Role of murine double minute 2 and retinoblastoma protein in cellular senescence and muscle cell differentiation
Walsh, Erica Marlene
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Murine double minute 2 (MDM2) is an ubiquitin E3-ligase critical for proteasome-mediated degradation of p53 and retinoblastoma protein (Rb). It has been shown that the central acid domain (CAD) of MDM2 is indispensable for p53 ubiquitination and degradation. The MDM2 CAD is targeted for phosphorylation by several protein kinases, thereby modulating its function. Previous work showed that MDM2 binds to Rb through the CAD and facilitates Rb protein degradation. Notably, the Rb-binding segment in the MDM2 CAD contains an S256XXXS260 motif, a site preferentially phosphorylated by glycogen synthase kinase 3-β (GSK3β). This study demonstrates that a Ser-256-Ala substitution leads to reduced MDM2 activity for p53 ubiquitination and degradation. Moreover, this single point mutation also reduces the ability of MDM2 to bind to Rb, resulting in Rb stabilization. Also, wild-type MDM2, but not MDM2(S256A), is able to inhibit Rb-induced cellular senescence, as evidenced by an increase in large, flat cell morphology and senescence-associated β-galactosidase staining. Together, these data indicate that phosphorylation of MDM2 CAD is critical for inhibiting p53 and Rb, and suggest that GSK3β may play an important role in modulating the p53 and Rb pathways. The effect of nutlin-3 on muscle cell differentiation was also investigated. Nutlin-3 is a potent small molecule that activates p53. We show that nutlin-3 effectively inhibits muscle cell differentiation in vitro, accompanied by lack of induction of myogenin and myosin heavy chain expression, two markers of muscle cell differentiation. Nutlin-3 leads to an accumulation of MDM2 protein levels, and inhibits the differentiation-induced increase in Rb expression. These data suggest that deregulation of MDM2 and Rb expression strikingly impacts muscle cell differentiation.
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