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.