Interplay between 14-3-3gamma and MDMX in regulating the p53 pathway

描述(申请人提供)：本提案重点研究14-3-3的作用？在调节MDM2-MDMX-P53反馈通路以响应低氧引起的细胞应激中的作用。缺氧在肿瘤的发生，特别是实体肿瘤的发展中起着至关重要的作用，因为在快速生长的肿瘤中，由于血液供应不足，所有实体肿瘤的缺氧区都显示出0%-8%的氧气浓度。在这一病理过程中，肿瘤抑制基因P53被诱导和激活，通过诱导细胞凋亡和细胞生长停滞以及减缓血管生成来防止肿瘤的生长。虽然P53在低氧反应中抑制肿瘤进展的作用已被很好地证实，但低氧触发P53诱导的确切机制仍不清楚。生物化学和遗传学研究表明，泛素介导的蛋白酶体周转和抑癌基因P53的活性受MDM2和MDMX两种癌蛋白的严格控制。这两种蛋白作为p53的负反馈调节蛋白协同工作，因为MDM2也是p53的转录靶点，而MDMX则充当MDM2的伙伴。因此，解开这个反馈环对于激活P53以防止细胞在各种压力下发生转化和肿瘤是至关重要的。然而，目前尚不清楚低氧是否通过影响这一环而诱导P53。我们最近研究的线索表明，14-3-3？可能在低氧-P53途径中发挥作用。根据我们最近的工作以及其他人的研究，我假设缺氧可能导致14-3-3？与被ATR-Chk1级联磷酸化的MDMX结合，从而抑制MDMX活性并导致P53激活。因此，本研究将通过三个具体的目标来验证这一假说：1.确定ATR和Chk1是否在缺氧反应中调节14-3-3？-MDMX相互作用；2.阐明缺氧诱导14-3-3-MDMX相互作用和P53激活的机制；3.确定14-3-3？在细胞培养和动物体内减轻P53对低氧的反应。这些研究的成功实施不仅将阐明一种新的机制，即P53被诱导对低氧的反应，而且还将识别14-3-3？作为细胞生长控制和肿瘤发生的另一个重要参与者。

DESCRIPTION (provided by applicant): This proposal focuses on studying the role of 14-3-3? in regulating the MDM2-MDMX-p53 feedback pathway in response to cellular stress caused by hypoxia. Hypoxia plays a crucial role in tumorigenesis and particularly in the progression of solid tumors, as all solid tumors display 0%-8% oxygen concentrations in their hypoxic regions due to insufficient blood supply in rapidly growing tumors. During this pathological period, the tumor suppressor p53 is induced and activated to prevent the growth of the tumors by inducing apoptosis and cell growth arrest as well as slowing down angiogenesis. Although the role of p53 in suppressing tumor progression in response to hypoxia has been well established, the precise mechanism underlying hypoxia-triggered induction of p53 remains poorly understood. Biochemical and genetic studies have demonstrated that the ubiquitin-mediated proteasomal turnover and activity of the p53 tumor suppressor are tightly controlled by two oncoproteins called MDM2 and MDMX. Both of the proteins work together as p53 negative feedback regulators, because MDM2 is also a transcriptional target for p53 and MDMX acts as an MDM2 partner. Thus, untying this feedback loop is essential for activating p53 in order to prevent cells from undergoing transformation and neoplasia in response to various stresses. However, it is still unclear whether hypoxia induces p53 by affecting this loop. Clues from our recent studies suggest that 14-3-3? may play a role in the hypoxia-p53 pathway. In light of our recent work as well as studies by others, I hypothesize that hypoxia may induce the association of 14-3-3? with MDMX that is phosphorylated by the ATR-ChK1 cascade, consequently suppressing MDMX activity and leading to p53 activation. Therefore, this proposal will examine this hypothesis by addressing three specific aims: 1. To determine if ATR and ChK1 play a role in regulating the 14-3-3?-MDMX interaction in response to hypoxia; 2. To elucidate the mechanisms underlying hypoxia-induced 14-3-3?-MDMX interaction and p53 activation; 3. To determine if loss of 14-3-3? alleviates p53 response to hypoxia in cell culture and in animals. Successful implementation of these proposed studies will not only elucidate a novel mechanism by which p53 is induced in response to hypoxia, but would also identify 14-3-3? as another important player in cell growth control and tumorigenesis.