Cubillos-Rojas Monica, Amair-Pinedo Fabiola, Peiró-Jordán Roser, Bartrons Ramon, Ventura Francesc, Rosa Jose Luis
From the Departament de Ciències Fisiològiques II, Campus de Bellvitge, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona 08907, Spain.
From the Departament de Ciències Fisiològiques II, Campus de Bellvitge, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona 08907, Spain
J Biol Chem. 2014 May 23;289(21):14782-95. doi: 10.1074/jbc.M113.527978. Epub 2014 Apr 9.
The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292-2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling.
肿瘤抑制因子p53是一种转录因子，可协调细胞对多种应激的反应。p53失活是肿瘤进展的重要步骤。p53的寡聚化对于其翻译后修饰以及调节抑制肿瘤生长所需靶基因转录的能力至关重要。在此，我们报告HECT E3泛素连接酶HERC2与p53相互作用。这种相互作用涉及HERC2的CPH结构域（Cul7、PARC和HERC2蛋白中的保守结构域）和p53的最后43个氨基酸残基。通过这种相互作用，HERC2调节p53的活性。RNA干扰实验表明，HERC2的缺失如何降低p53的转录活性而不影响其稳定性。HERC2对p53活性的这种调节独立于蛋白酶体或MDM2活性。在这些条件下，观察到细胞生长上调和集落形成增加，表明HERC2-p53相互作用具有功能相关性。在化疗药物博来霉素引起的DNA损伤后，这种相互作用得以维持。在这些应激细胞中，HERC2敲低不会损害p53的磷酸化。有趣的是，影响其四聚化结构域的p53突变破坏了HERC2-p53相互作用，表明HERC2在p53寡聚化中起作用。使用交联试验证明了这种调节作用。因此，HERC2缺失后p53活性的抑制可归因于p53寡聚化的减少。HERC2（2292-2923位残基）的异位表达证实了这些观察结果。总之这些结果确定HERC2是p53信号通路的一种新型调节因子。
