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。 公共卫生相关性：c-Myc蛋白的高表达在许多不同的人类癌症中被广泛观察到。这项研究的目的是揭示控制这种有效蛋白质的活性和表达水平的新的细胞机制。了解增加或减少c-Myc活性和表达的机制对于未来针对这种促肿瘤蛋白的治疗的发展至关重要。

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. PUBLIC HEALTH RELEVANCE: Elevated expression of the c-Myc protein is widely observed in many different human cancers. The purpose of this research is to reveal new cellular mechanisms that control both the activity and expression level of this potent protein. Understanding mechanisms that increase or decrease c-Myc's activity and expression is critical to the development of future therapies targeting this tumor promoting protein.