Functional Regulation Of Apak, A Selective Regulator Of P53, In Response To Various Stresses And The Molecular Mechanisms

The tumor suppressor p53 is a transcriptional factor and lies at the center of a protein signaling network that responds to various types of cellular stresses including DNA damage, abnormal proliferation and oncogene activation, and p53 activation leads to either cell cycle arrest or apoptosis or both, dependent of the severity of the stress. The KRAB-type zinc-finger protein (KZNF) family is the single largest class of transcription factors in human genome, and Apak (ATM and p53 associated KZNF protein) belongs to this family. As a newly identified negative regulator of p53, Apak directly binds to p53 in unstressed cells, specifically downregulates proapoptotic genes, and remarkably suppresses p53-mediated apoptosis by attenuating acetylation of p53. By contrast, Apak had no significant effects on p53-mediated cell cycle arrest. Apak exhibits this inhibitory role by recruiting the KAP-1/HDAC1/ATM protein complex. In response to DNA damage, Apak is phosphorylated by ATM and dissociates from p53, resulting in p53 activation and apoptosis induction. However, little is known how Apak is regulated in response to other types of stresses, such as oncogene activation. In this study, we investigated the possible regulations of Apak in response to oncogene activation and a variety of DNA damage signals and elucidated the underlying molecular mechanisms.Oncogene activation belongs to one of the most important reasons during tumorigenesis and its signaling to p53 has been identified to be different from the DNA damage signaling. So far, the p53 activation in response to oncogene activation largely depends on the upregulation of tumor suppressor p14ARF.Here we showed functional regulation of Apak and the molecular mechanisms in response to oncogene and DNA damage.1 Functional regulation of Apak and the molecular mechanisms in response to oncogene activation1) In response to overexpression of c-MYC, E2F1 or Ras-G12V mutant mimicking the oncogene activation, Apak dissociated from p53 and the inhibition on p53 was released. Importantly, these processes depended on p14ARF but not ATM-mediated phosphorylation. 2) Apak interacted with p14ARF both in vivo and in vitro. p14ARF competed with p53 to bind to Apak, thus promoting the Apak-p53 dissociation. Interestingly, we observed the enhanced protein stability of Apak after these oncogene insults, although its inhibitory effects on p53 were attenuated by p14ARF.3) Apak is a short-lived protein and stabilized by oncogene activation also dependent of p14ARF. Furthermore, we showed that Apak could be ubiquitinated by E3 ubiquitin ligases HDM2 and ARF-BP1, and then degraded by the proteasome. p14ARF negatively regulated the E3 ligase activity of HDM2 and ARF-BP1, therefore inhibited the Apak degradation.4) Knockdown of HDM2 and ARF-BP1 interfered with the stabilization of Apak by p14ARF. Collectively, these results revealed a novel signaling through oncogene to p14ARF to Apak.2 Functional regulation of Apak and the molecular mechanisms in response to DNA damageWe previously showed that Apak is negatively regulated in response to MMS treatment. However, the mechanism by which Apak is regulated in response to other types of DNA damage signals remains unclear. In this study, we showed that four of seven types of DNA damage signals we examined (induction by cisplatin, doxorubicin, etoposide, and camptothecin treatment) resulted in significant Apak phosphorylation and dissociation of Apak from p53, releasing the inhibition of p53 transcriptional activity. In contrast, Apak was not phosphorylated at Ser68 after 5-fluorouracil orα-lipoic acid treatment and persistently inhibited p53 activity. These findings provided evidence that the Apak-p53 interaction was regulated differentially by various DNA damage signals.