Molecular Oncology Department, Moffitt Cancer Center, Tampa, Florida 33612, USA.
Mol Cell Biol. 2011 Dec;31(24):4951-63. doi: 10.1128/MCB.05553-11. Epub 2011 Oct 10.
MDM2 is a major regulator of p53 by acting as a ubiquitin E3 ligase. The central acidic domain and C-terminal RING domain of MDM2 are both indispensable for ubiquitination of p53. Our previous study suggested that ATM phosphorylation of MDM2 near the C terminus inhibits RING domain oligomerization, resulting in p53 stabilization after DNA damage. We present here evidence that these modifications allosterically regulate the functions of both acidic domain and RING domain of MDM2. Using chemical cross-linking, we show that the MDM2 RING domain forms oligomers including dimer and higher-order complexes in vivo. RING domain dimerization efficiency is negatively regulated by upstream sequence. ATM-mediated phosphorylation of the upstream sequence further inhibits RING dimerization. Forced oligomerization of MDM2 partially overcomes the inhibitory effect of phosphorylation and stimulates p53 ubiquitination. Furthermore, the ability of MDM2 acidic domain to bind p53 core domain and induce p53 misfolding are also suppressed by the same C-terminal ATM sites after DNA damage. These results suggest that the acidic domain and RING domain of MDM2 are both allosterically coupled to the intervening ATM sites, which enables the same modification to regulate multiple MDM2 functions critical for p53 ubiquitination.
MDM2 作为泛素 E3 连接酶，是 p53 的主要调节因子。MDM2 的中央酸性结构域和 C 端 RING 结构域对于 p53 的泛素化都是不可或缺的。我们之前的研究表明，ATM 在 MDM2 的 C 端附近对 MDM2 的磷酸化会抑制 RING 结构域寡聚化，导致 DNA 损伤后 p53 稳定。我们在这里提出的证据表明，这些修饰通过变构调节 MDM2 的酸性结构域和 RING 结构域的功能。通过化学交联，我们表明 MDM2 的 RING 结构域在体内形成寡聚体，包括二聚体和更高阶的复合物。RING 结构域二聚体效率受到上游序列的负调控。ATM 介导的上游序列磷酸化进一步抑制 RING 二聚体化。MDM2 的强制寡聚化部分克服了磷酸化的抑制作用，并刺激了 p53 的泛素化。此外，在 DNA 损伤后，相同的 C 端 ATM 位点还抑制了 MDM2 酸性结构域与 p53 核心结构域结合并诱导 p53 错误折叠的能力。这些结果表明，MDM2 的酸性结构域和 RING 结构域都与中间的 ATM 位点变构偶联，使相同的修饰能够调节多个对 p53 泛素化至关重要的 MDM2 功能。
