c-Abl Phosphorylates Hdmx and Regulates Its Interaction with p53
2008; Elsevier BV; Volume: 284; Issue: 6 Linguagem: Inglês
10.1074/jbc.m809211200
ISSN1083-351X
AutoresValentina Zuckerman, Kristiaan J. Lenos, Grzegorz M. Popowicz, Isabelle Silberman, Tamar Grossman, Jean‐Christophe Marine, Tad A. Holak, Aart G. Jochemsen, Ygal Haupt,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoUpon exposure to DNA damage the p53 tumor suppressor is accumulated and activated to stall cellular growth. For this to occur, p53 must be relieved from its major inhibitors, Mdm2 (Hdm2 in humans) and Mdmx (Mdm4; Hdmx in humans). A key mechanism controlling this relief is the post-translational modifications of p53 and its inhibitors. We have previously demonstrated that the stress-activated tyrosine kinase, c-Abl, contributes to the relief of p53 from Hdm2. Because Hdmx is the major inhibitor of p53 activity, the additional possibility that c-Abl protects p53 through targeting Hdmx was explored in this study. c-Abl was found to interact with and to phosphorylate Hdmx. This phosphorylation was enhanced in response to DNA damage. Importantly, we mapped the sites of phosphorylation to the p53 binding domain of Hdmx. One of these phosphorylations, on tyrosine 99, inhibited Hdmx interaction with p53. This inhibition is consistent with the predicted role of this residue in the interaction with p53 based on the crystal structure of the interaction site. Our results show that c-Abl not only targets Hdm2, but also Hdmx, which together contribute to p53 activation in response to DNA damage. Upon exposure to DNA damage the p53 tumor suppressor is accumulated and activated to stall cellular growth. For this to occur, p53 must be relieved from its major inhibitors, Mdm2 (Hdm2 in humans) and Mdmx (Mdm4; Hdmx in humans). A key mechanism controlling this relief is the post-translational modifications of p53 and its inhibitors. We have previously demonstrated that the stress-activated tyrosine kinase, c-Abl, contributes to the relief of p53 from Hdm2. Because Hdmx is the major inhibitor of p53 activity, the additional possibility that c-Abl protects p53 through targeting Hdmx was explored in this study. c-Abl was found to interact with and to phosphorylate Hdmx. This phosphorylation was enhanced in response to DNA damage. Importantly, we mapped the sites of phosphorylation to the p53 binding domain of Hdmx. One of these phosphorylations, on tyrosine 99, inhibited Hdmx interaction with p53. This inhibition is consistent with the predicted role of this residue in the interaction with p53 based on the crystal structure of the interaction site. Our results show that c-Abl not only targets Hdm2, but also Hdmx, which together contribute to p53 activation in response to DNA damage. Tight regulation of the p53 protein is critical for tumor suppression, for a proper cellular response to stress, and for mouse development (1Vousden K.H. Lane D.P. Nat. Rev. Mol. Cell Biol.. 2007; 8: 275-283Google Scholar). P53 stability and activity are regulated by two major inhibitors, Mdm2 and Mdmx (Mdm4; Hdmx in humans), respectively. The importance of the Mdm genes as p53 regulators was best exemplified by the demonstration that elimination of p53 fully rescues mdm2 and mdmx null embryos from lethality (2Marine J.C. Dyer M.A. Jochemsen A.G. J. Cell Sci.. 2007; 120: 371-378Google Scholar). Mdm2 acts as the direct E3 ligase of p53, promoting it for proteasomal degradation and nuclear export (3Michael D. Oren M. Semin. Cancer Biol.. 2003; 13: 49-58Google Scholar). Unlike Mdm2, Mdmx lacks detectable ubiquitin E3 ligase activity and, hence, does not target p53 for degradation but, rather, inhibits p53 transcriptional activity by binding to its transactivation domain (for review, see Ref. 4Marine J.C. Jochemsen A.G. Biochem. Biophys. Res. Commun.. 2005; 331: 750-760Google Scholar). Down-regulation or mutations in Mdmx increase the induction of p53 target genes without a change in p53 expression levels (4Marine J.C. Jochemsen A.G. Biochem. Biophys. Res. Commun.. 2005; 331: 750-760Google Scholar, 5Marine J.C. Francoz S. Maetens M. Wahl G. Toledo F. Lozano G. Cell Death Differ.. 2006; 13: 927-934Google Scholar). The importance of Mdmx as a regulator of p53 is further demonstrated by its link to cancer development. Hdmx is overexpressed or amplified in 10-20% of human cancers including breast, stomach, colon, and lung cancers (for review, see Ref. 6Toledo F. Wahl G.M. Nat. Rev. Cancer.. 2006; 6: 909-923Google Scholar) and in 65% of retinoblastomas (7Laurie N.A. Donovan S.L. Shih C.S. Zhang J. Mills N. Fuller C. Teunisse A. Lam S. Ramos Y. Mohan A. Johnson D. Wilson M. Rodriguez-Galindo C. Quarto M. Francoz S. Mendrysa S.M. Guy R.K. Marine J.C. Jochemsen A.G. Dyer M.A. Nature.. 2006; 444: 61-66Google Scholar). Interestingly, in papillary thyroid carcinomas Hdmx expression is down-regulated, although certain Hdmx variants with p53 inhibitory properties were abnormally expressed in these tumors (8Prodosmo A. Giglio S. Moretti S. Mancini F. Barbi F. Avenia N. Di Conza G. Schunemann H.J. Pistola L. Ludovini V. Sacchi A. Pontecorvi A. Puxeddu E. Moretti F. J. Mol. Med.. 2008; 86: 585-596Google Scholar). Importantly, overexpression of Mdmx correlates well with a normal status of p53, supporting the notion that this inhibitory pathway is sufficient to suppress p53 and, thus, negates the need for p53 mutation. Thus, abrogating Mdmx function may be an important approach for reactivating p53 in cancer cells (for review, see Ref. 2Marine J.C. Dyer M.A. Jochemsen A.G. J. Cell Sci.. 2007; 120: 371-378Google Scholar).Mdm2 and Mdmx form homo- and heterodimers through their RING domains (9Tanimura S. Ohtsuka S. Mitsui K. Shirouzu K. Yoshimura A. Ohtsubo M. FEBS Lett.. 1999; 447: 5-9Google Scholar, 10Sharp D.A. Kratowicz S.A. Sank M.J. George D.L. J. Biol. Chem.. 1999; 274: 38189-38196Google Scholar); the ratio of these forms affect their protein stability and the extent of p53 inhibition (4Marine J.C. Jochemsen A.G. Biochem. Biophys. Res. Commun.. 2005; 331: 750-760Google Scholar, 11Gu J. Kawai H. Nie L. Kitao H. Wiederschain D. Jochemsen A.G. Parant J. Lozano G. Yuan Z.M. J. Biol. Chem.. 2002; 277: 19251-19254Google Scholar). An important link between the two proteins has been reported under stress conditions. After DNA damage, Mdmx accumulates in the nucleus (11Gu J. Kawai H. Nie L. Kitao H. Wiederschain D. Jochemsen A.G. Parant J. Lozano G. Yuan Z.M. J. Biol. Chem.. 2002; 277: 19251-19254Google Scholar, 12Li C. Chen L. Chen J. Mol. Cell. Biol.. 2002; 22: 7562-7571Google Scholar) where it inhibits p53 activity (4Marine J.C. Jochemsen A.G. Biochem. Biophys. Res. Commun.. 2005; 331: 750-760Google Scholar, 13Migliorini D. Lazzerini Denchi E. Danovi D. Jochemsen A. Capillo M. Gobbi A. Helin K. Pelicci P.G. Marine J.C. Mol. Cell. Biol.. 2002; 22: 5527-5538Google Scholar), and it is subsequently degraded in an Mdm2-dependent manner (14Pan Y. Chen J. Mol. Cell. Biol.. 2003; 23: 5113-5121Google Scholar, 15de Graaf P. Little N.A. Ramos Y.F. Meulmeester E. Letteboer S.J. Jochemsen A.G. J. Biol. Chem.. 2003; 278: 38315-38324Google Scholar, 16Kawai H. Wiederschain D. Kitao H. Stuart J. Tsai K.K. Yuan Z.M. J. Biol. Chem.. 2003; 278: 45946-45953Google Scholar). Mdmx degradation together with the self-degradation of Mdm2 contributes considerably to p53 activation and stabilization upon stress (for review, see Ref. 17Toledo F. Wahl G.M. Int. J. Biochem. Cell Biol.. 2007; 39: 1476-1482Google Scholar).An important mechanism leading to p53 activation in response to DNA damage involves post-translational modifications, which are mediated by several upstream positive regulators (for review, see Ref. 18Meek D.W. DNA Repair (Amst).. 2004; 3: 1049-1056Google Scholar). One such activator is the c-Abl non-receptor tyrosine kinase (19Levav-Cohen Y. Goldberg Z. Zuckerman V. Grossman T. Haupt S. Haupt Y. Biochem. Biophys. Res. Commun.. 2005; 331: 737-749Google Scholar). c-Abl has been implicated in the cellular response to stress by promoting cell cycle arrest or apoptosis (20Shaul Y. Ben-Yehoyada M. Cell Res.. 2005; 15: 33-35Google Scholar, 21Zhu J. Wang J.Y. Curr. Top. Dev. Biol.. 2004; 59: 165-192Google Scholar). c-Abl is critical for the accumulation of p53 in response to DNA damage (22Sionov R.V. Coen S. Goldberg Z. Berger M. Bercovich B. Ben-Neriah Y. Ciechanover A. Haupt Y. Mol. Cell. Biol.. 2001; 21: 5869-5878Google Scholar). c-Abl activates p53 by neutralizing the inhibitory effects of Hdm2 (22Sionov R.V. Coen S. Goldberg Z. Berger M. Bercovich B. Ben-Neriah Y. Ciechanover A. Haupt Y. Mol. Cell. Biol.. 2001; 21: 5869-5878Google Scholar, 23Sionov R.V. Moallem E. Berger M. Kazaz A. Gerlitz O. Ben-Neriah Y. Oren M. Haupt Y. J. Biol. Chem.. 1999; 274: 8371-8374Google Scholar, 24Goldberg Z. Vogt Sionov R. Berger M. Zwang Y. Perets R. Van Etten R.A. Oren M. Taya Y. Haupt Y. EMBO J.. 2002; 21: 3715-3727Google Scholar, 25Dias S.S. Milne D.M. Meek D.W. Oncogene.. 2006; 25: 6666-6671Google Scholar). Given the important inhibitory role of Mdmx in p53 regulation and the link between c-Abl and Hdm2, we asked whether c-Abl also protects p53 through targeting Hdmx. We report here a physical and biochemical link between c-Abl and Hdmx. c-Abl interacts with and phosphorylates Hdmx. These phosphorylations are enhanced in response to stress. Tyrosine phosphorylations of Hdmx were mapped to the p53 binding domain of Hdmx, and one of these phosphorylations inhibits p53 binding. Overall, our results reveal that Hdmx is a new target of c-Abl, which together with Hdm2 contributes to p53 activation in response to stress.EXPERIMENTAL PROCEDURESCell Lines, Transfection Assays, and Cell Treatments—H1299 lung adenocarcinoma cells, lacking p53 expression, were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. 293 and 293T human embryo kidney epithelial cells, MCF-7 breast carcinoma cells, and mdm2/p53 double null (2KO) fibroblasts were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected by the calcium phosphate method as described previously (26Haupt Y. Barak Y. Oren M. EMBO J.. 1996; 15: 1596-1606Google Scholar). Transfections using polyethyleneimine (PEI; Sigma) were done by mixing DNA with PEI reagent in serum-free medium for 5 min. 1 μl of polyethyleneimine (1 mg/ml) was used for 1 μg of DNA. The polyethyleneimine-DNA complex was applied to the cells for 5-7 h before the medium was replaced. Transfections with FuGENE 6 (Roche Applied Science) were performed in 9-cm dishes according to the manufacturer's instructions. The FuGENE 6-DNA complex was applied to the cells for 24 h before the medium was replaced. A constant amount of plasmid DNA in each sample was maintained by supplementing with empty plasmid. Usually, cells were transfected with 5-8 μg of the indicated plasmid for a 9-cm plate and with 2-5 μg for a 5-cm plate.For measuring the cellular response to stress, cells were treated with 3 mm H2O2 (Merck, Germany) together with 1 mm sodium orthovanadate (Sigma) for the indicated times. To induce DNA damage, cells were treated with either 2-2.5 μg/ml doxorubicin hydrochloride (Sigma) or 500 ng/ml neocarzinostatin (Sigma) in the dark. To inhibit proteasomal degradation, the proteasome inhibitor MG132 (Sigma) was added for 4 h.Plasmids, in Vitro Site-directed Mutagenesis, and Generation of Lentivirus—Expression plasmids used in this study were human wild-type (wt) 2The abbreviations used are: wt, wild type; HA, hemagglutinin; KD, kinase-defective; aa, amino acids; IP, immunoprecipitation; ATM, ataxia telangiectasia mutated; GFP, green fluorescent protein; BD, binding domain. p53 (pRC-CMV-p53), mouse wt nontagged and HA-tagged c-abl (pCMV-c-abl, type IV), mouse kinase-defective (KD) c-abl (pCMV-c-ablK290H, type IV) (24Goldberg Z. Vogt Sionov R. Berger M. Zwang Y. Perets R. Van Etten R.A. Oren M. Taya Y. Haupt Y. EMBO J.. 2002; 21: 3715-3727Google Scholar), p53-responsive cyclin G-luciferase (26Haupt Y. Barak Y. Oren M. EMBO J.. 1996; 15: 1596-1606Google Scholar), and an empty vector (pCMV-Neo-BamHI). Hdmx vectors were pcDNA3.1 human HA-hdmx, pcDNA3.1 human wt hdmx, HA-hdmx-C (amino acids (aa) 1-318 followed by 25 alternative aa), hdmx-D (aa 1-206 followed by 13 alternative aa), HA-hdmx-G (Δ27-124 aa). Hdmx deletion mutants were Myc-tagged hdmx ΔN (aa 101-490) and hdmx p53 binding domain (p53 BD)-His-Myc (aa 1-153). The Tyr to Phe substitutions within Hdmx were generated by using the QuikChange site-directed in vitro mutagenesis kit (Stratagene) on the full-length and the p53 BD hdmx forms. For the production of GFP-c-Abl lentivirus, the following vectors were used; GFP-c-Abl was cloned into SIN18.hEF1-WPRE vector using SmaI and XbaI sites, pCMVΔR8.91-packaging construct, and pMD2.VSVG envelope construct. For the production of lentivirus these three vectors were co-transfected into HEK293T cells, and lentivirus was collected from the supernatants after 48 h. For the production of si-3′UTR-Hdmx lentivirus (MISSION Library Sigma-Aldrich) the manufacturer's instructions were followed.Antibodies—Anti-Hdmx antibodies used were: rabbit polyclonal sera p55, p56 (27Ramos Y.F. Stad R. Attema J. Peltenburg L.T. van der Eb A.J. Jochemsen A.G. Cancer Res.. 2001; 61: 1839-1842Google Scholar), and 1328 3P. de Graaf and A. G. Jochemsen, unpublished data. , mouse monoclonal anti-bodies 6B1A, 11F4D, and 12G11G (28Stad R. Ramos Y.F. Little N. Grivell S. Attema J. van Der Eb A.J. Jochemsen A.G. J. Biol. Chem.. 2000; 275: 28039-28044Google Scholar), and mouse monoclonal MX-82 antibody (Sigma MX-82). Anti-c-Abl antibodies used were the rabbit polyclonal K-12 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and the monoclonal Abl-148 antibody (Sigma); the anti-phospho-Hdmx antibodies were phospho-Tyr-55 (Sigma PH-MDMX-55) and phospho-Tyr-99 monoclonal antibodies (Sigma PH-MDMX-169) and anti-phospho-Ser-367 rabbit polyclonal antibody (29Pereg Y. Lam S. Teunisse A. Biton S. Meulmeester E. Mittelman L. Buscemi G. Okamoto K. Taya Y. Shiloh Y. Jochemsen A.G. Mol. Cell. Biol.. 2006; 26: 6819-6831Google Scholar). Additional antibodies used were: anti-human p53 monoclonal antibodies PAb1801 and DO1; anti-p53 goat polyclonal antibody (FL-393 Santa Cruz Biotechnology); anti-p53 rabbit polyclonal antibody (FL-393, Santa Cruz Biotechnology); anti-phospho-p53 Ser-20 antibody (Cell Signaling Technology, Inc., Danvers, MA); anti-Hdm2 SMP14 and 2A9 monoclonal antibodies, anti-GFP (Roche Applied Science), anti-phosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY), anti-14-3-3 tau (BIO-SOURCE, Camarillo, CA), horseradish peroxidase-conjugated affinity-purified goat anti-mouse IgG (Jackson Immuno-Research Laboratories, Inc., West Grove, PA), and EnVision peroxidase anti-mouse or anti-rabbit IgG antibodies (DAKO Corp., Glostrup, Denmark).Immunoblotting, Immunoprecipitation, in Vivo Kinase Assay and Nickel Pulldown Assay—Western blot analysis and immunoprecipitation assay were performed as previously described (26Haupt Y. Barak Y. Oren M. EMBO J.. 1996; 15: 1596-1606Google Scholar), whereas the lysis buffer for immunoprecipitation consisted of 50 mm Tris, pH 8.0, 150-300 mm NaCl, 5 mm EDTA, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 4 mm NaF, and 1 mm sodium orthovanadate. Nickel pulldown and in vivo kinase assay were performed as described previously (24Goldberg Z. Vogt Sionov R. Berger M. Zwang Y. Perets R. Van Etten R.A. Oren M. Taya Y. Haupt Y. EMBO J.. 2002; 21: 3715-3727Google Scholar).Luciferase Assay—This assay was done essentially as previously described (24Goldberg Z. Vogt Sionov R. Berger M. Zwang Y. Perets R. Van Etten R.A. Oren M. Taya Y. Haupt Y. EMBO J.. 2002; 21: 3715-3727Google Scholar).Detection of Phosphorylated Tyr by Mass Spectrometry—Phosphorylated and non-phosphorylated Hdmx proteins were derived from in vivo kinase assay using wt or kinase-defective c-Abl. The proteins were digested using Asp N enzyme, and mass spectrometry was carried out with Qtof2 (Micromass, England) using nanospray attachment (30Wilm M. Mann M. Anal. Chem.. 1996; 68: 1-8Google Scholar). Data analysis was done using the biolynx package (Micromass, England), and data base searches were performed with the Mascot package (Matrix Science).RESULTSInteraction between c-Abl and Hdmx—To determine whether the protection of p53 by c-Abl involves relief from Hdmx, we examined a possible interaction between c-Abl and Hdmx proteins in cultured cells. HEK293 cells were transfected with expression plasmids for HA-Hdmx either alone or together with wt or a KD c-AblK290H mutant of c-Abl. Forty-eight hours post-transfection, cell extracts were subjected to immunoprecipitation (IP) using anti-c-Abl Abl-148 antibody followed by immunoblotting (IB) with anti-Hdmx antibodies. As shown in Fig. 1A, Hdmx was efficiently co-precipitated only in the presence of c-Abl, demonstrating an interaction between the two proteins in cultured cells. The interaction between Hdmx and the KD mutant of c-Abl appeared to be weaker. To further establish the interaction between Hdmx and c-Abl, we performed a reversal experiment where IP was done with anti-Hdmx 6B1A antibody and IB with anti-c-Abl antibody. Hdmx efficiently co-precipitated c-Abl (Fig. 1B). These results demonstrate an interaction between the two proteins in cultured cells. Next, we investigated whether endogenous Hdmx interacts with endogenous c-Abl. To answer this question, we performed co-immunoprecipitation experiments using MCF-7 cell line expressing relatively high endogenous Hdmx levels. c-Abl was immunoprecipitated from cell extracts using anti-c-Abl K-12 antibody followed by immunoblotting with anti-Hdmx antibody. As shown in Fig. 1C, Hdmx protein was co-immunoprecipitated by c-Abl, providing evidence for an interaction between the two endogenous proteins.Both c-Abl and Hdmx interact with p53 and Hdm2 (19Levav-Cohen Y. Goldberg Z. Zuckerman V. Grossman T. Haupt S. Haupt Y. Biochem. Biophys. Res. Commun.. 2005; 331: 737-749Google Scholar). We, therefore, investigated whether the c-Abl/Hdmx interaction is mediated by p53 and/or Hdm2. To address this question, HEK293 cells were transfected with expression plasmids for c-Abl in combination with different Hdmx deletion mutants. First, we tested the involvement of p53 by using Hdmx-G, a product of an alternative splice variant that lacks amino acids 27-124 encompassing the p53 binding domain of Hdmx (Ref. 15de Graaf P. Little N.A. Ramos Y.F. Meulmeester E. Letteboer S.J. Jochemsen A.G. J. Biol. Chem.. 2003; 278: 38315-38324Google Scholar and Fig. 1D). Twenty-four hours post-transfection cell extracts were subjected to immunoprecipitation assay. As shown in Fig. 1E, Hdmx-G (ΔN) bound efficiently to c-Abl, suggesting that p53 is not essential for c-Abl-Hdmx interaction under these conditions. Next we investigated the contribution of Hdm2 to the interaction by using another Hdmx alternative splice variant, Hdmx-D, which lacks the C terminus (amino acids 207-490) including the Hdm2 binding region and contains an additional 13 alternative amino acids after amino acid 206 (Fig. 1D). As shown in Fig. 1F, HdmxD (ΔC) efficiently co-precipitated with c-Abl, implying that the Hdm2 binding domain is not critical for c-Abl-Hdmx interaction. To further define the interaction region of c-Abl within Hdmx, a series of additional deletion mutants of Hdmx (Fig. 1D) were tested using the same assay. All the Hdmx deletion mutants tested were able to bind c-Abl (supplemental Fig. S1), implying that c-Abl may bind Hdmx at more than one site and/or that additional proteins are involved in the interaction with some of these mutants. c-Abl Phosphorylates Hdmx within the p53 Binding Domain—Because c-Abl is a tyrosine kinase, we asked whether Hdmx is phosphorylated by c-Abl. There are nine Tyr residues within Hdmx, five of which are clustered within the p53 BD (Fig. 2A) and are conserved among mice, rat, and human (Fig. 2B). In view of the possible link to p53 regulation, we examined the ability of c-Abl to phosphorylate an Hdmx deletion mutant containing the N-terminal p53 BD (amino acids 1-153) in an in vivo kinase assay. HEK293 cells were transfected with expression plasmids for the Myc- and His-tagged hdmx p53 BD either alone or in combination with wt or c-Abl KD. Twenty-four hours post-transfection Hdmx was isolated from cell extracts using nickel resin, and Tyr phosphorylation of Hdmx was monitored with anti-phosphotyrosine antibody. As shown in Fig. 2C, Tyr phosphorylation of Hdmx p53 BD was detected only in the presence of wt c-Abl, supporting a role for c-Abl in Tyr phosphorylation of Hdmx within the p53 BD.FIGURE 2c-Abl-dependent tyrosine phosphorylation of Hdmx. A, schematic representation of the tyrosine residues in Hdmx. The cluster of the tyrosine residues within p53 binding domain of Hdmx is highlighted. B, alignment of the Mdmx amino acid sequence from rat, mouse, and human. The conserved tyrosine residues within Mdmx are highlighted. C, HEK293 cells were transfected with expression plasmids for c-abl or c-abl KD either alone or together with His- and Myc-tagged hdmx p53 BD (binding domain; amino acids 1-153). Twenty-four hours post-transfection cells were harvested, and Hdmx p53 BD was isolated from cell extracts using nickel resin. Phosphorylation was detected with anti-phosphotyrosine antibody (4G10), and Hdmx levels were monitored by blotting (IB) with anti-Myc antibody. The expression level of c-Abl was monitored with anti-c-Abl antibody (ABL-148).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tyr-99 and Tyr-55 Are the Major Sites within the p53 BD of Hdmx—To identify the c-Abl Tyr phosphorylation site(s) within the p53 BD of Hdmx, we employed mass spectrometric analysis along with site-directed mutagenesis. Hdmx p53 BD was phosphorylated in vivo by c-Abl (Fig. 2C) and then subjected to mass spectrometry analysis. Tyr-55 was identified as a potential Tyr phosphorylation site (data not shown). Additionally, we generated substitution mutants from Tyr(Y) to Phe(F) for each Tyr residue in the p53 BD of Hdmx, either alone or in various combinations. The effect of these substitutions on Hdmx phosphorylation by c-Abl was measured in phosphorylation assays in vivo. HEK293 cells were transfected with expression plasmids for wt hdmx p53 BD or phosphorylation mutants together with wt c-Abl or c-Abl KD. Twenty-four hours post-transfection, Hdmx was precipitated, and Tyr phosphorylation analysis revealed that substitutions of Y99F and Y55F, but not the other Tyr residues within the p53 binding domain, reduced the extent of Hdmx phosphorylation (Fig. 3A and data not shown). Tyr phosphorylation was almost abolished in the Hdmx Y55F,Y99F double mutant (Fig. 3A), supporting the notion that Tyr-55 and Tyr-99 are the major c-Abl phosphorylation sites within the p53 BD of Hdmx. Among these two sites, Y99F substitution had a greater effect on Hdmx phosphorylation (Fig. 3A); hence, it was assumed to be a more dominating site. To further verify the phosphorylation of Hdmx on Tyr-99 and Tyr-55, anti-phospho-monoclonal antibodies were raised. To determine the specificity of these antibodies, HEK293 cells were transfected with expression plasmids for the Myc and His-tagged hdmx p53 BD in combination with wt c-Abl or c-Abl KD. Specific c-Abl-induced Hdmx phosphorylations on Tyr-55 and Tyr-99 were detected with the corresponding anti-phospho antibodies (Fig. 3B). A similar result was obtained when the experiment was repeated with full-length non-tagged wt Hdmx (Fig. 3C).FIGURE 3c-Abl phosphorylates Hdmx on Tyr-99 and Tyr-55 in vivo. A, HEK293 cells were transfected with the indicated plasmids. Twenty-four hours later Hdmx p53 BD was isolated by nickel resin from cell extracts, and the protein complex was subjected to Western blotting (IB) using the indicated antibodies as described in Fig. 2C. B and C, HEK293 cells were transfected with the indicated plasmids. Twenty-four (B) or forty-eight (C) hours later cell extracts were analyzed by Western blotting using anti-phospho-Tyr-55 antibody and anti-phospho-Tyr-99 antibody. Total Hdmx levels were monitored by blotting with anti-Hdmx 1328 polyclonal antibody. The position of Tyr-55 and Tyr-99-phosphorylated Hdmx and total Hdmx is indicated. A dividing line in B indicates that lanes were merged from different parts of the same gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Phospho-Tyr-99 Affects the Phosphorylation of Tyr-55—Because the phosphorylation sites reside within the same domain of Hdmx, it was of interest to test a potential dependence between the two sites. To examine this possibility, HEK293 cells were transfected with expression plasmids for Myc and His-tagged wt hdmx p53 BD or phosphorylation mutants either alone or in combination with wt c-Abl or c-Abl KD. The phosphorylation of Tyr-99 was detected in wt Hdmx and the Y55F substitution mutant (Fig. 4A), suggesting that the Tyr-99 phosphorylation is independent of Tyr-55 phosphorylation. Interestingly, the phosphorylation of Tyr-55 was reduced not only in the Y55F mutant but partially also in the Y99F mutant (Fig. 4B). A similar effect was observed with full-length non-tagged Hdmx (Fig. 4C). This suggests that phosphorylation on Tyr-99 is required at least partially for an efficient phosphorylation of Tyr-55. An alternative explanation is that the Y99F substitution impairs the interaction between Hdmx and c-Abl. To test this possibility HEK293 cells were transfected with expression plasmids for c-Abl either alone or together with Myc-His-tagged wt or Y99F hdmx p53 BD mutant. Hdmx was immunoprecipitated using anti-Myc antibody, and the amount of co-precipitated c-Abl was determined by IB using anti-c-Abl antibody (Fig. 4D). As shown in Fig. 4D, the Y99F substitution did not reduce the c-Abl-Hdmx interaction but, rather, increased it.FIGURE 4Tyr-99 is required for the efficient phosphorylation of Tyr-55, and both tyrosine phosphorylations are independent of Ser-342 and Ser-367 phosphorylation. A-C, HEK293 cells were transfected with the indicated plasmids. Twenty-four (A and B) or forty-eight hours (C) later cell extracts were subjected to Western blotting using either anti-phospho-Tyr-55 antibody (B and C) or anti-phospho-Tyr-99 antibody (A and C). Total Hdmx levels were monitored using anti-Hdmx 1328 polyclonal antibody. The position of Tyr-55 and Tyr-99-phosphorylated Hdmx and total Hdmx is indicated. Hdmx p53 BD mutant was described in Fig. 2C. D, HEK293 cells were transfected with the expression plasmid for c-abl alone or together with Myc and His-tagged hdmx p53 BD either wt or Y99F mutant. Twenty-four hours post-transfection Hdmx was immunoprecipitated using anti-Myc antibody. The level of the co-precipitated c-Abl was monitored by blotting with anti-c-Abl antibody (ABL-148). The expression level of the Hdmx was investigated by using anti-Myc antibody. E, HEK293-T cells were transfected with expression vectors for HA-tagged hdmx or hdmx-S342/367A or empty vector together with c-abl. Cells were either non-treated or treated with 3 mm H2O2 for 45 min together with tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4; 1 mm) for 5 h before harvest. Twenty-nine hours post-transfection cell extracts were subjected to IP with anti-HA rabbit polyclonal antibody. Immunoprecipitates were analyzed by Western blotting with anti-phospho-Tyr-55-Hdmx, anti-phospho-Y99-Hdmx, and anti-Hdmx MX-82 to monitor total Hdmx levels. To show the absence of Ser-367 phosphorylation of the mutant, the same protein extracts were used in immunoprecipitation with anti-HA monoclonal antibody, and immunoprecipitates were analyzed on Western blot with anti-phospho-Ser-367-Hdmx or anti-Hdmx antibody (BL1258).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interplay between c-Abl-mediated Phosphorylation of Hdmx and the DNA Damage-induced Hdmx Phosphorylation on Ser-342, Ser-367, and Ser-403 and 14-3-3 Binding—It has been shown that Hdmx is independently phosphorylated on Ser-342, Ser-367, and Ser-403 in response to double strand breaks by ATM (ataxia telangiectasia mutated) or by its target Chk2 (29Pereg Y. Lam S. Teunisse A. Biton S. Meulmeester E. Mittelman L. Buscemi G. Okamoto K. Taya Y. Shiloh Y. Jochemsen A.G. Mol. Cell. Biol.. 2006; 26: 6819-6831Google Scholar, 31LeBron C. Chen L. Gilkes D.M. Chen J. EMBO J.. 2006; 25: 1196-1206Google Scholar). Each of these phosphorylations contributes to the increased ubiquitination and degradation of Hdmx after DNA damage, caused by dissociation of the herpes virus-associated ubiquitin-specific protease (HAUSP) from both Hdmx and Hdm2 and promotion of nuclear accumulation of Hdmx (12Li C. Chen L. Chen J. Mol. Cell. Biol.. 2002; 22: 7562-7571Google Scholar, 32Meulmeester E. Maurice M.M. Boutell C. Teunisse A.F. Ovaa H. Abraham T.E. Dirks R.W. Jochemsen A.G. Mol. Cell.. 2005; 18: 565-576Google Scholar). Phosphorylation of Ser-342 and Ser-367 was shown to be essential for creating a binding site for several isoforms of the 14-3-3 protein, leading to nuclear localization of Hdmx, and promoting the degradation of Hdmx (29Pereg Y. Lam S. Teunisse A. Biton S. Meulmeester E. Mittelman L. Buscemi G. Okamoto K. Taya Y. Shiloh Y. Jochemsen A.G. Mol. Cell. Biol.. 2006; 26: 6819-6831Google Scholar, 33Chen L. Gilkes D.M. Pan Y. Lane W.S. Chen J. EMBO J.. 2005; 24: 3411-3422Google Scholar, 34Okamoto K. Kashima K. Pereg Y. Ishida M. Yamazaki S. Nota A. Teunisse A. Migliorini D. Kitabayashi I. Marine J.C. Prives C. Shiloh Y. Jochemsen A.G. Taya Y. Mol. Cell. Biol.. 2005; 25: 9608-9620Google Scholar). Because c-Abl is also a target for ATM-dependent phos
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