Chemical Approaches to Cell Signaling Enzymes

摘要： 这项长期竞争的续订申请CA74305涉及我们的申请计划 细胞信号传递中的化学和生化方法，以增强我们对 蛋白质翻译后修饰(PTM)及其涉及的途径。在下一个 周期中，我们计划研究三种关键酶PTEN、WWP2和 AKT1与促进癌症的磷脂PIP3(磷脂酰基)有关 3，4，5-三磷酸)。PTEN是一种最常见的突变肿瘤抑制基因 基因，催化PIP3转化为PIP2，并以这种方式拮抗 PI3-激酶致癌信号通路的作用。WWP2是Hect域E3 连接酶(泛素转移酶)，靶向PTEN进行破坏，被认为是 致癌基因。AKT1也是一种癌基因，是一种丝氨酸/苏氨酸蛋白激酶，由 PIP3和促进肿瘤的形成，是一个被广泛研究的癌症治疗靶点。 PTEN、WWP2和AKT1各自经历多个磷酸化，这些磷酸化 被提议具有重要的监管影响。我们的研究计划将继续 适应和使用蛋白质半合成方法，包括新描述的 酶催化的表达蛋白连接(EPL)产生这些信号酶 包含必要的PTM和适当的模拟来询问这些 磷酸化影响这些与癌症相关的关键分子的结构和功能 酵素。我们的三个具体目标是：1.优化酶催化表达的蛋白质 结论：1.连接(EPL)用于蛋白质半合成，并应用于PTEN调控的研究； 阐明WWP2 E3泛素连接酶调控的分子基础；3.测定 AKT1 C端磷酸化对其结构、活性、作用机制的影响 调节、底物专一性和抑制剂敏感性。针对特定站点进行修改 信号酶，我们将结合动力学分析，X射线结晶学， 蛋白质微阵列和基于细胞的研究，以阐明关键的调控和信号转导 功能。在完成这项研究工作后，我们应该会有一个更加清晰的 了解PIP3信令网络。此外，这些研究应该为 抗PTEN-AKT1通路失调的新治疗策略 癌症。

Abstract: This longstanding competing renewal application CA74305 concerns our plans to apply chemical and biochemical approaches in cell signaling to enhance our understanding of protein post-translational modifications (PTMs) and the pathways involved. In the next cycle, we plan to investigate the regulation of three key enzymes, PTEN, WWP2, and AKT1 that are connected to the cancer promoting phospholipid, PIP3 (phosphatidyl 3,4,5-triphosphate). PTEN is one of the most commonly mutated tumor suppressor genes and catalyzes the conversion of PIP3 to PIP2 and in this way antagonizes the action of the PI3-kinase oncogenic signaling pathway. WWP2 is a HECT domain E3 ligase (ubiquitin transferase) that targets PTEN for destruction and is considered to be an oncogene. AKT1, also an oncogene, is a protein Ser/Thr kinase that is activated by PIP3 and drives tumor formation and is an intensively studied cancer therapeutic target. PTEN, WWP2, and AKT1 are each subjected to multiple phosphorylations that are proposed to have important regulatory implications. Our research program will continue to adapt and employ protein semisynthetic methods including the newly described enzyme-catalyzed expressed protein ligation (EPL) to generate these signaling enzymes containing the requisite PTMs and appropriate mimics to interrogate how these phosphorylations influence the structure and function of these key cancer-related enzymes. Our three specific aims are: 1. Optimize enzyme-catalyzed expressed protein ligation (EPL) for protein semisynthesis and apply to the study of PTEN regulation; 2. Elucidate the molecular basis of WWP2 E3 ubiquitin ligase regulation; 3. Determine the role of C-terminal phosphorylation of AKT1 on its structure, activity, mechanism of regulation, substrate specificity, and inhibitor sensitivity. With site-specifically modified signaling enzymes in hand, we will integrate kinetic analysis, X-ray crystallography, protein microarrays, and cell-based studies to clarify key regulatory and signaling features. Upon completion of this research effort, we should have a much clearer understanding of the PIP3 signaling network. Moreover, these studies should pave the way for new therapeutic strategies to combat PTEN-AKT1 pathway dysregulation in cancer.