity and gain of oncogenic properties. This was mainfested by genomic instability (defective checkpoints and abnormal centrosome amplification) that perhaps contributes to the initiation and progression of tumorigenicity. Finally, this study underscores the potential functional 126 importance of the association of these two highly altered molecules (CK2 and p53) human cancer, which clearly merits further investigation. 127 Spindle damage 4 • #p38 MAPK Serine 392 p53 phosphorylation I I ? Mitotic arrest Apoptosis Antisense DRB (gain of function) Mitotic Checkpoint Override / Enhanced cell survival ADAPTATION \ ? Error Correction Cytokinesis Multiple eccentric centrosomes Figure 25: Schematic of events following nocodazole-induced spindle damage. Following activation of p38 M A P K there is a translocation and activation of CK2 to the centrosomes and damaged spindle. This is associated with serine 392 p53 phosphorylation which can be attenuated by pharmacological (DRB) means and antisense depletion of CK2a. Although mitotic arrest is independent of p53, subsequent override is dependent upon p53-mediated cyclin B degradation that requires CK2. Black solid arrows (direct), broken arrows (indirect), indicate events following spindle damage, grey arrows indicate those events following depletion/inhibition of CK2. 128 7. FUTURE STUDIES I propose that the holoenzyme CK2 functions as a tumor suppressor. However, it may also fulfill the definition of an oncogene i f is mutated, dysregulated or mis-expressed. Based on my findings I recommend experiments to identify the critical residues mediating the protein-protein interaction between p38 M A P kinase and protein kinase CK2. This work can be carried out by mapping the interacting binding residues between C K 2 and p38 M A P kinase. We have shown that the p38 M A P kinase inhibitor SB203580 interferes with the CK2-p38 complex formation, and it was previously known to disrupt the protein-protein interaction between p38 M A P kinase and its substrates at the A T P binding pocket of p38. Thus, by using site specific mutagenesis, it should be possible to construct a series of deletion mutants as well as defined mutations in the ATP binding pocket of p38 M A P kinase. I also recommend experiments to investigate the involvement of CK2 in p53-dependent apoptotic response to genotoxic stress (microtubule inhibitors, U V and y-radiation). This could include assessment of p53-induced genes mediating apoptosis such as Bax. Miyashita and Reed (1995) have shown that p53 is a direct transcriptional activator of the human Bax gene. This work would depend upon the creation of corresponding mutations in protein kinase CK2. The functional significance of this will depend upon assays such as p53 phosphorylation and transcription of p53 dependent genes (this can be done using luciferase-based promoter activation of genes such as Bax). 129 Several studies from different laboratories including ours indicate that the CK2 catalytic subunit a is disproportionately overexpressed compared with the regulatory (3 subunit in human cancer. I strongly believe that the aberrant expression of the stable holoenzyme CK2 (or the catalytic component) may play an integral role in human cancer particularly in colon cancer. Therefore, I suggest further investigation whether CK2 subunits are mutated, truncated or genetically mis-expressed in colon cancer. Experiments could be performed by generating transgenic mice that carry the wild-type CK2 subunits, using an intestine-specific promoter. This could determine whether it possesses oncogenic function within this system. 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