M. rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 P276-00 protein rather than as a direct E3 ligase in rules of p53 degradation. in mice results in early embryonic lethality, and deficiency rescues the lethal phenotype completely (11, 12). In physiological conditions, MDM2 maintains the p53 protein at an adequate level. However, MDM2 is definitely reported to be regularly amplified or overexpressed in human being cancers, many of which lack mutations in the gene (13). The part of overexpressed MDM2 in human being cancers is definitely believed to be functionally equivalent to p53 mutation (14). MDM2 is definitely a RING (really interesting fresh gene) finger website E3 ligase and directly interacts with p53 during the ubiquitylation process. The activity of MDM2 is definitely regulated by several mechanisms. First, amounting evidence is definitely demonstrated that co-factors are involved in the rules of MDM2-mediated p53 degradation. For example, ARF is definitely reported to interfere with the MDM2-p53 connection, therefore to inhibit MDM2-mediated p53 ubiquitylation (15,C18). YY1 and PACT are demonstrated to enhance the degradation of p53 by advertising the connection of MDM2 with p53 (19, 20). Conversely, binding of the ribosomal proteins L5, L11, and L23 to MDM2 inhibits the activity of MDM2 and takes on a crucial part in p53 activation upon ribosomal stress (21,C23). Importantly, MDM2 activity is also controlled by a protein degradation mechanism. MDM2 is definitely a short lived protein (9, 10). The stability of MDM2 is definitely tightly regulated by different mechanisms. Although it has been reported that MDM2 protein stability is definitely regulated by additional E3 ligases (for example, PCAF) P276-00 (24), the degradation of MDM2 is mainly induced by a self-ubiquitylation or autodegradation mechanism (25, 26). Furthermore, the self-ubiquitylation or autodegradation is definitely mediated by homodimerization of MDM2 itself. MDMX (also known as MDM4) has been shown to play a critical role in avoiding MDM2 self-ubiquitylation or autodegradation. MDMX is found to interact with MDM2 to form a heterodimer. The heterodimerization of MDM2 with MDMX blocks the homodimerization of MDM2 and inhibits MDM2 self-ubiquitylation (27,C29). In such a way, MDMX regulates the activity of MDM2 and furthers the level of p53. However, it remains unclear how the heterodimerization of MDM2 and MDMX happens. In this study, we shown the HECT-type E3 ubiquitin ligases Smurf1 and -2 enhance the heterodimerization of MDM2 and MDMX and block the homodimerization of MDM2. We provide evidence that Smurf1 and -2 play an important part in maintenance of MDM2 stability, by such a way Smurf1 P276-00 and -2 inhibit p53 activity and block apoptosis. MATERIALS AND METHODS Plasmid Constructs Full-length, truncated, and point mutations of Smurf1, Smurf2, MDM2, MDMX, and Smad5 P276-00 were constructed by inserting PCR-amplified fragments into the related vectors. Detailed construct information is usually available upon request. pCMV/p53, pCMV/MDM2, and pcDNA3/poly-HA-tagged ubiquitin were gifts from Dr. Yue Xiong. 6Myc-Smurf1 wild type, 6Myc-Smurf1-C699A, and FLAG-Smurf1 were provided by Dr. Kohei Miyazono. HA-MDMX was provided by Dr. Geoffrey M. Wahl. Constructs of other Nedd4 family members were kindly provided by Dr. Wesley I. Sundquist. Cell Culture and Transfection Human embryonic kidney HEK293T cells, human breast malignancy MCF7 cells, human colon cancer HCT116 cells (ubiquitylation assay, cells were treated with MG132 (20 m; Sigma) for 8 h before harvesting. The cell lysis were prepared in altered RIPA lysis buffer (10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5 mm EDTA, 1% (v/v) Nonidet P-40, 1% sodium deoxycholate, 0.025% SDS, protease inhibitors), immunoprecipitated with the indicated antibody, and detected by immunoblotting. For ubiquitylation assay, E1, UbcH5b, UbcH5c P276-00 (E2), HA-Ub (all from Boston Biochem), GST-MDM2, and Smurf1 were incubated at 30 C for 2 h and terminated with sample buffer. RNA Interference The siRNAs against Smurf1 (5-GCAUCGAAGUGUCCAGAGAAG-3), Smurf2 (5-CCUUCUGUGUUGAACAUAA-3), MDM2 (5-UGGUUGCAUUGUCCAUGGC-3), MDMX (5-UCAAUCAGGUACGACCAAA-3), NEDL1 (5-GAUGCCAGCUCGUACUUUG-3), NEDL2 (5-GAUUGGACUUUAUCAUAUA-3), and nontargeting control (5-UUCUCCGAACGUGUCACGU-3) were synthesized by Shanghai GenePharm. The siRNAs were transfected with a FuGENE HD (Roche Applied Science) reagent, and the interference efficiency was assessed by Pdgfra immunoblotting. Reporter Assays The luciferase reporter plasmid pG13-Luc (a gift from Dr. Bert Vogelstein) was transfected into the cells, and the reporter assays were performed as described (30). Real Time Quantitative PCR Assay Real time quantitative PCR was performed as described previously (31). Sequences of primers of the p53 targeted genes used in quantitative PCR assays are listed in supplemental Table S1..
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