Cellular Mechanisms Controlling Myc Protein Stability

描述(申请人提供)：c-Myc癌蛋白是细胞功能的关键调节因子，其过度表达与人类癌症密切相关。C-Myc的表达受到包括蛋白质稳定性在内的多个水平的调控。一个复杂的信号通路通过丝氨酸62(S62)和苏氨酸58(T58)这两个高度保守的位点的顺序磷酸化来影响c-Myc蛋白的稳定性。这些磷酸化位点对c-Myc的稳定性有相反的影响，其中S62的磷酸化可以稳定c-Myc，随后T58的磷酸化促进c-Myc泛素依赖的蛋白分解。丝裂原刺激通过包括MAPKs和CDKs在内的一系列激酶诱导S62的磷酸化，从而允许c-Myc在细胞生长反应后的瞬时稳定。然后，蛋白质水平通过GSK3介导的T58磷酸化而下调。然后，c-Myc的双磷酸化形式被磷酸化导向的Pro异构酶Pin1识别，该酶催化Pro 63上的顺式到反式异构化。这使得反式特异性蛋白磷酸酶PP2A-B56能够去除稳定的S62磷酸。然后，T58磷酸化的c-Myc成为多泛素化的底物，并被E3泛素连接酶SCFFBW7降解。最近的研究表明，Axin1支架蛋白协调这一c-Myc降解途径。重要的是，这一过程在人类癌症中可能会受到损害，因为多个测试样本显示S62磷酸化增强，T58磷酸化降低，c-Myc稳定性增加；在c-Myc稳定的人类癌症中发现了Axin1的损伤。此外，Axin1存在于Myc靶基因启动子上，这表明c-Myc的活性和降解可能是耦合的。新的数据表明，Pin1在调节c-Myc方面具有双重作用，既增强了c-Myc的转录活性，又刺激了其周转。这一提议的中心假设是，Pin1通过增强其对启动子的招募来增加S62磷酸化的c-Myc的转录活性，在启动子中，它随后被包含GSK3、PP2A-B56和Pin1的Axin1核破坏复合体关闭，并且这一过程可以在癌细胞中解除调控，从而增强Myc的致癌活性。我们将通过以下三个具体目标来检验这一假说：1)检测Pin1在协调c-Myc转录活性与Axin1介导的破坏中的作用；2)分析在未转化细胞和癌细胞中Axin1-Myc破坏复合体的调节；以及3)研究Pin1介导的c-Myc激活和Axin1介导的c-Myc降解在人类癌症中的生物学相关性，并在体外和体内建立模型。这些目标的完成将揭示控制c-Myc活性和表达的新的分子机制，涉及肿瘤抑制蛋白Axin1和多功能Pin1 Pro-异构酶。总之，这些目标将提供关于调控c-Myc癌蛋白致瘤潜力的细胞机制的关键新信息，这可能极大地有助于寻找c-Myc靶向疗法来治疗癌症患者。

DESCRIPTION (provided by applicant): The c-Myc oncoprotein is a critical regulator of cellular function and its overexpression has been tightly linked to human cancer. Expression of c-Myc is regulated at many levels including protein stability. A complex signaling pathway affects c-Myc protein stability through the sequential phosphorylation of two highly conserved sites, Serine 62 (S62) and Threonine 58 (T58). These phosphorylation sites have opposing effects on c-Myc stability, where phosphorylation at S62 can stabilize c-Myc; subsequent phosphorylation at T58 promotes c-Myc ubiquitin-dependent proteolysis. Mitogen stimulation induces S62 phosphorylation through a number of kinases including MAPKs and CDKs to allow transient stabilization of c-Myc following a cell growth response. Protein levels are then downregulated through T58 phosphorylation, mediated by GSK3 . The dually phosphorylated form of c-Myc is then recognized by a phosphorylation-directed prolyl isomerase, Pin1, which catalyzes a cis to trans isomerization at Proline 63. This allows the trans-specific protein phosphatase PP2A-B56 to remove the stabilizing S62 phosphate. T58 phosphorylated c-Myc is then a substrate for poly- ubiquitination and degradation by the E3 ubiquitin ligase SCFFBW7. Recent research demonstrates that the Axin1 scaffold protein coordinates this c-Myc degradation pathway. Importantly, this process can be impaired in human cancer as multiple tested samples show enhanced S62 phosphorylation, reduced T58 phosphorylation, and increased c-Myc stability; and lesions in Axin1 have been identified in human cancers with stabilized c-Myc. Moreover, Axin1 is present at Myc target gene promoters suggesting that c-Myc activity and degradation may be coupled. New data demonstrates that Pin1 plays a dual role in regulating c-Myc, both enhancing its transcriptional activity and stimulating its turnover. The central hypothesis of this proposal is that Pin1 increases the transcriptional activity of S62 phosphorylated c-Myc by enhancing its recruitment to promoters, where it is subsequently shut off at the promoter by an Axin1-nucleated destruction complex containing GSK3 , PP2A-B56 and Pin1, and this process can be deregulated in cancer cells potentiating Myc's oncogenic activity. This hypothesis will be tested with the following three specific aims: 1) examine a role for Pin1 in coordinating c-Myc transcriptional activity with Axin1-mediated destruction; 2) analyze regulation of the Axin1-Myc destruction complex in non-transformed and cancer cells; and 3) investigate the biological relevance of Pin1-mediated activation and Axin1-mediated degradation of c- Myc in human cancer and model this in vitro and in vivo. Completion of these aims will reveal novel molecular mechanisms that control c-Myc activity and expression involving the tumor suppressor protein, Axin1, and the multifunctional Pin1 prolyl-isomerase. Together, these aims will provide critical new information about cellular mechanisms that regulate the tumorigenic potential of the c-Myc oncoprotein, which could greatly aid in the search for c-Myc targeted therapy to treat cancer patients.