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的表达在许多水平上受到调节，包括蛋白质稳定性。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）的连续磷酸化影响c-Myc蛋白的稳定性。这些磷酸化位点对c-Myc稳定性有相反的影响，其中S62处的磷酸化可以稳定c-Myc;随后T58处的磷酸化促进c-Myc泛素依赖性蛋白水解。促分裂原刺激通过许多激酶（包括MAPK和CDK）诱导S62磷酸化，以允许c-Myc在细胞生长应答后的瞬时稳定。然后通过T58磷酸化下调蛋白水平，由GSK 3介导。然后，c-Myc的双重磷酸化形式被磷酸化指导的脯氨酰异构酶Pin 1识别，Pin 1催化脯氨酸63处的顺式至反式异构化。这允许反式特异性蛋白磷酸酶PP 2A-B56去除稳定的S62磷酸。然后，T58磷酸化的c-Myc是E3泛素连接酶SCFFBW 7的聚泛素化和降解的底物。最近的研究表明，Axin 1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，因为多个测试样品显示S62磷酸化增强，T58磷酸化减少，c-Myc稳定性增加;并且在具有稳定c-Myc的人类癌症中已经鉴定出Axin 1中的病变。此外，Axin 1存在于Myc靶基因启动子处，表明c-Myc活性和降解可能是偶联的。新的数据表明，Pin 1在调节c-Myc中起着双重作用，既增强其转录活性，又刺激其周转。该提议的中心假设是Pin 1通过增强其向启动子的募集来增加S62磷酸化的c-Myc的转录活性，随后在启动子处通过含有GSK 3、PP 2A-B56和Pin 1的Axin 1有核破坏复合物关闭，并且该过程可以在增强Myc的致癌活性的癌细胞中去调节。该假设将通过以下三个具体目标进行检验：1）检查Pin 1在协调c-Myc转录活性与Axin 1介导的破坏中的作用; 2）分析非转化细胞和癌细胞中Axin 1-Myc破坏复合物的调节;以及3）研究Pin 1介导的c-10活化和Axin 1介导的c-10降解的生物学相关性。Myc在人类癌症中的作用以及体外和体内模型。这些目标的完成将揭示新的分子机制，控制c-Myc的活性和表达，涉及肿瘤抑制蛋白，Axin 1，和多功能Pin 1脯氨酰异构酶。总之，这些目标将提供有关调节c-Myc癌蛋白致瘤潜力的细胞机制的重要新信息，这将极大地有助于寻找c-Myc靶向治疗癌症患者的方法。 公共卫生相关性：在许多不同的人类癌症中广泛观察到c-Myc蛋白表达升高。这项研究的目的是揭示控制这种有效蛋白的活性和表达水平的新细胞机制。了解增加或减少c-Myc活性和表达的机制对于开发针对这种肿瘤促进蛋白的未来疗法至关重要。

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.