Cell Cycle-regulated Expression, Phosphorylation, and Degradation of p55Cdc
1997; Elsevier BV; Volume: 272; Issue: 45 Linguagem: Inglês
10.1074/jbc.272.45.28501
ISSN1083-351X
Autores Tópico(s)Cancer-related Molecular Pathways
Resumop55Cdc is a mammalian protein that shows high homology to the cell cycle proteins Cdc20p of Saccharomyces cerevisiae and the product of the Drosophila fizzy(fzy) gene, both of which contain WD repeats and are thought to be required for the metaphase-anaphase transition. Thefzy mutants exhibit a metaphase arrest phenotype, which is accompanied by stabilization of cyclins A and B, leading to the hypothesis that fzy function is required for cell cycle-regulated ubiquitin-mediated proteolysis. p55Cdc expression was initiated at the G1/S transition and steady state levels of p55Cdc were highest at M and lowest in G1. Inhibition of the 26 S proteasome prevented both mitotic exit and loss of p55Cdc at the M/G1 transition, suggesting that p55Cdc degradation was mediated by the cell cycle-regulated proteolytic pathway. Immune complexes of p55Cdc obtained at different cell cycle stages showed a variety of proteins with dramatic differences observed in the pattern of associated proteins during the transition from G2 to M. Immunolocalization of p55Cdc demonstrated dynamic changes in p55Cdc localization as the cells transit mitosis. p55Cdc appears to act as a regulatory protein interacting with several other proteins, perhaps via its seven WD repeats, at multiple points in the cell cycle. p55Cdc is a mammalian protein that shows high homology to the cell cycle proteins Cdc20p of Saccharomyces cerevisiae and the product of the Drosophila fizzy(fzy) gene, both of which contain WD repeats and are thought to be required for the metaphase-anaphase transition. Thefzy mutants exhibit a metaphase arrest phenotype, which is accompanied by stabilization of cyclins A and B, leading to the hypothesis that fzy function is required for cell cycle-regulated ubiquitin-mediated proteolysis. p55Cdc expression was initiated at the G1/S transition and steady state levels of p55Cdc were highest at M and lowest in G1. Inhibition of the 26 S proteasome prevented both mitotic exit and loss of p55Cdc at the M/G1 transition, suggesting that p55Cdc degradation was mediated by the cell cycle-regulated proteolytic pathway. Immune complexes of p55Cdc obtained at different cell cycle stages showed a variety of proteins with dramatic differences observed in the pattern of associated proteins during the transition from G2 to M. Immunolocalization of p55Cdc demonstrated dynamic changes in p55Cdc localization as the cells transit mitosis. p55Cdc appears to act as a regulatory protein interacting with several other proteins, perhaps via its seven WD repeats, at multiple points in the cell cycle. In eukaryotic cells, different complexes of kinases and their associated activating or inhibitory proteins control progression through discrete steps of the cell cycle. The best understood complexes known to play a central role in cell cycle progression are the cyclins in association with their cyclin-dependent kinases (Cdks) 1The abbreviations used are: Cdk, cyclin-dependent kinase; CENP, centromere protein; LLnL,N-acetylleucylnorleucinal; E64d, (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methyl-butane ethyl ester; DCB, dihydrocytochalasin B; S100, 100,000 ×g supernatant; P100, 100,000 × g pellet; Plk, Polo-like kinase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TD, telophase disc; FITC, fluorescein isothiocyanate. (reviewed in Refs.1Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2955) Google Scholar, 2Sherr C.J. Cell. 1994; 79: 551-555Abstract Full Text PDF PubMed Scopus (2603) Google Scholar, 3Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3227) Google Scholar). In addition to regulation by phosphorylation and association with Cdk inhibitors, the cyclin-Cdk complexes are subject to a more irrevocable form of regulation, i.e. the degradation of cyclin via the cell cycle-regulated ubiquitin-mediated proteolytic pathway (4Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-137Crossref PubMed Scopus (1914) Google Scholar, 5Hershko A. Ganoth D. Pehrson J. Palazzo R.E. Cohen L.H. J. Biol. Chem. 1991; 266: 16376-16379Abstract Full Text PDF PubMed Google Scholar, 6Holloway S.L. Glotzer M. King R.W. Murray A.W. Cell. 1993; 73: 1393-1402Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 7King R.W. Deshaies R.J. Peters J.M. Kirschner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1122) Google Scholar). The work of a number of laboratories has recently converged to identify three tetratricopeptide proteins, CDC16, CDC23, and CDC27, as components of the E3 complex that becomes activated during mitosis (8Sudakin V. Ganoth D. Dahan A. Heller H. Hershko J. Luca F.C. Ruderman J.V. Hershko A. Mol. Biol. Cell. 1995; 6: 185-198Crossref PubMed Scopus (649) Google Scholar) and catalyzes the mitosis specific conjugation of ubiquitin to B type cyclins in yeast (9Irniger S. Piatti S. Michaelis C. Nasmyth K. Cell. 1995; 81: 269-278Abstract Full Text PDF PubMed Scopus (473) Google Scholar) Xenopus (10King R.W. Peters J.M. Tugendreich S. Rolfe M. Hieter P. Kirschner M.W. Cell. 1995; 81: 279-288Abstract Full Text PDF PubMed Scopus (832) Google Scholar), and humans (11Tugendreich S. Tomkiel J. Earnshaw W. Hieter P. Cell. 1995; 81: 261-268Abstract Full Text PDF PubMed Scopus (317) Google Scholar). These three proteins can form a large complex whose function is required for the metaphase to anaphase transition (12Lamb J.R. Michaud W.A. Sikorski R.S. Hieter P.A. EMBO J. 1994; 13: 4321-4328Crossref PubMed Scopus (214) Google Scholar). Many of the genes encoding tetratricopeptide proteins have been reported to functionally interact with members of the WD repeat family (13Goebl M. Yanagida M. Trends Biochem. Sci. 1991; 16: 173-177Abstract Full Text PDF PubMed Scopus (378) Google Scholar, 14Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1310) Google Scholar). Recent investigations have demonstrated that other cell cycle-regulated proteins undergo proteolysis at defined cell cycle stages, and that proteins other than cyclins must be degraded to allow the metaphase to anaphase transition (6Holloway S.L. Glotzer M. King R.W. Murray A.W. Cell. 1993; 73: 1393-1402Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 7King R.W. Deshaies R.J. Peters J.M. Kirschner M.W. Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1122) Google Scholar). Centromereprotein (CENP)-E and CENP-F are mammalian kinetochore localized proteins whose expression peaks at mitosis, exhibit dynamic changes in their localization at different mitotic stages, and are rapidly degraded after mitosis (15Brown K.D. Coulson R.M.R. Yen T.J. Cleveland D.W. J. Cell Biol. 1994; 125: 1303-1312Crossref PubMed Scopus (119) Google Scholar, 16Liao H. Winkfien R.J. Mack G. Rattner J.B. Yen T.J. J. Cell Biol. 1995; 130: 507-518Crossref PubMed Scopus (303) Google Scholar). In Aspergillus nidulans mitotic exit apparently requires the destruction of the cell cycle-regulated NIMA protein kinase (17Pu R.T. Osmani S.A. EMBO J. 1995; 14: 995-1003Crossref PubMed Scopus (74) Google Scholar), and inDrosophila the separation of sister chromatids has been reported to require the product of the pimples gene, a protein that is degraded after the metaphase-anaphase transition (18Stratmann R. Lehner C.F. Cell. 1996; 84: 25-35Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Most of these proteins do not have the cyclin destruction box motif, which targets the A and B type cyclins for destruction (4Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-137Crossref PubMed Scopus (1914) Google Scholar). We have (19Weinstein J. Jacobsen F.W. Hsu-Chen J. Wu T. Baum L.G. Mol. Cell. Biol. 1994; 14: 3350-3363Crossref PubMed Scopus (111) Google Scholar) identified a protein, p55Cdc, which appears to be a mammalian counterpart of products of the Saccharomyces cerevisiae CDC20 (20Sethi N. Monteagudo M.C. Koshland D. Hogan E. Burke D.J. Mol. Cell. Biol. 1991; 11: 5592-5602Crossref PubMed Google Scholar), Drosophila fzy (21Dawson I.A. Roth S. Artavanis-Tsakonas S. J. Cell Biol. 1995; 129: 725-737Crossref PubMed Scopus (165) Google Scholar), and the newly described fission yeast slp1 (22Matsumoto T. Mol. Cell. Biol. 1997; 17: 742-750Crossref PubMed Scopus (75) Google Scholar) genes, due to the strong homologies within their WD repeats (14Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1310) Google Scholar). cdc20 mutants arrest in mitosis at the nonpermissive temperature after the formation of a complete short spindle and nuclear migration to the neck between the mother cell and a large bud (23Byers B. Goetsch L. Cold Spring Harbor Symp. Quant. Biol. 1974; 38: 123-131Crossref PubMed Scopus (228) Google Scholar). It has been suggested that the cdc20p is required for modulation of microtubule structure, either by altering the surface of microtubules or by promoting disassembly (20Sethi N. Monteagudo M.C. Koshland D. Hogan E. Burke D.J. Mol. Cell. Biol. 1991; 11: 5592-5602Crossref PubMed Google Scholar,24O'Toole E.T. Mastronarde D.N. Giddings T.M. Winey M. Burke D.J. McIntosh J.R. Mol. Biol. Cell. 1997; 8: 1-11Crossref PubMed Scopus (46) Google Scholar). The fzy mutants have demonstrated that the failure to degrade cyclins A and B, and the failure of sister chromatids to separate, is due to a lack of functional Fizzy (Fzy) protein (21Dawson I.A. Roth S. Artavanis-Tsakonas S. J. Cell Biol. 1995; 129: 725-737Crossref PubMed Scopus (165) Google Scholar, 25Sigrist S. Jacobs H. Stratmann R. Lehner C.F. EMBO J. 1995; 14: 4827-4838Crossref PubMed Scopus (257) Google Scholar). Dawson et al. (21Dawson I.A. Roth S. Artavanis-Tsakonas S. J. Cell Biol. 1995; 129: 725-737Crossref PubMed Scopus (165) Google Scholar) have postulated that Fzy function is required for cell cycle-regulated ubiquitin-mediated proteolysis.slp1 mutants are defective in chromosome separation and recovery from DNA damage arrest (22Matsumoto T. Mol. Cell. Biol. 1997; 17: 742-750Crossref PubMed Scopus (75) Google Scholar). The high degree of homology between p55Cdc, Fzy, Cdc20p, and slp1 protein, as well as their essential role in cell cycle, has prompted the proposal that they are orthologous members of a gene family within the highly degenerate WD repeat superfamily (21Dawson I.A. Roth S. Artavanis-Tsakonas S. J. Cell Biol. 1995; 129: 725-737Crossref PubMed Scopus (165) Google Scholar, 22Matsumoto T. Mol. Cell. Biol. 1997; 17: 742-750Crossref PubMed Scopus (75) Google Scholar). Mammalian p55Cdc exhibits several properties that are consistent with its role in the cell cycle. p55Cdc, not expressed in differentiated or quiescent cells, is readily detectable in dividing cells. p55Cdc appears to be essential for cell division, since transfection of antisense p55Cdc cDNA into Chinese hamster ovary cells results in isolation of only those cells that exhibit a compensatory increase in p55Cdc transcripts in the sense orientation (19Weinstein J. Jacobsen F.W. Hsu-Chen J. Wu T. Baum L.G. Mol. Cell. Biol. 1994; 14: 3350-3363Crossref PubMed Scopus (111) Google Scholar). Immune complexes of p55Cdc exhibited a protein kinase activity that was higher in actively proliferating cells than in quiescent cells and fluctuated with the cell cycle. Overexpression of p55Cdc in myeloid cells inhibited granulocytic differentiation and accelerated apoptosis, suggesting that p55Cdc regulation is critical for normal cell cycle control during myeloid cell proliferation and differentiation (26Kao C.T. Lin M. O'Shea-Greenfield A. Weinstein J. Sakamoto K.M. Oncogene. 1996; 13: 1221-1229PubMed Google Scholar). This study was undertaken to examine the expression, phosphorylation, degradation, and localization of p55Cdc at different stages of the mammalian cell cycle. The mitotic Cdk, p34 cdc2, was analyzed in parallel and served as an additional control in the course of this work. Pulse labeling experiments with 35S or32P showed that both the biosynthesis and phosphorylation of p55Cdc were low at G1 and peaked at G2, with a significant drop in phosphorylation during the G2 to M transition. Immunecomplexes, obtained from different cell cycle stages and subcellular fractions, showed distinct p55Cdc-associated proteins. A steady accumulation of p55Cdc during the course of the cell cycle culminated in rapid loss at the M to G1 transition. This loss could only be prevented by a 26 S proteasome inhibitor, suggesting that p55Cdc may be another cell cycle protein to be degraded by the cell cycle-regulated ubiquitin-mediated proteolytic pathway. Localization of p55Cdc by indirect immunofluorescence displayed dynamic changes during mitosis, from the centromeres at prometaphase, mitotic spindle in metaphase, to the spindle equator in anaphase. These observations indicate a role for p55Cdc in G2 and/or M. HeLa and IMR-90 cells were grown in α-minimum essential medium or minimum essential medium, respectively, supplemented with 10% fetal bovine serum, glutamine, and nonessential amino acids (Life Technologies, Inc.). HeLa cells were synchronized at the beginning of S (G1/S) by the double thymidine/aphidicolin block (27Heintz N. Sive H.L. Roeder R.G. Mol. Cell. Biol. 1983; 3: 539-550Crossref PubMed Scopus (314) Google Scholar). S phase cells were harvested 4 h, G2 cells were harvested 8 h, and mitotic cells were obtained by shake off 10–11 h following release from the aphidicolin block. The remaining adherent monolayer was the late G2population. To obtain a G1 population, the nonadherent pseudo-mitotic (G2/M) cells collected following 12–14 h of nocodazole treatment (0.1 μg/ml) were washed and replated in media without nocodazole. Approximately 60% of these cells had completed mitosis 2 h later, and the daughter cells had adhered to the plates by 3 h. At this stage, the nonadherent cells were rinsed off. The cells were used for G1 analysis 4–8 h following replating. To obtain cells at the M/G1 transition, pseudo-mitotic cells were harvested 7 h after nocodazole treatment. The washed cells were replated in media without drugs (carrier dimethyl sulfoxide, Me2SO), or with media containing the following additions: 50 μm N-acetylleucylnorleucinal (LLnL), 50 μg/ml (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methyl-butane ethyl ester (E64d), 0.1 μg/ml staurosporine, or 5 μg/ml dihydrocytochalasin B (DCB), (Sigma Chemical Co). Following a 2.5-h incubation, the cells were analyzed for DNA content by flow cytometer (DNA QC Particles kit, Becton Dickinson), and for p55Cdc, cyclin B, and p34 cdc2 by Western analysis. An identical analysis was performed on cells that had been treated for 15 h with 5 μg/ml aphidicolin to obtain a population of cells accumulated in G1 and S phase. IMR-90 cells were arrested in G0 by growing the cells for 3 days in low serum (0.5%). The quiescent cells were activated by addition of 20% serum to the media, and the 35S-labeled cells were harvested at 4 h intervals following activation. Cells were preincubated with methionine- and cysteine-free media containing 5% dialyzed serum for 30 min, followed by 1 h in the same medium containing 100 μCi/ml Tran35S-label (ICN Biomedicals Inc.). The media for the 0 time point contained only 0.5% dialyzed serum. 32P labeling was done for 1 h after a 30-min incubation in phosphate-deficient medium containing 5% dialyzed serum. The label32Pi (ICN Biomedicals) was present at a concentration of 0.5 mCi/ml. Subcellular fractions were obtained by lysing the cells in a hypotonic buffer (20 mm Hepes, pH 7.4, 5 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, 0.1% Nonidet P-40, 50 mm NaF, 100 mmNa3VO4, 20 μg/ml aprotinin, 1 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, and 50 μg/ml phenylmethylsulfonyl fluoride) after a quick rinse in cold distilled water. All protease inhibitors were purchased from Sigma. The cells were left on ice for 30 min with intermittent swirling and then scraped off the plates with a cell scraper. The nuclei were pelleted by a 1000 × g spin in a swinging bucket rotor for 10 min. The supernatant was separated into S100 (cytosol) and P100 (particulate) fractions by spinning for 1 h at 100,000 ×g. The nuclear and P100 pellets were resuspended in radioimmunoprecipitation assay buffer, described previously (19Weinstein J. Jacobsen F.W. Hsu-Chen J. Wu T. Baum L.G. Mol. Cell. Biol. 1994; 14: 3350-3363Crossref PubMed Scopus (111) Google Scholar). Nonidet P-40 and NaCl were added to the S100 fraction to make its composition closer to the other two fractions. The resuspended P100 and nuclear fractions were spun for 30 min at 20,000 × gto obtain a clarified lysate for immunoprecipitation experiments. Preparation of a total cell lysate has been described previously (19Weinstein J. Jacobsen F.W. Hsu-Chen J. Wu T. Baum L.G. Mol. Cell. Biol. 1994; 14: 3350-3363Crossref PubMed Scopus (111) Google Scholar). All lysates were stored at −80 °C until needed. The preparation of the antibody against a glutathioneS-transferase-p55Cdc fusion construct has been described (19Weinstein J. Jacobsen F.W. Hsu-Chen J. Wu T. Baum L.G. Mol. Cell. Biol. 1994; 14: 3350-3363Crossref PubMed Scopus (111) Google Scholar). Rabbit antibodies were generated against the carboxyl-terminal end of p55Cdc (CKSSLIHQGIR), conjugated to keyhole limpet hemocyanin antigen. Antisera were affinity-purified by passage through a Sulfolink (Pierce) column to which the peptide had been coupled. The column was washed with 10 column volumes of PBS, and the affinity-purified antibodies were eluted with 0.1 m glycine, pH 2.8, into tubes containing 1 m Tris, pH 8. The pH of the pooled fractions was adjusted to neutral, and they were stored in aliquots at −80 °C. Protein concentrations were estimated with the bicinchoninic acid reagent (Pierce). The protocol for immunoprecipitations has been described (19Weinstein J. Jacobsen F.W. Hsu-Chen J. Wu T. Baum L.G. Mol. Cell. Biol. 1994; 14: 3350-3363Crossref PubMed Scopus (111) Google Scholar). The concentration of antibody was increased to 5 μg/100 μg of lysate for the glutathioneS-transferase-p55Cdc fusion protein antibody and to 15 μg/100 μg of lysate for the carboxyl-terminal peptide antibody. The concentration of the control adsorbed antisera was always 5–10-fold higher than that of the affinity-purified antibodies. Antibodies to p34 cdc2 (monoclonal antibody 17), cyclin A (BF683), cyclin E (E-19), and goat polyclonal antibody against the carboxyl-terminal peptide of human lamin B were purchased from Santa Cruz Biotechnology Inc. The monoclonal antibody to human cyclin B1 (GNS-1) was purchased from Pharmingen, and the monoclonal antibody to human lactate dehydrogenase was from Sigma. The band intensity was quantitated by PhosphorImager analysis (Molecular Dynamics). Total cell lysates were prepared in 1.5 × SDS sample loading buffer (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (208448) Google Scholar) at a concentration of 106 cells/0.15 ml. The subcellular fractions were prepared exactly as described above, except that the S100 fraction was concentrated so that normalization was by cell number for all three fractions. The proteins were transferred to Hybond nitrocellulose (Amersham Corp.) for 2 h at 80 mA with the Pharmacia Multiphor II apparatus (Pharmacia Biotech Inc.). The transferred proteins were visualized by the ECL detection system (Amersham Corp). For indirect immunofluorescence experiments, an asynchronous population of HeLa cells was fixed in −20 °C methanol, 2 mm EGTA for 10 min, blocked, and permeabilized for 7 min in PBS containing 5% donkey serum and 0.1% Triton X-100 and rinsed briefly in PBS, 0.1% BSA. The cells were incubated with 10 μg/ml p55Cdc affinity-purified antibody and 1/50 dilution of β-tubulin monoclonal antibody from Amersham Corp. or 50 μg/ml control adsorbed antisera for 75 min. The slides were washed twice in PBS, 0.1% BSA and blocked again with 5% donkey serum in PBS. Incubation with fluorescence-labeled secondary antibodies (1/400 diluted Cy3 conjugated to AffiniPure F(ab′)2 fragment donkey anti rabbit IgG, 1/100 diluted FITC-conjugated AffiniPure F(ab′) fragment donkey anti rabbit IgG and 1/100 diluted FITC-conjugated AffiniPure F(ab′)2 fragment donkey anti mouse IgG, Jackson ImmunoResearch Labs) was for 1 h. Slides were washed twice in PBS, 0.1% BSA and once in PBS containing 2.5 μg/ml Hoescht 33342 (Molecular Probes). The slides were rinsed in distilled water and mounted in Vectashield (Vector Laboratories). Results were observed under fluorescence with a Nikon Microphot-FXA equipped with a Plan Apo 50× oil immersion lens (Nikon Inc). Cells at various mitotic stages were photographed under identical conditions with the appropriate filters. All experiments were performed reproducibly at least twice. Previous work demonstrated that p55Cdc expression was readily detectable in a rapidly proliferating, but not quiescent population of Rat1 fibroblasts (19Weinstein J. Jacobsen F.W. Hsu-Chen J. Wu T. Baum L.G. Mol. Cell. Biol. 1994; 14: 3350-3363Crossref PubMed Scopus (111) Google Scholar). To identify the stage of the cell cycle at which p55Cdc expression is initiated, a normal diploid untransformed human fibroblast cell line was examined. IMR-90 cells were arrested at G0 by serum starvation and induced to enter the cell cycle by the addition of 20% serum to the medium. IMR-90 cells enter S phase 16 h following serum stimulation and show peak DNA synthesis at 24 h (29Pagano M. Pepperkok R. Lukas J. Baldin V. Ansorge W. Bartek J. Draetta G. J. Cell Biol. 1993; 121: 101-111Crossref PubMed Scopus (285) Google Scholar). At 4-h intervals following serum activation the cells were labeled with 35S and immunoprecipitated with p55Cdc antibody or p34 cdc2 antibody for comparison. The results in Fig. 1 show the appearance of p55Cdc 16 h following growth stimulation and a barely detectable band at 12 h, coincidental with the biosynthesis of p34 cdc2 . The p34 cdc2 immunoprecipitates consistently show a band of slightly higher mobility than p55Cdc, which could be due to cyclin A. At 24 h both p55Cdc and p34 cdc2 synthesis were still increasing. Activation of p34 cdc2 synthesis has been shown to occur at the G1/S transition (30Lee M.G. Norbury C.J. Spurr N.K Nurse P. Nature. 1988; 333: 676-679Crossref PubMed Scopus (134) Google Scholar, 31Welch P.J. Wang J.Y.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3093-3097Crossref PubMed Scopus (101) Google Scholar, 32Furukawa Y. Piwnica-Worms H. Ernst T.J. Kanakura Y. Griffin J.D. Science. 1990; 250: 805-808Crossref PubMed Scopus (160) Google Scholar). To determine the half-life of p55Cdc, an asynchronous population of HeLa cells was labeled for 1 h with35S and total cell lysates or subcellular fractions were prepared after 0, 1, 2, 6, or 21 h of chase. Lanes 3,5, 7, 9, and 12 in Fig.2 a show the amount of p55Cdc at 0, 1, 2, 6, and 21 h following the labeling pulse. PhosphorImager analysis revealed the half-life of p55Cdc to be 2 h, followed by a slower loss so that 35% of the label remained with p55Cdc at 6 h (data not shown). p34 cdc2 (Fig.2 a, lanes 2, 4, 6,8, and 10) showed no significant loss of radioactivity over the time course of the experiment but showed a conversion to the slower mobility phosphorylated forms over time. This result is generally in agreement with the reported long half-life of 18 h for this protein (31Welch P.J. Wang J.Y.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3093-3097Crossref PubMed Scopus (101) Google Scholar). PhosphorImager analysis of the half-life of p55Cdc at different subcellular locations showed it to be unique for each site. Comparison of p55Cdc detection of the S100 fraction (Fig. 2 b, lanes 3, 5,7, 9, and 12) showed the half-life in this fraction to be approximately 1 h. The P100 fraction had a half-life of 6 h (Fig. 2 c), while the nuclear fraction still retained 70% of its radioactivity at this time (Fig.2 d). Apparently, the newly synthesized p55Cdc was rapidly degraded or transferred from the cytosol to the cytoskeletal membranes and nucleus, where it had a longer half-life. Interestingly, p34 cdc2 showed a steadily increasing accumulation over time in the P100 (Fig. 2 c, lanes 2, 4,6, 8, and 10) and nuclear (Fig.2 d, lanes 2, 4, 6,8, and 10) fractions of the slower mobility hyperphosphorylated form while the cytosol fraction (Fig.2 b) showed a steady loss of the high mobility form. This would be consistent with the phosphorylation of p34 cdc2 prior to or immediately following nuclear entry. To analyze the cell cycle-regulated expression, distribution and phosphorylation status of p55Cdc, HeLa cells were synchronized and labeled at various stages of the cell cycle (Fig. 3 a). At indicated times, the cells were solubilized and the lysates immunoprecipitated. Since p55Cdc is known to be associated with protein kinase(s) activity, which fluctuates with the cell cycle (19Weinstein J. Jacobsen F.W. Hsu-Chen J. Wu T. Baum L.G. Mol. Cell. Biol. 1994; 14: 3350-3363Crossref PubMed Scopus (111) Google Scholar) and partitions to different subcellular locations, 2J. Weinstein, unpublished observations. it would be of interest to observe p55Cdc immune complexes from the cytosol (S100), particulate (P100), and nuclear fractions. Biosynthesis of p55Cdc in HeLa cells was detectable in G1 and increased steadily through the cell cycle to peak at G2 (Fig. 3, band c). The regulation of p55Cdc biosynthesis was similar to that of p34 cdc2, being very low at G1 and peaking at G2. The p55Cdc immunoprecipitates showed the presence of a 100-kDa protein in the P100 and nuclear fractions through the entire cell cycle. The p34 cdc2 immunoprecipitates showed a faint band that appeared to comigrate with p55Cdc and could be due to cyclin A and/or B. A 10-fold increase in the rate of p34 cdc2 synthesis has been observed as HeLa cells progress from G1 to G2 (33McGowan C.H. Russel P. Reed S.I. Mol. Cell. Biol. 1990; 10: 3847-3851Crossref PubMed Scopus (80) Google Scholar). Although the bulk of newly synthesized p34 cdc2 was found in the S100 fraction, p55Cdc had the highest relative concentration in the P100 fraction (Fig. 3 c) and this pattern did not change with the cell cycle. As expected, the nuclear p34 cdc2 in the G2 samples was primarily the slower mobility (inactive) form, while that in the S100 and P100 fraction of mitotic cells (Fig. 3 b) was of the faster mobility (active) form. The results of labeling HeLa cells with 32P for 1 h at different stages of the cell cycle are shown in Fig.4. The late G2 cells represent the adherent monolayer after the mitotic cells have been harvested by repeated pipetting. This population also has cells that have already exited mitosis and entered G1. p55Cdc phosphorylation is undetectable in any of the fractions obtained from a G1 population of cells, followed by progressive increase in phosphorylation of p55Cdc as the cells proceed from G1 to G2. The p55Cdc immunoprecipitates from P100 fractions (Fig.4 b, lanes 2 and 3) contain a unidentified band of 35 kDa that co-migrates with the slower mobility form of p34 cdc2, and is undetectable in the mitotic P100 fraction. The S100 fractions (Fig. 4 a, lanes 2and 3) showed a diffuse band at longer exposures (data not shown), which could represent different phosphorylated forms of p55Cdc. Immune complexes obtained from the nuclear fraction with antibody against glutathione S-transferase-p55Cdc fusion protein (Fig. 4 c, lane 2) also showed a slower migrating band around 58 kDa that was not detected by the p55Cdc COOH-terminal antibody (Fig. 4 c, lane 3). Since the nuclear membrane has disintegrated in the mitotic cells, only the S100 and P100 fractions were obtained. The mitotic S100 fraction showed a unique profile of p55Cdc-associated phosphorylated proteins in the immune complex, including the appearance of proteins of 72 and 140 kDa. The same profile was obtained by two different antibodies to p55Cdc (Fig.4 a, lanes 2 and 3). The rate of p34 cdc2 phosphorylation in the various subcellular fractions from different stages in the cell cycle (Fig. 4, a,b, and c, lane 4) paralleled that of p55Cdc, although unlike p55Cdc, phosphorylated p34 cdc2 was detectable during G1. Enhancement in the rate of p34 cdc2 phosphorylation at G2 has been observed previously (34Draetta G. Beach D. Cell. 1988; 54: 17-26Abstract Full Text PDF PubMed Scopus (527) Google Scholar). The data obtained in Fig. 4 were subjected to PhosphorImager analysis, and the results are shown in Fig. 5. Except for the late G2 population, both proteins exhibit the highest concentration of phosphorylated protein in the P100 fraction. This is noteworthy since 35S labeling detected most p34 cdc2 in the cytosol fraction and similar results were obtained for the distribution of p34 cdc2 in the various subcellular fractions (Fig. 6,lanes 1–3), although longer exposure showed detectable low mobility form of p34 cdc2 in the membrane fraction (data not shown). Thus, the p34 cdc2 associated with cell membranes must be in a highly phosphorylated state. This is consistent with the report that myt1, the inhibitory kinase that phosphorylates p34 cdc2 on both threonine 14 and tyrosine 15, is a membrane-associated kinase (35Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (548) Google Scholar,36Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar). In this context, it is interesting that human cyclin B2 has been localized primarily to the Golgi apparatus (37Jackman M. Firth M. Pines J. EMBO J. 1995; 14: 1646-1654Crossref PubMed Scopus (222) Google Scholar). The phosphorylation rate of both p55Cdc (Fig. 5 a) and p34 cdc2 (Fig.5 b) peak at G2 and show a dramatic dephosphorylation at M. However, although p55Cdc showed an overall dephosphorylatio
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