p85/p110 PI3 Kinase-Structure, function and Physiology

说明(申请人提供)：磷脂酰肌醇3-激酶(PI-3-激酶)是细胞增殖、运动和细胞凋亡的重要调节因子。IA类PI3-激酶是一种二聚体，含有不同的调节亚基(P85)和催化亚基(P110)，这两个亚基在人类癌症中都是经常突变的。虽然学术界和工业界已经将大量的精力应用于这些酶的研究，但PI 3-激酶调控的一些关键方面仍然知之甚少。例如，尽管p110催化亚基已经结晶，但关于p85的不同结构域如何在空间定位，或者p85的突变如何导致PI 3-激酶调节丧失的信息很少。P110催化亚基在PTEN缺失的前列腺癌细胞增殖和肿瘤发生中的具体作用已被确定，但调控这些细胞中p110的机制尚不清楚。虽然已经在结肠癌和胶质母细胞瘤中发现了新的P85突变，但这些突变对体内肿瘤发生和转移的影响尚不清楚。目前的提案同时使用生化和遗传方法来解决有关PI3-激酶信号在癌症中的基本问题。目的1使用单分子FRET测量来生成p85调节亚基的空间图谱，并确定PI 3-激酶激活剂的结合如何改变p85的构象以及与p110催化亚基的相互作用。目的2研究G亚基激活p110的机制。将使用遗传和生物化学方法确定p110-G相互作用的位置。然后，这些位点将发生突变，以测试PTEN缺失的前列腺癌细胞中p110对G结合信号的要求。目的3研究P85突变体在结肠癌和胶质母细胞瘤细胞系中的信号转导，以及最近发现的突变体所在的肿瘤类型。慢病毒方法将被用来击倒内源P85，并用野生型或突变型P85拯救。这些突变体对体外细胞生长和运动以及体内肿瘤发生和转移的影响将被研究。这一提议将定义PI-3-激酶信号和调控的新方面，并将提供更好的了解可用作药物靶点的结构特征。

DESCRIPTION (provided by applicant): Phosphoinositide 3-kinases (PI 3-kinases) are critical regulators of proliferation, motility, and apoptosis. The Class IA PI 3-kinase is a dimer containing distinct regulatory (p85) and catalytic (p110) subunits, both of which are frequently mutated in human cancer. While a large amount of energy in academia and industry has been applied to the study of these enzymes, there are critical aspects of PI 3-kinase regulation that remain poorly understood. For example, although the p110 catalytic subunit has been crystallized, there is little information as to how the distinct domains of p85 are oriented in space, or how mutants of p85 lead to a loss of PI 3-kinase regulation. A specific role of p110 catalytic subunit in the proliferation and tumorigenesis of PTEN null prostate cancer cells has been identified, but the mechanisms that regulate p110 in these cells are not clear. Although novel mutations of p85 have been identified in colon cancer and glioblastoma, the effects of these mutations on tumorigenesis and metastasis in vivo are not known. The current proposal uses both biochemical and genetic approaches to address fundamental questions concerning PI 3-kinase signaling in cancer. Aim 1 uses single molecule FRET measurements to produce a spatial map of the p85 regulatory subunit, and to determine how the binding of PI 3-kinase activators alters p85 conformation and interactions with the p110 catalytic subunit. Aim 2 examines the mechanism of p110 activation by G subunits. Sites of p110-G interactions will be defined using genetic and biochemical methods. These sites will then be mutated, in order to test the requirement for G binding in signaling by p110 in PTEN null prostate cancer cells. Aim 3 will study the signaling by p85 mutants in colon cancer and glioblastoma cell lines, the tumor types in which the recently described mutants were identified. Lentivirus methods will be used to knock down endogenous p85 and rescue with wild type or mutant p85. The effects of these mutants on in vitro cell growth and motility, and in vivo tumorigenesis and metastasis, will be studied. This proposal will define novel aspects of PI 3-kinase signaling and regulation, and will provide a better understanding of structural features that could be exploited as drug targets.