Cellular Mechanisms Controlling Myc Protein Stability

描述（由申请人提供）：c-Myc癌蛋白是细胞功能的关键调节因子，其过表达与人类癌症密切相关。c-Myc的表达在包括蛋白质稳定性在内的许多水平上受到调节。一个复杂的信号通路通过两个高度保守位点丝氨酸62 （S62）和苏氨酸58 （T58）的顺序磷酸化影响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位点的磷酸化可以稳定c-Myc；随后T58位点的磷酸化促进c-Myc泛素依赖性蛋白水解。丝裂原刺激通过包括mapk和CDKs在内的许多激酶诱导S62磷酸化，从而允许c-Myc在细胞生长反应后短暂稳定。然后通过GSK3介导的T58磷酸化下调蛋白水平。c-Myc的双磷酸化形式随后被磷酸化导向的脯氨酸异构酶Pin1识别，该酶催化脯氨酸63的顺式到反式异构化。这允许跨特异性蛋白磷酸酶PP2A-B56去除稳定的S62磷酸盐。T58磷酸化的c-Myc随后成为E3泛素连接酶SCFFBW7的多泛素化和降解的底物。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，这一过程可能在人类癌症中受损，因为多个测试样本显示S62磷酸化增强，T58磷酸化减少，c-Myc稳定性增加；在c-Myc稳定的人类癌症中发现了Axin1的病变。此外，Axin1存在于Myc靶基因启动子上，表明c-Myc活性和降解可能是耦合的。新的数据表明，Pin1在调节c-Myc中起双重作用，既增强其转录活性，又刺激其周转。该建议的中心假设是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，随后在启动子处被含有GSK3， PP2A-B56和Pin1的axin1核破坏复合体关闭，这一过程可以在癌细胞中解除调控，增强Myc的致癌活性。这一假设将通过以下三个特定目的进行验证：1)检查Pin1在协调c-Myc转录活性与axin1介导的破坏中的作用；2)分析未转化细胞和癌细胞中Axin1-Myc破坏复合体的调控；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活性和表达的新分子机制，包括肿瘤抑制蛋白Axin1和多功能的Pin1脯氨酸异构酶。总之，这些目标将提供关于调节c-Myc癌蛋白致瘤潜能的细胞机制的重要新信息，这可能极大地有助于寻找c-Myc靶向治疗癌症患者的方法。

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.