Abstract
The critical tumor suppressor p53 is mutated or functionally inactivated in nearly all cancers. We have shown previously that the MDM2-MDMX complex functions as an integral unit in targeting p53 for degradation. Here we identify the small protein 14-3-3 as a binding partner of MDMX, which binds at the C terminus (Ser367) in a phosphorylation-dependent manner. Importantly, we demonstrate that the serine/threonine kinase Akt mediates phosphorylation of MDMX at Ser367. This phosphorylation leads to stabilization of MDMX and consequent stabilization of MDM2. Previous studies have shown that Akt phosphorylates and stabilizes MDM2. Our data suggest that stabilization of MDMX by Akt may be an alternative mechanism by which Akt up-regulates MDM2 protein levels and exerts its oncogenic effects on p53 in tumor cells.
Highlights
A close homologue of MDM2, MDMX, has been shown to be another critical regulator of p53 activity [3, 4]
A fundamental process in the development of cancer is the inactivation of the p53 tumor suppressor pathway
Amplifications of the MDM2 gene or stabilization of the MDM2 protein can result in the inactivation of p53 by increasing its degradation and inhibiting p53 transcriptional activity [25, 30, 48]
Summary
Preparation of Plasmids and Proteins—pcDNA3MDMX(S367A) and pcDNA3-MDMX(P369R) mutants were generated by two-step site-directed PCR mutagenesis utilizing the following primers: Ser367 to Ala, 5Ј-TGTCGAAGAACCATTGCGGCTCCTGTCGTT and 3Ј-TCTAACGACAGGAGCCGCAATGGTTCTTCG; and Pro369 to Arg, 5Ј-ATTTCGGCTCGAGTCGTTAGA and 3Ј-TCTAACGACTCGAGCCGAAAT. Cell lysates were prepared in 1% Triton X-100 lysis buffer and incubated with preconjugated agarose beads with anti-FLAG antibodies (Sigma). After boiling in Laemmli buffer, lysates were resolved by 12.5% SDS-PAGE. For experiments with the PI 3-kinase inhibitor, cells grown in serum-free medium were pretreated with 10 M LY294002 (Sigma) for 30 min and treated with IGF-1 as described above. The samples were incubated at 95 °C for 10 min and resolved by SDS-PAGE. Cell lysates were prepared in 0.5% Triton X-100 lysis buffer and incubated with anti-FLAG M5 beads (Sigma) for an additional 2 h. MDMX immunoprecipitates were incubated with lysates expressing constitutively active or dominant-negative Akt in kinase assay buffer in the presence of 10 mM [␥-32P]ATP and incubated at 30 °C for 30 min.
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