Title: Akt Enhances Mdm2-mediated Ubiquitination and Degradation of p53
Abstract: p53 plays a key role in DNA damage-induced apoptosis. Recent studies have reported that the phosphatidylinositol 3-OH-kinase-Akt pathway inhibits p53-mediated transcription and apoptosis, although the underlying mechanisms have yet to be determined. Mdm2, a ubiquitin ligase for p53, plays a central role in regulation of the stability of p53 and serves as a good substrate for Akt. In this study, we find that expression of Akt reduces the protein levels of p53, at least in part by enhancing the degradation of p53. Both Akt expression and serum treatment induced phosphorylation of Mdm2 at Ser186. Akt-mediated phosphorylation of Mdm2 at Ser186 had little effect on the subcellular localization of Mdm2. However, both Akt expression and serum treatment increased Mdm2 ubiquitination of p53. The serum-induced increase in p53 ubiquitination was blocked by LY294002, a phosphatidylinositol 3-OH-kinase inhibitor. Moreover, when Ser186 was replaced by Ala, Mdm2 became resistant to Akt enhancement of p53 ubiquitination and degradation. Collectively, these results suggest that Akt enhances the ubiquitination-promoting function of Mdm2 by phosphorylation of Ser186, which results in reduction of p53 protein. This study may shed light on the mechanisms by which Akt promotes survival, proliferation, and tumorigenesis. p53 plays a key role in DNA damage-induced apoptosis. Recent studies have reported that the phosphatidylinositol 3-OH-kinase-Akt pathway inhibits p53-mediated transcription and apoptosis, although the underlying mechanisms have yet to be determined. Mdm2, a ubiquitin ligase for p53, plays a central role in regulation of the stability of p53 and serves as a good substrate for Akt. In this study, we find that expression of Akt reduces the protein levels of p53, at least in part by enhancing the degradation of p53. Both Akt expression and serum treatment induced phosphorylation of Mdm2 at Ser186. Akt-mediated phosphorylation of Mdm2 at Ser186 had little effect on the subcellular localization of Mdm2. However, both Akt expression and serum treatment increased Mdm2 ubiquitination of p53. The serum-induced increase in p53 ubiquitination was blocked by LY294002, a phosphatidylinositol 3-OH-kinase inhibitor. Moreover, when Ser186 was replaced by Ala, Mdm2 became resistant to Akt enhancement of p53 ubiquitination and degradation. Collectively, these results suggest that Akt enhances the ubiquitination-promoting function of Mdm2 by phosphorylation of Ser186, which results in reduction of p53 protein. This study may shed light on the mechanisms by which Akt promotes survival, proliferation, and tumorigenesis. phosphatidylinositol 3-OH-kinase constitutively active kinase-negative mitogen-activated protein kinase phosphate-buffered saline reverse transcription mitogen-activated protein kinase/extracellular signal-regulated kinase kinase hemagglutinin wild type Growth factors, cytokines, and certain oncogenes have been shown to be effective inhibitors of apoptosis, and in many cases, their anti-apoptotic effects are mediated by the phosphatidylinositol 3-OH-kinase (PI3K)1-induced activation of Akt (1Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3755) Google Scholar, 2Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar). For instance, Ras activation of the PI3K-Akt pathway confers protection from apoptosis in fibroblasts in response to DNA damage or oncogenic Myc (3Sklar M.D. Science. 1988; 239: 645-647Crossref PubMed Scopus (369) Google Scholar, 4Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1078) Google Scholar). In this respect, the PI3K-Akt pathway-mediated survival contributes to the ability of Ras to function as an oncogene (1Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3755) Google Scholar, 2Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1408) Google Scholar). Although several Akt targets have been reported, it is not fully understood how Akt promotes survival (5Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4474) Google Scholar, 6Datta S.R. Dudek H. Tao X. 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Central to this process is Mdm2 (murine double minute), a ubiquitin ligase that targets p53 for ubiquitination and allows export of p53 from the nucleus to the cytoplasm, where p53 degradation by proteasomes takes place (16Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3795) Google Scholar, 17Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1627) Google Scholar, 18Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2881) Google Scholar, 19Fuchs S.Y. Adler V. Buschmann T., Wu, X. Ronai Z. Oncogene. 1998; 17: 2543-2547Crossref PubMed Scopus (213) Google Scholar, 20Boyd S.D. Tsai K.Y. Jacks T. Nat. Cell Biol. 2000; 2: 563-568Crossref PubMed Scopus (291) Google Scholar, 21Geyer R.K., Yu, Z.K. Maki C.G. Nat. Cell Biol. 2000; 2: 569-573Crossref PubMed Scopus (302) Google Scholar). Under normal circumstances, p53 is maintained at very low levels by continuous ubiquitination and degradation. Activation of p53 in response to cellular stresses is mediated partly by inhibition of Mdm2 and rapid stabilization of p53 protein (22Woods D.B. Vousden K.H. Exp. Cell. Res. 2001; 264: 56-66Crossref PubMed Scopus (292) Google Scholar). The deregulated activation of mitogenic signals, caused by the oncogenic activation of Ras or Myc for example, leads to the activation of p53, which provides a mechanism to prevent the abnormal proliferation associated with tumor development (23Zindy F. Eischen C.M. Randle D.H. Kamijo T. Cleveland J.L. Sherr C.J. Roussel M.F. Genes Dev. 1998; 12: 2424-2433Crossref PubMed Scopus (1072) Google Scholar, 24Vousden K.H. Cell. 2000; 103: 691-694Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). However, this activation of p53 by mitogenic signals must be suppressed during normal cell proliferation to prevent p53 from inducing cell cycle arrest or apoptosis. Therefore, it appears reasonable to assume that mitogenic signals elicit both p53-activating and -inactivating signals. Recent studies have indeed shown that Ras can inhibit or activate p53, depending on the cellular contexts and the duration of Ras activation (24Vousden K.H. Cell. 2000; 103: 691-694Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar, 25Ries S. Biederer C. Woods D. Shifman O. Shirasawa S. Sasazuki T. McMahon M. Oren M. McCormick F. Cell. 2000; 103: 321-330Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). The Raf-MEK-MAPK pathway has been shown to mediate Ras activation of p53 (26Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (779) Google Scholar), most likely through induction of p19ARF, which in turn inactivates Mdm2. The PI3K-Akt pathway has recently been reported to inhibit the transcriptional activity of p53 and reduce the pro-apoptotic functions of p53 (27Sabbatini P. McCormick F. J. Biol. Chem. 1999; 274: 24263-24269Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar,28Yamaguchi A. Tamatani M. Matsuzaki H. Namikawa K. Kiyama H. Vitek M.P. Mitsuda N. Tohyama M. J. Biol. Chem. 2001; 276: 5256-5264Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). 2Y. Ogawara and Y. Gotoh, unpublished data. Therefore, it is possible that the PI3K-Akt pathway opposes the MAPK pathway in activation of p53. However, it has yet to be determined how Akt suppresses p53. Here we show that Akt does not affect the mRNA levels of p53 but promotes ubiquitination and degradation of p53 protein. We confirmed very recent studies showing that Mdm2 serves as a good substrate for Akt (29Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11598-11603Crossref PubMed Scopus (973) Google Scholar, 30Zhou B.P. Liao Y. Xia W. Zou Y. Spohn B. Hung M.C. Nat. Cell Biol. 2001; 3: 973-982Crossref PubMed Scopus (798) Google Scholar). Although they have shown that Akt promotes nuclear translocation of Mdm2, we could not detect any effect of Akt on Mdm2 subcellular localization. Instead, we found that Akt facilitates the functions of Mdm2 to promote p53 ubiquitination by phosphorylation of Ser186. These findings may explain how mitogenic signal and Ras inhibit p53 during normal cell proliferation and may also provide a mechanism by which Akt promotes survival. Human p53 cDNA, human Mdm2 cDNA, and p53-responsive luciferase reporter plasmid (PG13-Luc) are kind gifts from Dr. B. Vogelstein. FLAG-tagged and HA-tagged p53 cDNA were cloned into the KpnI-BamHI sites of pcDNA3.1(+) (Invitrogen) (FLAG-p53 and HA-p53). Mdm2 mutant S186A was generated by QuikChange (Stratagene) by utilizing primers 5′-CGCCACAAAGCTGATAGTATTTCCC-3′ and 5′-GGGAAATACTATCAGCTTTGTGGCG-3′. Mdm2 mutant S166A/S186A was generated by utilizing primers 5′-GGAGAGCAATTGCTGAGACAGAAG-3′ and 5′-CTTCTGTCTCAGCAATTGCTCTCC-3′ to mutate Ser166 into Ala of S186A Mdm2. FLAG-tagged wild type (WT), S186A, and S166A/S186A Mdm2 were cloned into theKpnI-XhoI sites of pcDNA3.1(+). For bacterial expression, WT and S186A Mdm2 were cloned into the EcoRI and XhoI sites of pGEX-6P-1 (Amersham Biosciences). Human WT Akt and a constitutively active (CA) Akt were kindly provided by Dr. D. Alessi and Dr. R. Roth (31Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence J.C., Jr. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), respectively. A kinase-negative (KN) Akt was made by mutating Lys179 into Ala as described (12Masuyama N. Oishi K. Mori Y. Ueno T. Takahama Y. Gotoh Y. J. Biol. Chem. 2001; 276: 32799-32805Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). A dominant-negative Akt (3A Akt) was made by mutating Lys179, Thr308, and Ser473 into Ala. CA Akt, WT Akt, KN Akt, and 3A Akt were cloned into BamHI site of pcDNA3.1(+). FLAG-tagged ubiquitin was cloned into theKpnI-BamHI sites of pcDNA3.1(+). The antibodies used in this study include polyclonal anti-HA antibody Y-11 (Santa Cruz Biotechnology, Inc.), monoclonal anti-α-tubulin antibody DM 1A (Sigma), monoclonal anti-topoisomerase II α antibody 8D2 (Medical & Biological Laboratories), monoclonal anti-MEK1 antibody 25 (Transduction Laboratories), monoclonal anti-FLAG antibody M2 (Sigma), polyclonal anti-Akt antibody (Cell Signaling), monoclonal anti-p53 antibody DO7 (Oncogene), and monoclonal anti-Mdm2 antibody IF2 (Calbiochem). To generate anti-Ser(P)186 Mdm2, a phosphopeptide corresponding to the amino acid sequence of human Mdm2 178–193 (CRQRKRHKpSDSISLSF) was synthesized and coupled to keyhole limpet hemocyanin (Sawady Technology). This antigen was injected into Japanese White rabbits, from which serum was collected approximately every 2 weeks. The serum was affinity purified by passing over a thiopropyl-Sepharose 6B column (Amersham Biosciences) coupled with a synthetic peptide of the sequence CRQRKRHKpSDSISLSF, and the bound antibodies were eluted. The elutes containing phosphopeptide-specific antibodies were then passed through a column coupled with the unphosphorylated peptide (i.e. CRQRKRHKSDSISLSF) to deplete antibodies that react with unphosphorylated Mdm2. MCF-7, Saos-2, and 293T cells were grown in Dulbecco's modified Eagle's medium containing penicillin-streptomycin and 10% fetal bovine serum. For MCF-7 and Saos-2 cells, transfection was carried out by using LipofectAMINE Plus reagent (Invitrogen) in 6-well plates or 10-cm dishes with 5 × 105 cells/dish and 3 × 106 cells/dish, respectively. For 6-well plates, the cells were transfected with 1–3 μg of total DNA together with 6 μl of LipofectAMINE Plus reagent and 4 μl of LipofectAMINE reagent/well. For 10-cm dishes, the cells were transfected with 4–6 μg of total DNA together with 20 μl of LipofectAMINE Plus reagent and 30 μl of LipofectAMINE reagent/dish. For 293T cells, transfection was carried out by using FuGENE6 transfection reagent (Roche Molecular Biochemicals) in 6-cm dishes (2 × 106 cells, 5 μg of total DNA, and 12 μl of FuGENE6 transfection reagent/dish). For luciferase assay for p53 transcriptional activity, the cells were transfected with PG13-Luc together with various constructs and a β-galactosidase expression plasmid. The β-galactosidase expression was driven by a cytomegalovirus promoter, and used for a standard to normalize transfection efficiency. Luciferase and β-galactosidase activities were assessed 24 h after transfection. Total RNA was isolated from MCF-7 cells using the TRIzol reagent (Invitrogen) and transcribed using ReverTra Ace (Toyobo) with oligo(dT) primers, according to the manufacturer's instructions. The aliquots of cDNA corresponding to 100 ng of total RNA were used for PCR amplification in a 50-μl solution containing 1× KOD Dash buffer (Toyobo), 0.2 mm dNTPs, 1.25 units of KOD Dash (Toyobo), and 0.4 μm of each primer performed with a PerkinElmer Life Sciences DNA thermal cycler. The primers used to amplify p53 were 5′-TCTGGGACAGCCAAGTCTGT-3′ (forward) and 5′-GGAGTCTTCCAGTGTGATGA-3′ (reverse). The primers for glyceraldehyde-3-phosphate dehydrogenase were 5′-CATTGACCTCAACTACATGG-3′ (forward) and 5′-TTGCCCACAGCCTTGGCAGC-3′ (reverse). The PCR parameters consisted of an initial cycle of 95 °C for 30 s followed by 28 cycles of 95 °C for 10 s, 60 °C for 10 s, and 74 °C for 20 s and final extension for 1 min at 74 °C. The amplified PCR products were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining. The products of constitutively expressed glyceraldehyde-3-phosphate dehydrogenase mRNA served as a control. All of the products were assayed in the linear range of the RT-PCR amplification process. MCF-7 cells were transfected with FLAG-p53 (0.1 μg) and the indicated amounts of either CA Akt or KN Akt for 22 h or treated with LY294002 (10 μm) for 5 h and then treated with 80 μg/ml of cycloheximide for 0, 30, 60, 90, or 120 min as indicated. The cell lysates were subjected to Western blot analysis with anti-FLAG antibody or anti-p53 antibody, and the relative intensity of each band was estimated using densitometry. For bacterial expression of Mdm2, BL21-Gold (DE3) cells (Stratagene) were transformed with Mdm2 pGEX-6P-1, cultured in LB medium containing 50 μg/ml of ampicillin, and induced with isopropyl-β-d-thiogalactopyranoside (1 mm) for 5 h. The cells were harvested, resuspended in a sonication buffer (50 mm Tris-HCl, pH 8.0, 50 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol, and 1 mm phenylmethylsulfonyl fluoride), and lysed by sonication. Triton X-100 was added to a final concentration of 1%, and the lysates were centrifuged at 20,000 × g for 20 min at 4 °C. The crude lysate was filtered (0.45-μm pore size; Millipore, Bedford, MA) and loaded onto a 2-ml Glutathione-SepharoseTM 4B (Amersham Biosciences) column equilibrated with 5 volumes of PBS. The column was washed with 10 volumes of Cleavage Buffer (Amersham Biosciences). The glutathione-Sepharose was mixed with PreScission protease (Amersham Biosciences) and incubated at 4 °C for 12 h, and Mdm2 was eluted. Mdm2 was dialyzed against a dialysis buffer containing 20 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 0.1% (v/v) aprotinin. Recombinant active Akt and kinase-negative Akt were prepared as described previously (32Laine J. Kunstle G. Obata T. Sha M. Noguchi M. Mol. Cell. 2000; 6: 395-407Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 33Obata T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Recombinant Akt was incubated with substrates (1 μg of recombinant Mdm2 protein) in 15 mm MgCl2, 1 mmdithiothreitol, 100 μm ATP, and 20 mmTris-HCl, pH 7.5, for 30 min at 37 °C in the presence of [γ-32P]ATP (2 μCi) (Amersham Biosciences). The reaction was stopped by the addition of Laemmli's sample buffer. The samples were subsequently resolved by SDS-PAGE and analyzed by autoradiography. The phosphorylation reaction was also carried out without radiolabeled ATP, and the samples were resolved by SDS-PAGE and subjected to Western blot analysis with anti-Ser(P)186 Mdm2 antibody. The cells grown on coverslips were fixed for 10 min in PBS containing 3.7% formaldehyde. The fixed coverslips were permeabilized in PBS containing 0.1% Triton X-100 for 10 min, washed twice in PBS (5 min), and incubated in a blocking buffer (PBS containing 0.2% bovine serum albumin) for 30 min. The cells were then incubated in the blocking buffer containing the primary antibody for 1 h and washed three times in PBS (5 min) before incubation with the appropriate fluorescein-conjugated secondary antibody plus Hoechst 33258 (Molecular Probes, Inc.) for a further 30 min. The cells were washed three times in PBS (5 min) and washed in water. The stained cells were mounted on glass slides and examined under a fluorescent microscope (Nikon). The cells were trypsinized, rinsed with PBS, and collected by centrifugation. The cells were then suspended in 300 μl of a hypotonic buffer (50 mm Tris, pH 7.5, 5 mm EDTA, 10 mm NaCl, and 0.005% Nonidet P-40) and placed on ice for 15 min. The cells were then homogenized and spun at 500 × g for 5 min before the supernatant (cytoplasmic fraction) was collected. The remaining pellet was washed with 300 μl of the hypotonic buffer, resuspended in 100 μl of radioimmune precipitation buffer (2 mm Tris, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1.0% Nonidet P-40, 1.0% deoxycholate, and 0.025% SDS), sonicated, and spun at 15,000 ×g for 15 min to remove debris and collect the supernatant (nuclear fraction). We confirmed the separation of the cytoplasmic and nuclear fractions by Western blotting of MEK1 (a cytoplasmic marker) (34Fukuda M. Gotoh Y. Nishida E. EMBO J. 1997; 16: 1901-1908Crossref PubMed Scopus (335) Google Scholar) and topoisomerase II α (a nuclear marker) (21Geyer R.K., Yu, Z.K. Maki C.G. Nat. Cell Biol. 2000; 2: 569-573Crossref PubMed Scopus (302) Google Scholar), respectively. The cells were rinsed with PBS and scraped into 400 μl of radioimmune precipitation buffer. The cells were then sonicated and spun at 15,000 × g for 15 min to remove cellular debris. The supernatants were used as cell lysates. For immunoprecipitation, the cell lysates were incubated with antibody for 1 h on ice and then with protein A-Sepharose (AmershamBiosciences) beads for 1 h at 4 °C. The beads were washed four times with radioimmune precipitation buffer and then eluted in Laemmli's SDS sample buffer. The elutes were subjected to SDS-PAGE and Western blot analysis. The cells were transfected with HA-p53 and FLAG-tagged ubiquitin together with various constructs. The cells were exposed to β-lactone (5 μm) (Calbiochem) for 2 h before the preparation of cell lysates to inhibit proteasome-mediated degradation of ubiquitinated proteins. The cell lysates were immunoprecipitated with anti-HA antibody. Immunoprecipitates were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. For detection of p53, the blot was probed with anti-p53 antibody. For detection of ubiquitinated p53, the blot was probed with anti-FLAG antibody or anti-p53 antibody. Akt has been shown to suppress p53-dependent apoptosis triggered by hypoxia (28Yamaguchi A. Tamatani M. Matsuzaki H. Namikawa K. Kiyama H. Vitek M.P. Mitsuda N. Tohyama M. J. Biol. Chem. 2001; 276: 5256-5264Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), etoposide, γ-irradiation (data not shown), or ectopic expression of p53 (Ref. 28Yamaguchi A. Tamatani M. Matsuzaki H. Namikawa K. Kiyama H. Vitek M.P. Mitsuda N. Tohyama M. J. Biol. Chem. 2001; 276: 5256-5264Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar and data not shown). Previous reports have shown that Akt is capable of inhibiting the transcriptional activity of p53 (28Yamaguchi A. Tamatani M. Matsuzaki H. Namikawa K. Kiyama H. Vitek M.P. Mitsuda N. Tohyama M. J. Biol. Chem. 2001; 276: 5256-5264Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 29Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11598-11603Crossref PubMed Scopus (973) Google Scholar). We confirmed that expression of active Akt reduced the transcriptional activity of a p53 reporter plasmid in MCF-7 cells (Fig.1A). However, the underlying mechanisms of Akt inhibition of p53 remain unclear. To dissect the mechanisms by which Akt inhibits the transcriptional activity of p53, we first investigated whether Akt expression has any effect on the protein and mRNA levels of p53. To examine this, we transfected MCF-7 cells with Akt constructs (the transfection efficiency was about 70%). The amounts of endogenous p53 protein were markedly reduced by expression of active Akt (Fig. 1B). In contrast, the mRNA levels of p53 detected by RT-PCR were unchanged by expression of active Akt (Fig. 1C). These results indicate that Akt reduces the levels of p53 protein but not p53 mRNA in MCF-7 cells. Because it is well established that the level of p53 protein is regulated largely by stability, we then asked whether the stability of p53 was affected by Akt. FLAG-tagged p53 was ectopically expressed in MCF-7 cells along with an Akt plasmid. Titration of the amount of co-transfected Akt plasmid showed that increasing amounts of active Akt correlated with decreased levels of p53 protein (Fig. 1D). The stability of p53 protein was then assessed by the addition of cycloheximide, a translational inhibitor. Two hours of cycloheximide treatment decreased p53 protein by 40% in control cells, whereas the same treatment decreased p53 protein by 80% in active Akt-expressing cells (Fig. 2A), indicating that the degradation rate of p53 protein was greater in active Akt-expressing cells. The amounts of p53 protein were then estimated by densitometry. As shown in Fig. 2B, p53 decayed faster when active Akt was expressed. The degradation enhanced by Akt was blocked by treatment with MG132, a proteasome inhibitor (data not shown). When MCF-7 cells were treated with LY294002, a PI3K inhibitor, the stability of endogenous p53 protein increased (Fig. 2C). These results suggest that the PI3K-Akt pathway accelerates p53 degradation. We asked whether Akt might regulate p53 stability by a direct phosphorylation of p53. We found that immunoprecipitated active Akt was not able to phosphorylate p53 in vitro (data not shown), suggesting an indirect regulation of p53 by Akt. The major way in which p53 is degraded is by Mdm2-mediated ubiquitination. Mdm2 is phosphorylated at multiple sites in vivo (35Hay T.J. Meek D.W. FEBS Lett. 2000; 478: 183-186Crossref PubMed Scopus (51) Google Scholar). Interestingly, analysis of human Mdm2 sequence revealed two sites (Ser166 and Ser186) that conform to the consensus site phosphorylated by Akt (RXRXX(S/T)), and recent studies have shown that Mdm2 can be phosphorylated by Akt at these sitesin vitro and in insulin-like growth factor-1-treated cells (29Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11598-11603Crossref PubMed Scopus (973) Google Scholar, 30Zhou B.P. Liao Y. Xia W. Zou Y. Spohn B. Hung M.C. Nat. Cell Biol. 2001; 3: 973-982Crossref PubMed Scopus (798) Google Scholar). The first site (Ser166) is not conserved across species; however the second site (Ser186) is conserved among species as far as we know, suggesting its possible functional importance. We confirmed that active Akt, but not kinase-negative Akt, was capable of inducing Mdm2 phosphorylation in vitro (Fig.3A). To further examine whether Akt phosphorylates Mdm2 at Ser186, we generated a polyclonal antibody that specifically recognizes phosphorylated Ser186 of Mdm2. Upon Western blot analysis, this antibody detected Mdm2 that had been phosphorylated by Akt in vitro(Fig. 3B). The specificity of this antibody was confirmed by its failure to recognize the Mdm2 mutant in which Ser186was mutated into Ala (S186A Mdm2) (Fig. 3B). By the use of anti-Ser(P)186 Mdm2 antibody, we found that serum stimulation increased Ser186 phosphorylation (Fig. 3C). The increase in Ser186phosphorylation was blocked by LY294002, suggesting that PI3K is required for serum induction of Ser186 phosphorylation (Fig. 3C). We also found that active Akt expression was sufficient for inducing Ser186 phosphorylation of Mdm2in vivo. In addition, expression of kinase-negative Akt blocked the serum induction of Ser186 phosphorylation (Fig.3D). These results strongly support the possibility that the PI3K-Akt pathway mediates Mdm2 phosphorylation at Ser186in vivo. We next asked whether Akt phosphorylation of Mdm2 at Ser186has any impact on Mdm2. We first examined whether Akt regulates the stability of Mdm2 protein. Western blot analysis indicated that expression of active Akt did not affect the levels of ectopically expressed Mdm2 (Fig. 4A). In addition, the levels of wild type and S186A mutant of Mdm2 were almost the same when expressed in MCF-7 cells (Fig. 4B). Therefore, Ser186 phosphorylation does not appear to alter the stability of Mdm2 protein. Because Ser186 is located close to the nuclear localization sequence and nuclear export signal of Mdm2 (residues 178–185 and 191–199, respectively), we asked whether Akt phosphorylation of Mdm2 alters its subcellular localization. In MCF-7 cells, endogenous Mdm2 was localized mainly in the nucleus and slightly in the cytoplasm (Fig.5A). In subcellular fractionation experiments, more than 90% of Mdm2 was found in the nuclear fraction (Fig. 5B). Serum and LY294002 treatment did not change the amounts of Mdm2 protein in the nuclear and cytoplasmic fractions (Fig. 5B). The localization of Mdm2 did not change upon serum or LY294002 treatment in immunostaining experiments either (Fig. 5A). Expression of active Akt did not induce nuclear translocation of Mdm2 as shown in Fig. 5 (B–D). Furthermore, we found that expression of a dominant-negative Akt (3A Akt) did not change the localization of endogenous Mdm2 in MCF-7 cells (Fig. 5C). Importantly, the localization of S186A Mdm2 as well as S166A/S186A Mdm2 was mainly in the nucleus, indistinguishable from that of wild type Mdm2 when expressed in Saos-2 cells (Fig.5D). Therefore, we conclude that Akt does not induce nuclear translocation of Mdm2 in our system, in apparent contradiction to the previous reports (29Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11598-11603Crossref PubMed Scopus (973) Google Scholar, 30Zhou B.P. Liao Y. Xia W. Zou Y. Spohn B. Hung M.C. Nat. Cell Biol. 2001; 3: 973-982Crossref PubMed Scopus (798) Google Scholar) (see “Discussion”). We then tested the possibility that Ser186 phosphorylation regulates the function of Mdm2. Mdm2 is known to promote p53 degradation by facilitating ubiquitination (19Fuchs S.Y. Adler V. Buschmann T., Wu, X. Ronai Z. Oncogene. 1998; 17: 2543-2547Crossref PubMed Scopus (213) Google Scholar). Because serum treatment increased Ser186phosphorylation (Fig. 3C), we examined the ability of Mdm2 to promote ubiquitination of p53 in the presence or absence of serum. To detect ubiquitination of p53, MCF-7 cells were transfected with FLAG-tagged ubiquitin and HA-tagged p53 and treated with a proteasome inhibitor for 2 h. p53 was immunoprecipitated and subjected to Western blot analysis with both anti-FLAG antibody and anti-p53 antibody to visualize the ubiquitination of p53. As shown in Fig.6 (A and B), serum treatment markedly enhanced the ubiquitination-inducing effect of Mdm2. This enhancement of p53 ubiquitination was reduced by LY294002 treatment (Fig. 6B), suggesting that serum enhancement of p53 ubiquitination is PI3K-dependent. We examined whether Akt is sufficient to enhance p53 ubiquitination. MCF-7 cells wer