CARP 1/2 synergize with Mdm2 to regulate p53 Free

2535

We reported that CARPs, as E3 ligases, target p53 and phospho-p53 for degradation in an Mdm2-independent manner. Because Mdm2 is a primary negative regulator of p53, we determined whether Mdm2 is involved in CARP-mediated regulation of p53. Exogenous CARP protein stabilized exogenous and endogenous Mdm2 protein levels in several cell lines, and this stabilization was not observed with RING-deleted Mdm2 mutants. In addition, CARP gene silencing destabilized Mdm2. In several cell lines (H460, MCF-7, PA-1 and U2OS with wild type p53), hypoxic or hypoxia-mimetic conditions (0.2% oxygen or DFX) led to a rapid rise in HIF-1α and p53 levels, and a reduction of CARP2 and Mdm2 levels. Growth factor IGF-1 increased the level of CARP2 as well as Mdm2. We found that the N-terminus of Mdm2 (1-50 aa) and the RING domain of CARP1 or CARP2 are responsible for binding. Co-transfection of CARP, Mdm2 with p53 into HCT116 (p53-/-) cells, more significantly enhanced the degradation of p53 and then reduced apoptosis as compared with transfection of CARPs or Mdm2 with p53, suggesting that CARPs and Mdm2 collaboratively target p53 for degradation. Because the E3 activity of Mdm2 is sequestrated by ARF, we overexpressed CARPs in p53-/-Mdm2-/- cells and this led to ARF degradation that was impaired by the proteasome inhibitor MG132, suggesting that CARPs target ARF for degradation by the proteasome. CARPs through Mdm2 binding disrupted the interaction of ARF and Mdm2, thereby releasing the sequestering inhibition of ARF on Mdm2. These data suggest that CARPs maintain an intact E3 activity of Mdm2 by inhibiting ARF functions. Taken together, CARPs, as a family of newly defined p53-targeting E3 ligases, modulate the integrity of Mdm2 activities by regulating the level and function of MDM2 to synergistically down-regulate p53.