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• W2015432978 abstract "The tumor suppressor p53 is commonly inhibited under conditions in which the phosphatidylinositide 3′-OH kinase/protein kinase B (PKB)Akt pathway is activated. Intracellular levels of p53 are controlled by the E3 ubiquitin ligase Mdm2. Here we show that PKB inhibits Mdm2 self-ubiquitination via phosphorylation of Mdm2 on Ser166 and Ser188. Stimulation of human embryonic kidney 293 cells with insulin-like growth factor-1 increased Mdm2 phosphorylation on Ser166 and Ser188 in a phosphatidylinositide 3′-OH kinase-dependent manner, and the treatment of both human embryonic kidney 293 and COS-1 cells with phosphatidylinositide 3′-OH kinase inhibitor LY-294002 led to proteasome-mediated Mdm2 degradation. Introduction of a constitutively active form of PKB together with Mdm2 into cells induced phosphorylation of Mdm2 at Ser166 and Ser188 and stabilized Mdm2 protein. Moreover, mouse embryonic fibroblasts lacking PKBα displayed reduced Mdm2 protein levels with a concomitant increase of p53 and p21Cip1, resulting in strongly elevated apoptosis after UV irradiation. In addition, activation of PKB correlated with Mdm2 phosphorylation and stability in a variety of human tumor cells. These findings suggest that PKB plays a critical role in controlling of the Mdm2·p53 signaling pathway by regulating Mdm2 stability. The tumor suppressor p53 is commonly inhibited under conditions in which the phosphatidylinositide 3′-OH kinase/protein kinase B (PKB)Akt pathway is activated. Intracellular levels of p53 are controlled by the E3 ubiquitin ligase Mdm2. Here we show that PKB inhibits Mdm2 self-ubiquitination via phosphorylation of Mdm2 on Ser166 and Ser188. Stimulation of human embryonic kidney 293 cells with insulin-like growth factor-1 increased Mdm2 phosphorylation on Ser166 and Ser188 in a phosphatidylinositide 3′-OH kinase-dependent manner, and the treatment of both human embryonic kidney 293 and COS-1 cells with phosphatidylinositide 3′-OH kinase inhibitor LY-294002 led to proteasome-mediated Mdm2 degradation. Introduction of a constitutively active form of PKB together with Mdm2 into cells induced phosphorylation of Mdm2 at Ser166 and Ser188 and stabilized Mdm2 protein. Moreover, mouse embryonic fibroblasts lacking PKBα displayed reduced Mdm2 protein levels with a concomitant increase of p53 and p21Cip1, resulting in strongly elevated apoptosis after UV irradiation. In addition, activation of PKB correlated with Mdm2 phosphorylation and stability in a variety of human tumor cells. These findings suggest that PKB plays a critical role in controlling of the Mdm2·p53 signaling pathway by regulating Mdm2 stability. The phosphatidylinositide 3′-OH kinase (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositide 3′-OH kinase; PKB, protein kinase B; m/p-PKB, myristoylated/palmitoylated PKB; Mdm2, murine double minute 2; HEK cells, human embryonic kidney cells; IGF-1, insulin-like growth factor-1; GST, glutathione S-transferase; MEF, mouse embryonic fibroblast; TRITC, tetramethylrhodamine isothiocyanate; E1, ubiquitin-activating enzyme; E3, ubiquitin ligase; BrdUrd, bromodeoxyuridine; HA, hemagglutinin; PBS, phosphate-buffered saline; WT, wild type; KD, kinase dead; GFP, green fluorescent protein.-PKB/Akt pathway is a key component of growth factor-induced cell survival. This pathway has been implicated in suppressing apoptosis in a number of cell types in response to a variety of stimuli, including growth factor withdrawal, cell cycle discordance, loss of cell adhesion, DNA damage, and treatment with anti-Fas antibody or transforming growth factor β (1Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar, 2Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar, 3Brunet A. Datta S.R. Greenberg M.E. Curr. Opin. Neurobiol. 2001; 11: 297-305Crossref PubMed Scopus (1016) Google Scholar). A number of PKB substrates that are components of the intrinsic cell death machinery have been identified, including glycogen synthase kinase-3, the Bcl2 family member BAD, the protease caspase-9, and Forkhead transcription factors (1Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar, 2Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (876) Google Scholar, 3Brunet A. Datta S.R. Greenberg M.E. Curr. Opin. Neurobiol. 2001; 11: 297-305Crossref PubMed Scopus (1016) Google Scholar, 4Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar). In each case, phosphorylation of these proteins by PKB suppresses their pro-apoptotic function. Stimulation of cells with survival factors causes activation and nuclear translocation of PKB in target cells (5Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar, 6Meier R. Alessi D.R. Cron P. Andjelkovic M. Hemmings B.A. J. Biol. Chem. 1997; 272: 30491-30497Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), suggesting that PKB may modulate nuclear targets. Several recent studies show that p53-mediated apoptosis is inhibited under conditions in which the PI3K/PKB pathway is activated (7Sabbatini P. McCormick F. J. Biol. Chem. 1999; 274: 24263-24269Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 8Yamaguchi 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 (210) Google Scholar, 9Hong M. Lai M.D. Lin Y.S. Lai M.Z. Cancer Res. 1999; 59: 2847-2852PubMed Google Scholar, 10Mazzoni I.E. Said F.A. Aloyz R. Miller F.D. Kaplan D. J. Neurosci. 1999; 19: 9716-9727Crossref PubMed Google Scholar, 11Gottlieb T.M. Leal J.F. Seger R. Taya Y. Oren M. Oncogene. 2002; 21: 1299-1303Crossref PubMed Scopus (383) Google Scholar). The p53 gene product is a tumor suppressor that mediates growth arrest, senescence, and apoptosis in response to several cellular stresses (12Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5850) Google Scholar). Protein levels of p53 are the most important determinant of its functions. In normal unstressed cells, p53 is an unstable protein with a half-life of less than 20 min, which is kept at very low cellular levels because of continuous degradation mediated largely by Mdm2 (13Momand J. Wu H.H. Dasgupta G. Gene (Amst.). 2000; 242: 15-29Crossref PubMed Scopus (530) Google Scholar, 14Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 15Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3750) Google Scholar, 16Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2860) Google Scholar, 17Piette J. Neel H. Maréchal V. Oncogene. 1997; 15: 1001-1010Crossref PubMed Scopus (241) Google Scholar, 18Moll U.M. Petrenko O. Mol. Cancer Res. 2003; 1: 1001-1008PubMed Google Scholar). Mdm2 was originally identified on double-minute chromosomes of spontaneous transformed mouse 3T3 fibroblasts (19Cahilly-Snyder L. Yang-Feng T. Francke U. George D.L. Somatic Cell Mol. Genet. 1987; 13: 235-244Crossref PubMed Scopus (306) Google Scholar). Mdm2 harbors a self- and p53-specific E3 ubiquitin ligase activity within its evolutionarily conserved C-terminal RING finger domain, which mediates p53 ubiquitination and rapid degradation by the 26 S proteasome (20Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (618) Google Scholar, 21Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). The current model places p53 and Mdm2 in an autoregulatory feedback loop; Mdm2 transcription is induced by p53, and Mdm2 in turn binds to the N-terminal transactivation domain of p53, thereby inactivating p53 transcriptional activity (12Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5850) Google Scholar, 13Momand J. Wu H.H. Dasgupta G. Gene (Amst.). 2000; 242: 15-29Crossref PubMed Scopus (530) Google Scholar, 14Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 17Piette J. Neel H. Maréchal V. Oncogene. 1997; 15: 1001-1010Crossref PubMed Scopus (241) Google Scholar, 18Moll U.M. Petrenko O. Mol. Cancer Res. 2003; 1: 1001-1008PubMed Google Scholar, 20Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (618) Google Scholar, 21Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar, 22Michael D. Oren M. Curr. Opin. Genet. Dev. 2002; 12: 53-59Crossref PubMed Scopus (244) Google Scholar, 23Lahav G. Rosenfeld N. Sigal A. Geva-Zatorsky N. Levine A.J. Elowitz M.B. Alon U. Nat. Genet. 2004; 36: 147-150Crossref PubMed Scopus (822) Google Scholar). Low levels of Mdm2 activity induce mono-ubiquitination and nuclear export of p53, whereas high levels promote polyubiquitination and nuclear degradation of p53 (24Li M. Brooks C.L. Wu-Baer F. Chen D. Baer R. Gu W. Science. 2003; 302: 1972-1975Crossref PubMed Scopus (643) Google Scholar). The levels of Mdm2 expression, the association of Mdm2 with p53, and the E3 ubiquitin ligase activity of Mdm2 that down-regulates p53 are regulated by phosphorylation (25Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar), oligomerization (26Maki C.G. J. Biol. Chem. 1999; 274: 16531-16535Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and binding to other factors such as p19/p14ARF (27Kamijo T. Weber J.D. Zambetti G. Zindy F. Roussel M.F. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8292-8297Crossref PubMed Scopus (789) Google Scholar), MdmX (28Sharp D.A. Kratowicz S.A. Sank M.J. George D.L. J. Biol. Chem. 1999; 274: 38189-38196Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), and hHR23A (29Brignone C. Bradley K.E. Kisselev A.F. Grossman S.R. Oncogene. 2004; 23: 4121-4129Crossref PubMed Scopus (61) Google Scholar). More recent data showed that Mdm2 is directly regulated by a deubiquitinase (HAUSP), which reveals a dynamic role of ubiquitination and deubiquitination of Mdm2 in the Mdm2·p53 pathway (30Li M. Brooks C.L. Kon N. Gu W. Mol. Cell. 2004; 13: 879-886Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). Expression of active or kinase-defective PKB has little or no effect on the phosphorylation status of p53 (8Yamaguchi 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 (210) Google Scholar), suggesting that p53 does not serve as a direct substrate for PKB, thus raising the possibility that Mdm2 itself might be the principal target of the PI3K/PKB signaling pathway. Indeed, it has been reported that Mdm2 could be phosphorylated by PKB in vitro at Ser166 and Ser186, and this phosphorylation promotes the nuclear entry of Mdm2 (31Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11598-11603Crossref PubMed Scopus (960) Google Scholar, 32Zhou B.P. Liao Y. Xia W. Zou Y. Spohn B. Hung M.C. Nat. Cell Biol. 2001; 3: 973-982Crossref PubMed Scopus (788) Google Scholar). Here we report that PKB phosphorylates Mdm2 on a previously reported site, Ser166 (31Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11598-11603Crossref PubMed Scopus (960) Google Scholar, 32Zhou B.P. Liao Y. Xia W. Zou Y. Spohn B. Hung M.C. Nat. Cell Biol. 2001; 3: 973-982Crossref PubMed Scopus (788) Google Scholar), and on a novel site, Ser188, in vitro and in vivo. Phosphorylation of Mdm2 by PKB leads to inhibition of Mdm2 self-ubiquitination and stabilization of Mdm2 by protecting Mdm2 from proteasome-dependent degradation. Thus, our results suggest that PKB-dependent phosphorylation of Mdm2 leads to the increase of Mdm2 stability and a consequent down-regulation of p53. Materials and Chemicals—Chemicals and venders were as follows: [γ-32P]ATP (Amersham Biosciences); ATP bromodeoxyuridine (BrdUrd) and cycloheximide (Sigma); E1 ubiquitin-activating enzyme, LY-294002, and MG132 (Calbiochem); microcystin-LR (Alexis); insulin-like growth factor-1 (IGF-1) (Invitrogen); protein kinase A inhibitor peptide (Bachem); monoclonal antibody to Mdm2 (SMP-14, Santa Cruz Inc.); monoclonal antibody to p53 (PAb421) and polyclonal anti-p21Cip1 (Ab4) (Oncogene Science); monoclonal antibody to HA (12CA5, Roche Applied Science); polyclonal anti-phospho-Ser473 PKB antibody (Ser(P)473, Cell Signaling); polyclonal anti-PKB (Ab10) was described previously (5Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar); TRITC-conjugated anti-rabbit IgG antibody (Chemicon); fluorescein isothiocyanate-conjugated anti-BrdUrd antibody (BD Biosciences). Preparation of Plasmids and Proteins—pCMV5-HA-PKB expression vectors have been described (5Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). pGEX2T-Mdm2 was made by inserting the BamHI-EcoRI fragment of human Mdm2 cDNA released from pcDNA3-Mdm2 (a kind gift of Dr. C. G. Maki, University of Chicago) into pGEX2T vector (Amersham Biosciences), The same fragment was also ligated into the BglI-EcoRI sites of pEGFP-C1 (Invitrogen) vector to generate pEGFP-Mdm2. The Mdm2 mutants were generated using the QuikChange kit (Stratagene). pGEX2T-UbcH5 and pGEX2KT-ubiquitin were obtained from Dr. W. Krek (Eidgenössische Technische Hochschule, Zürich, Switzerland). All constructs were verified by DNA sequencing. GST fusion proteins were expressed in Escherichia coli BL21 strain and purified on glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions. Human ΔPH-PKBα cDNA (118–480) was released from pMV1 vector as a EcoRI-EcoRI fragment and was inserted into pFastBacHTc vector (Invitrogen) to generate pFastBacHTc.ΔPH-PKBα B. After transposition into the DH10Bac competent cells, the recombinant Bacmid DNA was isolated and then transfected into Sf9 cells. The resulting recombinant baculovirus particles were used to infect Sf9 cells, and the expressed His-tagged ΔPH-PKBα protein was purified on nickel nitrilotriacetic acid resin according to the manufacturer's instructions (Qiagen). Production of Antibodies—The peptides Ser160-Arg-Arg-Arg-Ala-Ile-Ser166-Glu-Thr-Glu-Glu-Asn-Ser-Asp173, Ser160-Arg-Arg-Arg-Ala-Ile-phospho-Ser166-Glu-Thr-Glu-Glu-Asn-Ser-Asp173, Lys182-Arg-His-Lys-Ser-Asp-Ser188-Ile-Ser-Leu-Ser-Phe-Asp-Glu195, and Lys182-Arg-His-Lys-Ser-Asp-phospho-Ser188-Ile-Ser-Leu-Ser-Phe-Asp-Glu195 were synthesized by Neosystem (Strasbourg, France). The rabbit polyclonal anti-phosphopeptide antibodies were prepared and affinity-purified using a nonphosphopeptide affinity column followed by a phosphopeptide affinity column. These antibodies are referred to as Ser(P)166 and Ser(P)188. The polyclonal antibody against Mdm2 was raised by immunizing rabbits with GST-Mdm2 (full-length of human Mdm2) and affinity-purified using a GST-coupled Sepharose column followed by GST-Mdm2-coupled Sepharose column. This antibody is referred to as anti-Mdm2. Cell Culture, Transfections, Immunoprecipitation, and Immunofluoresence—Generation of PKBα knockout mouse has been described in Yang et al. (33Yang Z.Z. Tschopp O. Hemmings-Mieszczak M. Feng J. Brodbeck D. Perentes E. Hemmings B.A. J. Biol. Chem. 2003; 278: 32124-32131Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). MEFs were prepared from E13.5 embryos generated by the Pkbα+/+ wild type (Pkbα+/+) or knockout (Pkbα–/–) intercrosses as described previously (33Yang Z.Z. Tschopp O. Hemmings-Mieszczak M. Feng J. Brodbeck D. Perentes E. Hemmings B.A. J. Biol. Chem. 2003; 278: 32124-32131Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 34Kamijo T. Zindy F. Roussel M.F. Quelle D.E. Downing J.R. Ashmun R.A. Grosveld G. Sherr C.J. Cell. 1997; 91: 649-659Abstract Full Text Full Text PDF PubMed Scopus (1390) Google Scholar). The genotype of each embryo was verified by PCR (data not shown). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Transfections were performed by calcium phosphate precipitation (5Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). Cells were lysed in Nonidet P-40 lysis buffer and immunoprecipitated exactly as described (5Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). For cell immunofluoresence, the cells were fixed with 3.7% formaldehyde and permeabilized with 0.2% Triton X-100. After blocking, cells were incubated with monoclonal anti-Mdm2 antibody (SMP14, Santa Cruz Inc., 2 μg/ml diluted in PBS) for 2 h followed with polyclonal Mdm2 phospho-antibodies (2 μg/ml diluted in PBS) for an additional 2 h. Cells were then extensively washed and incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG and TRITC-conjugated anti-rabbit IgG (both 1:100 dilution). After washing, the cells were visualized by confocal microscopy (Olympus BX61). Cell Cycle and Cell Death Analysis—Exponentially growing MEFs were labeled for 30 min with 10 μm BrdUrd (Sigma). Subsequently, the cells were exposed to 120 J/m2 UV-C irradiation (UV Stratalinker 1800; Stratagene) or left untreated, harvested at indicated intervals by trypsinization, and fixed with 70% ethanol at –20 °C. Cells were then (i) incubated in 2 n HCl containing 0.2 mg/ml porcine pepsin (Sigma) for 20 min at room temperature, (ii) neutralized with 0.1 m sodium tetraborate (Sigma) and washed twice in PBS, (iii) incubated for 1 h at room temperature in PBS containing 0.5% Tween 20, 1% bovine serum albumin, and 1:50 (v/v) fluorescein isothiocyanate-labeled anti-BrdUrd mouse monoclonal antibody (BD Biosciences), and (iv) washed in PBS and resuspended in PBS containing 20 μg/ml propidium iodide (Sigma). Bivariatic flow cytometry analysis was performed on fluorescence-activated cell analyzer (FACSCalibur; BD Biosciences) equipped with an air-cooled 15-milliwatt 488 nm argon ion laser. Collected data were analyzed for cell cycle distribution and cell death rate using the Cell Quest software (BD Biosciences). In Vitro PKB Kinase Assay—Phosphorylation of Mdm2 and mutants were carried out at 30 °C for different times in a final volume of 50 μl of kinase assay buffer containing 50 μg/ml GST-Mdm2 or mutants and PKB as indicated in each experiment. The reactions were started by the addition of 100 μm [γ-32P]ATP and terminated by the addition of SDS sample buffer and immediately boiled for 3 min. Samples were then subjected to 8% SDS-PAGE followed by autoradiography or 32P determination in excised gel slices at positions corresponding to Mdm2. To determine the effect of phosphorylation on Mdm2 self-ubiquitination, phosphorylation of Mdm2 was carried out under the similar conditions using nonradioactive ATP instead of [γ-32P]ATP. At each time point the reaction was terminated by the addition of 10 mm EDTA (final) and 20 μl of 50% glutathione-Sepharose 4B. After extensive washing, the bead-bound GST-Mdm2 was used for ubiquitination assay. In Vitro Ubiquitination Assay—32P-Labeling of GST-ubiquitin and ubiquitination assay were carried out as described previously (35Tan P. Fuchs S.Y. Chen A. Wu K. Gomez C. Ronai Z. Pan Z.Q. Mol. Cell. 1999; 3: 527-533Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Assay conditions were 30 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 1 mm dithiothreitol, 2 mm ATP, 15 ng of E1 ubiquitin-activating enzyme, 0.5 μg of GST-UbcH5, and 1.5 μg of GST-Mdm2 or mutants in a final volume of 50 μl. The reactions were started by the addition of 2 μg of 32P-labeled GST-ubiquitin and incubated at 37 °C for 1 h. GST-Mdm2 was then precipitated by glutathione-Sepharose 4B and eluted in SDS sample buffer. The samples were separated by 8% SDS-PAGE and then analyzed by autoradiography. Effect of Phosphorylation on Mdm2 Self-ubiquitination—We identified two major phosphorylation sites of Mdm2 on Ser166 and Ser188 by PKB in vitro (data not shown). Residue Ser166 has been previously described as a PKB site (31Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11598-11603Crossref PubMed Scopus (960) Google Scholar, 32Zhou B.P. Liao Y. Xia W. Zou Y. Spohn B. Hung M.C. Nat. Cell Biol. 2001; 3: 973-982Crossref PubMed Scopus (788) Google Scholar), whereas Ser188 was identified as a novel PKB site here for the first time. We also confirmed the sites of phosphorylation by in vitro kinase assay using peptides corresponding to the two sites as substrates (data not shown). We failed to detect phosphorylation on Ser186, which was previously described by other groups (31Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11598-11603Crossref PubMed Scopus (960) Google Scholar, 32Zhou B.P. Liao Y. Xia W. Zou Y. Spohn B. Hung M.C. Nat. Cell Biol. 2001; 3: 973-982Crossref PubMed Scopus (788) Google Scholar). The region surrounding Ser166 and Ser188 in human Mdm2 conforms to a consensus sequence motif for PKB phosphorylation, i.e. RXRXX(S/T) (36Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (552) Google Scholar). It was also found that Ser166 and Ser188 are the best predicted sites for PKB using the Scansite motif-profile scoring algorithm program generated by Yaffe and Cantley (Ref. 37Yaffe M.B. Leparc G.C. Lai J. Obata T. Volinia S. Cantley L.C. Nat. Biotechnol. 2001; 19: 348-353Crossref PubMed Scopus (465) Google Scholar, scansite.mit.edu). Because Mdm2 is capable of self-ubiquitination and the ubiquitin proteasome pathway-dependent degradation is an important mechanism for regulating Mdm2 levels in cells (20Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (618) Google Scholar, 21Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar), we asked whether PKB-induced phosphorylation of Mdm2 might have an effect on its ubiquitination. We examined the effect of phosphorylation on reconstituted Mdm2 self-ubiquitination in vitro. As shown in Fig. 1A, Mdm2 self-ubiquitination was observed only in the presence of both ubiquitin-activating enzyme E1 and the ubiquitin-conjugating enzyme E2, UbcH5. The degree of Mdm2 self-ubiquitination was reduced over time by PKB-mediated phosphorylation. To determine which phosphorylation site was responsible for this reduction of Mdm2 self-ubiquitination, two mutants, i.e. GST-Mdm2S166A and GST-Mdm2S188V (in which the serine residues were individually changed to alanine or valine), were first phosphorylated by recombinant ΔPH-PKBα and then subjected to the ubiquitination assay. As shown in Fig. 1B, the non-phosphorylated forms of Mdm2 and the mutants were all ubiquitinated in the presence of both E1 and UbcH5. Phosphorylation of wild-type GST-Mdm2 completely abolished Mdm2 self-ubiquitination. However, phosphorylation of either GST-Mdm2S166A or GST-Mdm2S188V only partially inhibited Mdm2 self-ubiquitination. The Mdm2S188A and Mdm2S166A/S188V double mutant were highly degraded in E. coli and could not be tested. Three Asp mutants (GST-Mdm2S166D, GST-Mdm2S188D, and GST-Mdm2S166D/S188D) were also generated and tested in this in vitro ubiquitination assay. As shown in Fig. 1C, all three Asp mutants were poorly ubiquitinated, further supporting the idea that the protective effect of PKB on Mdm2 self-ubiquitination may be due primarily to the phosphorylation of Mdm2 at Ser166 and Ser188. Production and Characterization of Mdm2 Phospho-specific Antibodies—To monitor the phosphorylation of Mdm2 in vivo, two rabbit polyclonal antibodies that recognized phosphorylated Mdm2 at Ser166 and Ser188 were prepared. These antibodies are referred to as Ser(P)166 and Ser(P)188, respectively. The specificity of the purified antibodies was evaluated by immunoblotting. As shown in Fig. 2A, GST-Mdm2 phosphorylated by either pervanadate-activated HA-PKBα (WT, van) or recombinant ΔPH-PKBα was recognized by each antibody. Non-phosphorylated GST-Mdm2 or GST-Mdm2 phosphorylated by either untreated HA-PKBα (WT) or kinase-defective HA-PKBα (KD) was not detected. The Ser(P)166 antibody detected both GST-Mdm2 and GST-Mdm2S188V phosphorylated by ΔPH-PKBα but did not detect GST-Mdm2S166A (Fig. 2A). Similarly, Ser(P)188 could also detect GST-Mdm2 and GST-Mdm2S166A but not GST-Mdm2S188V when phosphorylated by ΔPH-PKBα. These results suggest that each phospho antibody specifically detected phosphorylation of Mdm2 by PKB at Ser166 and Ser188, respectively. IGF-1 Induces in Vivo Phosphorylation of Mdm2 at Ser166 and Ser188—To determine whether our phospho antibodies could detect Mdm2 phosphorylation in vivo, the effect of IGF-1 on HEK293 cells was examined. Serum-starved cells were incubated with 100 ng/ml IGF-1 for different times, and cell lysates were analyzed by immunoblotting using Ser(P)166 and Ser(P)188. After IGF-1 stimulation a dramatic increase in phosphorylation on Mdm2 residues Ser166 and Ser188 was observed, and a relatively high level of phosphorylation was maintained for the 30-min duration of the experiment (Fig. 2B). The IGF-1-induced phosphorylation of Mdm2 was suppressed by pretreatment with 50 μm LY-294002, an inhibitor of PI3K (Fig. 2B). These results suggest that the activation of PI3K/PKB pathway is required for Mdm2 phosphorylation at Ser166 and Ser188in vivo. PKB Promotes Mdm2 Phosphorylation in Vivo and Prevents Its Degradation—To test whether Mdm2 is phosphorylated in transfected cells, a pEGFP-Mdm2 was co-transfected with pCMV5-HA-m/p-PKBα (m/p-PKBα, a constitutively active membrane-targeted myristoylated/palmitoylated PKBα), pCMV5-HA-KD-PKBα (KD-PKBα, a kinase-deficient PKBα), or pCMV5 into COS-1 cells as described under “Experimental Procedures.” The expression of m/p-PKBα but not KD-PKBα caused an increase in the phosphorylation level of GFP-Mdm2 at both Ser166 and Ser188 (Fig. 3A), indicating that PKB kinase activity is required for the phosphorylation of Mdm2 at Ser166 and Ser188in vivo. Significantly, the level of GFP-Mdm2 expression was lower in the mock or KD-PKBα-transfected cells but remained high in the m/p-PKBα-transfected cells (Fig. 3A, left panel). To explore whether the lower levels of Mdm2 were due to degradation of protein, transfected cells were exposed to the proteasome inhibitor MG132. As shown in the right panel of Fig. 3A, the reduction of Mdm2 protein levels in both mock and KD-PKBα-transfected cells was reversed upon MG132 treatment and reached a similar level to that of the m/p-PKBα-transfected cells. The efficiency of transfection was monitored by co-expression of GFP as indicated. Lysates were probed with anti-phospho-Ser473 antibody (Ser(P)473) to confirm the activation status of PKB. These data suggested that transfected GFP-Mdm2 is a labile protein that is a target for degradation (20Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (618) Google Scholar, 21Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar) and, furthermore, that PKB phosphorylation is required for protecting Mdm2 from proteasome-dependent destruction, thus increasing Mdm2 stability. To further confirm the hypothesis that PKB activity is sufficient to stabilize Mdm2, GFP-Mdm2 was co-transfected with m/p-PKBα, KD-PKBα, or pCMV5 empty vector in COS-1 cells. The transfected cells were treated with a protein synthesis inhibitor cycloheximide for the indicated period of time before harvesting. Levels of GFP-Mdm2 were markedly decreased in mock or KD-PKBα-cotransfected cells but remained relatively high in m/p-PKBα co-transfected cells over time (Fig. 3B). This was not a result of variations in transfection efficiency as shown by the levels of GFP control. These data suggest that maintenance of a higher level of GFP-Mdm2 in the m/p-PKBα-cotransfected cells may be primarily due to prevention of Mdm2 protein degradation by PKB rather than activation of transcriptional machinery. To investigate whether phosphorylation of Mdm2 by PKB was sufficient to increase its stability, three Ser to Asp phosphorylation site mutants of Mdm2 were constructed and transfected into COS-1 cells. As shown in Fig. 3C, wild-type GFP-Mdm2 (WT) was rapidly degraded in the absence of MG132 but markedly accumulated in the presence of this inhibitor. Substitution of Ser166 to Asp (GFP-Mdm2S166D) and Ser188 to Asp (GFP-Mdm2S188D) resulted in increased Mdm2 stability even in the absence of MG132. A double mutant in which both Ser166 and Ser188 were changed to Asp (GFP-Mdm2S166D/S188D, DD) was tested and was as stable as each single mutant. These results indicate that phosphorylation of Mdm2 at Ser166 and Ser188 b" @default.
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• W2015432978 title "Stabilization of Mdm2 via Decreased Ubiquitination Is Mediated by Protein Kinase B/Akt-dependent Phosphorylation" @default.
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