Negative regulation of p53 family protein p73 by transcription factor E2F-1

我们首次发现E2F-1具有促进COS7和SAOS-2细胞中p73泛素依赖性蛋白水解降解的能力。在H1299、U2OS、H4和A549细胞中也得到了类似的结果。缺失分析显示E2F-1的转激活功能是p73降解所必需的。我们还证明了一种名为NEDL2的新型hect型E3泛素蛋白连接酶与p73的PY基序相互作用，从而诱导其泛素化。出乎意料的是，nedl2介导的p73泛素化导致其半衰期增加，并增强了其转录活性。这些观察结果强烈提示泛素化存在非蛋白水解调节功能。通过基于酵母的双杂交筛选，我们确定了RanBPM是p73的结合伙伴。RanBPM结合到p73的极cooh末端区域，并通过抑制p73的泛素化水平来增加其稳定性。cooh末端的赖氨酸残基很可能是泛素化的靶标。此外，我们发现u -box型E3/E4泛素蛋白连接酶UFD2a通过与p73的物理相互作用诱导蛋白酶体依赖性蛋白水解降解p73。值得注意的是，在我们的实验条件下没有检测到ufd2a介导的p73泛素化。因此，我们目前的研究结果表明，p73不仅受泛素化依赖的降解途径的调节，而且还受与UFD2a以泛素化独立的方式进行功能相互作用的调节。

We have found for the first time that E2F-1 has an ability to promote the ubiquitin-dependent proteolytic degradation of p73 in COS7 and SAOS-2 cells. Similar results were also obtained in H1299, U2OS, H4 and A549 cells. Deletion analysis revealed that the transactivation function of E2F-1 is required for the degradation of p73. We also demonstrated that a novel HECT-type E3 ubiquitin protein ligase termed NEDL2 interacts with the PY motif of p73, and thereby inducing its ubiquitination. Unexpectedly, NEDL2-mediated ubiquitination of p73 resulted in an incre ase in its half-life, and enhanced its transcriptional activity. These observations strongly suggest that there exists a non-proteolytic regulatory function of the ubiquitination. By using a yeast-based two-hybrid screening, we have identified RanBPM as a binding partner of p73. RanBPM bound to the extreme COOH-terminal region of p73, and increased its stability by inhibiting the ubiquitination levels of p73. It is likely that the COOH-terminal Lys residues could be target(s) for ubiquitination. In addition, we have found that U-box-type E3/E4 ubiquitin protein ligase termed UFD2a induces the proteasome-dependent proteolytic degradation of p73 through the physical interaction with p73. Of note, UFD2a-mediated ubiquitination of p73 was not detected under our experimental conditions. Thus, our present results indicate that p73 is regulated not only by the ubiquitination-dependent degradation pathway but also by the functional interaction with UFD2a in a ubiquitination-independent manner.