Cyclin A-CDK Phosphorylation Regulates MDM2 Protein Interactions
2001; Elsevier BV; Volume: 276; Issue: 32 Linguagem: Inglês
10.1074/jbc.m011326200
ISSN1083-351X
Autores Tópico(s)Microtubule and mitosis dynamics
ResumoThe product of theMDM2 gene interacts with and regulates a number of proteins, in particular the tumor suppressor p53. The MDM2 protein is likely to be extensively modified in vivo, and such modification may regulate its functions in cells. We identified a potential cyclin-dependent kinase (CDK) site in murine MDM2, and found the protein to be efficiently phosphorylated in vitro by cyclin A-containing complexes (cyclin A-CDK2 and cyclin A-CDK1), but MDM2 was either weakly or not phosphorylated by other cyclin-containing complexes. Moreover, a peptide containing a putative MDM2 cyclin recognition motif specifically inhibited phosphorylation by cyclin A-CDK2. The site of cyclin A-CDK2 phosphorylation was identified as Thr-216 by two-dimensional phosphopeptide mapping and mutational analysis. Phosphorylation of MDM2 at Thr-216 both weakens its interaction with p53 and modestly augments its binding to p19ARF. Interestingly, an MDM2-specific monoclonal antibody, SMP14, cannot recognize MDM2 phosphorylated at Thr-216. Changes in SMP14 reactivity of MDM2 in staged cell extracts indicate that phosphorylation of MDM2 at Thr-216 in vivo is most prevalent at the onset of S phase when cyclin A first becomes detectable. The product of theMDM2 gene interacts with and regulates a number of proteins, in particular the tumor suppressor p53. The MDM2 protein is likely to be extensively modified in vivo, and such modification may regulate its functions in cells. We identified a potential cyclin-dependent kinase (CDK) site in murine MDM2, and found the protein to be efficiently phosphorylated in vitro by cyclin A-containing complexes (cyclin A-CDK2 and cyclin A-CDK1), but MDM2 was either weakly or not phosphorylated by other cyclin-containing complexes. Moreover, a peptide containing a putative MDM2 cyclin recognition motif specifically inhibited phosphorylation by cyclin A-CDK2. The site of cyclin A-CDK2 phosphorylation was identified as Thr-216 by two-dimensional phosphopeptide mapping and mutational analysis. Phosphorylation of MDM2 at Thr-216 both weakens its interaction with p53 and modestly augments its binding to p19ARF. Interestingly, an MDM2-specific monoclonal antibody, SMP14, cannot recognize MDM2 phosphorylated at Thr-216. Changes in SMP14 reactivity of MDM2 in staged cell extracts indicate that phosphorylation of MDM2 at Thr-216 in vivo is most prevalent at the onset of S phase when cyclin A first becomes detectable. cyclin-dependent kinase glutathioneS-transferase monoclonal antibody fluorescence-activated cell sorter cyclin recognition motif enzyme-linked immunosorbent assay human MDM2 adenosine 5′-(β,γ-imino)triphosphate The mdm2 gene was cloned originally from a spontaneously transformed mouse cell line 3T3DM (1Cahilly-Snyder L. Yang-Feng T. Francke U. George D.L. Somatic Cell Mol. 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The uncoupling of S phase has been correlated with elevated cyclin A but not cyclin E or cyclin D mRNA levels in MDM2-overexpressing cells (38Lundgren K. Montes de Oca Luna R. McNeill Y.B. Emerick E.P. Spencer B. Barfield C.R. Lozano G. Rosenberg M.P. Finlay C.A. Genes Dev. 1997; 11: 714-725Crossref PubMed Scopus (212) Google Scholar). MDM2 expression was reported to lead to the activation of the cyclin A promoter (10Leveillard T. Wasylyk B. J. Biol. Chem. 1997; 272: 30651-30661Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). This may be related to interactions of MDM2 with components of the basal transcription factor TFIID; the MDM2 C-terminal RING finger domain interacts with TAFII250, and the region close to the acidic domain interacts with the TATA-binding protein TBP (10Leveillard T. Wasylyk B. J. Biol. Chem. 1997; 272: 30651-30661Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 11Thut C.J. Goodrich J.A. Tjian R. 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More than one third of the amino acids on the MDM2 protein are either serine or threonine residues, and the MDM2 protein is phosphorylated at multiple sites in vivo, especially in the N terminus and the central acidic domain (51Hay T.J. Meek D.W. FEBS Lett. 2000; 478: 183-186Crossref PubMed Scopus (51) Google Scholar). It has been reported that ataxia telangiectasia mutated kinase (ATM) (52Khosravi R. Maya R. Gottlieb T. Oren M. Shiloh Y. Shkedy D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14973-14977Crossref PubMed Scopus (353) Google Scholar), DNA-dependent protein kinase (53Mayo L.D. Turchi J.J. Berberich S.J. Cancer Res. 1997; 57: 5013-5016PubMed Google Scholar), and casein kinase 2 (54Guerra B. Gotz C. Wagner P. Montenarh M. Issinger O.G. Oncogene. 1997; 14: 2683-2688Crossref PubMed Scopus (63) Google Scholar,55Gotz C. Kartarius S. Scholtes P. Nastainczyk W. Montenarh M. Eur. J. Biochem. 1999; 266: 493-501Crossref PubMed Scopus (33) Google Scholar) can phosphorylate MDM2 proteins and modulate MDM2 functions. Murine MDM2 has at least one potential site for CDK phosphorylation. We report here that cyclin A-CDK complexes are unique in their ability to efficiently phosphorylate MDM2 and that this phosphorylation affects the interactions of MDM2 with proteins. A construct expressing GST-MDM2 was provided kindly by M. Oren (Weizmann Institute). Mutant GST-MDM2 T216A and T384A constructs were generated from wild-type GST-MDM2 using the Stratagene QuickChange® mutagenesis kit. Bacterially expressed GST-MDM2 proteins were purified from glutathione-Sepharose 4B columns (Life Technologies, Inc.). Complexes containing cyclin E and CDK2 (300 ng of cyclin E and 600 ng of CDK2 in 10 µl), cyclin A and CDK2 (200 ng of cyclin A and 300 ng of CDK2 in 10 µl), cyclin B and CDK1 (12 ng of cyclin B and 25 ng of CDK1 in 10 µl), or cyclin A and CDK1 (10 ng of cyclin A and 150 ng of CDK1 in 10 µl) were purified from extracts of insect Sf9 cells that had been co-infected with these respective pairs of recombinant baculoviruses as described previously (50Wang Y. Prives C. Nature. 1995; 376: 88-91Crossref PubMed Scopus (326) Google Scholar). A purified complex containing cyclin D1 and CDK4 was provided generously by Y. Taya (56Kitagawa M. Higashi H. Jung H.K. Suzuki-Takahashi I. Ikeda M. Tamai K. Kato J. Segawa K. Yoshida E. Nishimura S. Taya Y. EMBO J. 1996; 15: 7060-7069Crossref PubMed Scopus (534) Google Scholar). The murine p53 protein was isolated from extracts of baculovirus-infected insect cells by immunoaffinity purification over a PAb 421 column as described previously (57Wang E.H. Friedman P.N. Prives C. Cell. 1989; 57: 379-392Abstract Full Text PDF PubMed Scopus (150) Google Scholar). A construct expressing His-tagged p19ARF-N-37 was provided kindly by the laboratory of C. Sherr and was purified from bacteria as described (58Weber J.D. Kuo M.L. Bothner B. DiGiammarino E.L. Kriwacki R.W. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 2000; 20: 2517-2528Crossref PubMed Scopus (244) Google Scholar). Kinase assay mixtures (30 µl) contained kinase buffer (50 mm Hepes (pH 7.5), 10 mm MgCl2, 1 mm dithiothreitol, 100 µm ATP, and 4 µCi of γ-[32P]ATP) and 100 ng of purified GST-MDM2, pRb, or histone H1 proteins as substrates. Cyclin-CDK complexes were added into reaction mixtures, which were incubated at 30 °C for 15 min, and then resolved on 10% SDS-polyacrylamide gels. The gels were either silver-stained or stained with Coomassie Blue to detect protein levels before exposure to x-ray film. Peptides used in the cyclin recognition motif experiments were synthesized by Synpep. 32P-labeled GST-MDM2 (1 µg) was phosphorylated by cyclin A-CDK2 as described above and then run on a 10% SDS-polyacrylamide gel and exposed to x-ray film. The phosphorylated GST-MDM2 full-length protein was then cut out and eluted from the gel slice in buffer containing 0.05 mNH4HCO3 (pH 7.3), 0.5% β-mercaptoethanol, and 0.1% SDS. The eluted proteins were acid-precipitated and oxidized as described (59Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar) and then digested with 10 µl of V8 protease (1 µg/µl) at 37 °C for 16 h in 50 µl of buffer containing 0.05 m NH4HCO3 (pH 7.3) (60Sorensen S.B. Sorensen T.L. Breddam K. FEBS Lett. 1991; 294: 195-197Crossref PubMed Scopus (66) Google Scholar). The proteolytically digested MDM2 was subjected to electrophoresis in pH 1.9 buffer in the first dimension and chromatography in phospho-buffer (N-butanol/pyridine/glacial acetic acid/H2O, 15:10:3:12) in the second dimension as described (59Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). Baculovirus-expressed murine wild-type p53 protein (50 ng) or bacterially expressed p19ARF-N-37 peptide (20 ng) was used to coat each well in a 96-well Pro-bind plate (Falcon) in 200 µl of PBS for 2 h in 4 °C. The wells were then washed three times in PBS containing 0.05% Tween and then incubated in blocking buffer (PBS containing 0.05% Tween and 1% bovine serum albumin) for 1 h at 4 °C. Phosphorylated or unphosphorylated GST-MDM2 (15–180 ng) in 200 µl of PBS was then added to each well and incubated for 1 h at 4 °C. Roscovitine (10 µm) was present in the PBS to prevent the kinase from phosphorylating the coating protein. The wells were washed briefly three times with PBS containing 0.05% Tween, at which time an anti-MDM2 monoclonal antibody and either a 1:1000 dilution of purified mAb SMP14 (200 µg/ml, Santa Cruz Biotechnology) or a 1:40 dilution of mAb 4B11 hybridoma supernatant (generously provided by A. Levine) in blocking buffer was added and incubated for 1 h at room temperature. After three washes, a 1:2000 dilution of monoclonal anti-mouse IgG antibody conjugated to alkaline phosphatase (Sigma) in blocking buffer was added and incubated at room temperature for 30 min. After five washes, 10 mm p-nitrophenol phosphate (Sigma) in 100 mm 2-amino-2-methyl-1,3-propanediol (Sigma) was added to the wells. Absorbance at 405 nm was measured at 10-min intervals using a Bio-Rad model 550 microplate reader. The amount of MDM2 bound to the coating protein was calculated from the change in absorbance at different time points. Each data point is an average of readings from triplicate wells. Swiss 3T3 mouse fibroblast cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Log-phase cells were starved in Dulbecco's modified Eagle's medium with 0.5% fetal bovine serum for 48 h before being released into Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cell cultures (1 plate each) were collected at different time points and washed twice in ice-cold PBS. The cells were scraped, centrifuged at 1500 rpm in 4 °C, and then lysed in extraction buffer (PBS containing 2% SDS and 10 mm iodoacetamide). Extracts were then heated at 95 °C for 10 min and sonicated for 10 min. Protein concentrations were determined by the BCA assay (Pierce), and 150 µg of each extract was run on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane (Schleicher & Schüell). MDM2 protein was detected by Western blot analysis using anti-MDM2 antibodies, either mAb SMP14 (Santa Cruz Biotechnology) or mAb 3G5 hybridoma supernatant (a gift from Dr. A. Levine). The same blots were also probed with an anti-cyclin A polyclonal antibody H437 or an anti-cyclin B monoclonal antibody Ab-2 (both from Santa Cruz Biotechnology). Duplicate plates of cells at each time point were collected and processed for FACS analysis. GST-tagged proteins in kinase reaction mixtures were incubated with glutathione-Sepharose 4B beads (Life Technologies, Inc.) in 200 µl of ice-cold TEGN washing buffer (10 mm Tris (pH 7.5), 1 mm EDTA, 150 mmNaCl, 10% glycerol, 0.5% Nonidet P-40, and 0.5 mm sodium orthovanadate) for 1 h at 4 °C. The beads were centrifuged at 2500 rpm, washed twice with TEGN buffer, and blocked in 200 µl of TEGN buffer containing 1% bovine serum albumin at 4 °C. Beads containing 100 ng of GST-tagged protein were rocked with mouse p53 protein in 200 µl of TEGN buffer at 4 °C for 1 h, washed five times with the same buffer, and then separated on a 10% SDS-polyacrylamide gel. GST-MDM2 and mouse p53 protein were detected by Western blot analysis with an anti-MDM2 rabbit polyclonal antibody or a mixture of mouse p53 monoclonal antibodies (PAb 242, PAb 246, and PAb 248). Examination of the sequence of murine MDM2 protein indicated that there are two possible CDK sites in the protein at residues 216 (TPSH) and 384 (TPLS). In fact, Thr-216–Pro-217 is conserved between murine and human (Thr-218–Pro-219) MDM2. Using bacterially expressed full-length murine GST-MDM2 as the substrate, we performed in vitro kinase assays with increasing amounts of purified cyclin A-CDK2 complex (Fig.1). GST-MDM2 was phosphorylated by cyclin A-CDK2 in a dose-dependent fashion. At the highest level of kinase complex added, the mobility of MDM2 protein was shifted completely to a slower migrating form, indicating that it had been efficiently phosphorylated (Fig. 1, lanes 6 and7). To ensure that the phosphorylation observed was the result of cyclin A-CDK2 activity rather than a contaminating kinase, we used a CDK2 inhibitor roscovitine (ID50 = 700 nm, specific for CDK1 and CDK2) (61Rudolph B. Saffrich R. Zwicker J. Henglein B. Muller R. Ansorge W. Eilers M. EMBO J. 1996; 15: 3065-3076Crossref PubMed Scopus (119) Google Scholar, 62Meijer L. Borgne A. Mulner O. Chong J.P. Blow J.J. Inagaki N. Inagaki M. Delcros J.G. Moulinoux J.P. Eur. J. Biochem. 1997; 243: 527-536Crossref PubMed Scopus (1209) Google Scholar). The incorporation of label from [32P]ATP was substantially decreased by roscovitine with a concomitant increase in electrophoretic mobility of the MDM2 polypeptide when compared with the phosphorylated form (Fig.1, compare lanes 7 and 8). We then investigated the abilities of other cyclin-CDK complexes to phosphorylate GST-MDM2. The activities of the complexes were normalized using either histone H1 or pRb as a positive control. Although the cyclin E-CDK2 complex has the same kinase component as cyclin A-CDK2, it only very weakly phosphorylated GST-MDM2 (Fig.2A, lanes 6–9). Cyclin D1-CDK4 was also impaired in phosphorylating GST-MDM2 when compared with its ability to phosphorylate pRb protein (Fig. 2 B,lanes 1 and 2 versus lanes 3 and4). It is interesting as well that the cyclin B-CDK1 complex phosphorylated GST-MDM2 more modestly than did cyclin A-CDK2 (Fig.2 C, lanes 5–8) even though, as expected, both kinases phosphorylated histone H1 to roughly the same extent. Because cyclin A has been shown to form a complex with CDK1 at the onset of mitosis (47Pagano M. Pepperkok R. Verde F. Ansorge W. Draetta G. EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1134) Google Scholar), we then compared the abilities of purified cyclin A-CDK1 and cyclin B-CDK1 complexes to phosphorylate GST-MDM2 in vitro (Fig. 2 D). Indeed, cyclin A-CDK1 phosphorylated GST-MDM2 and, similar to cyclin A-CDK2, did so more effectively than cyclin B-CDK1. Therefore, cyclin A-containing CDK complexes are the most effective in phosphorylating the MDM2 protein. To identify the CDK site(s) in MDM2, either Thr-216 or Thr-384 was mutated to alanine in full-length GST-MDM2, and wild-type and T216A or T384A MDM2 proteins were purified from bacteria and treated with cyclin A-CDK2 in mixtures containing [32P]ATP. We then performed two-dimensional mapping of phosphopeptides generated from V8 protease digestion of phosphorylated wild-type or mutant proteins. In the map of the phosphorylated wild-type protein, a predominant phosphopeptide was observed (Fig. 3 A,arrow). This spot was diminished when the CDK2-specific inhibitor roscovitine was added to the kinase reaction mixture (Fig.3 B), supporting the possibility that it is specifically phosphorylated by cyclin A-CDK2. Importantly, this phosphopeptide was absent in the two-dimensional map of the T216A mutant (Fig.3 C) but was present in the map of the T384A mutant (Fig.3 D). The additional minor phosphopeptides that were in the two-dimensional maps are most likely the result of cryptic phosphorylation by the CDK complex, because they are largely suppressed by roscovitine. We conclude that cyclin A-CDK2 phosphorylates predominantly Thr-216 in murine MDM2. Phosphorylation of a number of cyclin-dependent kinase substrates requires their interaction with cyclins mediated by sequences (CRMs) termed cyclin-CDK substrate recognition motifs (63Ewen M.E. Faha B. Harlow E. Livingston D.M. Science. 1992; 255: 85-87Crossref PubMed Scopus (164) Google Scholar, 64Krek W. Ewen M.E. Shirodkar S. Arany Z. 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Although we failed to detect a stable interaction between GST-MDM2 and cyclin A (alone or in complex with CDK2) in a GST pull-down assay (data not shown), the pho
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