p85/p110 PI3 Kinase--Structure, Function and Physiology

p85/p110 PI3 激酶——结构、功能和生理学

• 批准号: 7145342
• 负责人: Jonathan M. Backer
• 金额: $ 35.26万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7145342
• 关键词: biological signal transduction; cytoskeleton; electron spin resonance spectroscopy; endopeptidases; enzyme activity; enzyme structure; intracellular transport; liquid chromatography mass spectrometry; nuclear magnetic resonance spectroscopy; phosphatidylinositol 3 kinase; phosphopeptides; protein isoforms; protein protein interaction; protein transport

DESCRIPTION (provided by applicant): Phosphoinositide 3-kinases are critical regulators of proliferation, motility, apoptosis and vesicular trafficking, and are constitutively activated in human cancers. Class IA enzymes are composed of a regulatory subunit (p85) that inhibits a distinct catalytic subunit (p110). The minimal fragment of p85 required for regulation of p110 is a coiled-coil region, the iSH2 domain, linked to a single SH2 domain (the nSH2 domain). Isolated iSH2 domains are sufficient to bind to p110, but inhibition additionally requires the nSH2 domain linked to the iSH2 domain. How does the nSH2 domain exert its effects? Using site-directed cysteine mutagenesis and EPR spectroscopy, the iSH2 domain has been demonstrated to be a conformationally rigid coiled-coil consisting of 2 100 A-long antiparallel helices. This has led to a new model, in which the iSH2 domain binds p110, facilitating additional inhibitory contacts between the nSH2 domain and p110. This model is supported by recent structural studies defining a region of the distal iSH2 domain that makes a close contact with the nSH2 domain. Truncations of the iSH2 domain that remove this region abolish p85 inhibition of p110. The present proposal tests the hypothesis that the orientation of the nSH2 domain is a critical determinant of p110 activity, and that this orientation is maintained by specific contacts between the nSH2 domain and the iSH2 domain. Aim 1 will use NMR spectroscopy to define the structure of the nSH2-iSH2-cSH2 fragment of p85 in its basal state, when activated by phosphopeptides or oncogenic mutations, and when inhibited by serine autophosphorylation. Aim 2 uses EPR spectroscopy to examine the relative orientation of the nSH2 and iSH2 domains within the context of p85/p110 dimers, and to test the hypothesis that different activating inputs to p85 (binding of activated Cdc42 to the BCR domain versus binding of phosphopeptides to the SH2 domains) exert their effects through similar mechanisms. Aim 3 will use mass spectrometry-based oxidative foot printing to identify p85/p110 contacts, determine how these contacts are modulated by activators/inhibitors of p85/p110, and explore the tertiary organization of p85. Completion of these aims will greatly increase our understanding of the p85/p110 PI 3-kinase, and lead to mechanistic insights into its activation in human cancer.

描述（由申请人提供）：磷酸肌醇3-激酶是增殖，运动，凋亡和囊泡运输的关键调节剂，并且在人类癌症中被组成式激活。 IA类酶由抑制不同催化亚基的调节亚基（P85）组成（P110）。 p110调节所需的p85的最小片段是与单个SH2域（NSH2域）相关的ISH2域的盘绕螺旋区域。分离的ISH2域足以与p110结合，但是抑制作用还需要与ISH2域相关的NSH2域。 NSH2域如何发挥其影响？使用位置定向的半胱氨酸诱变和EPR光谱，ISH2结构域已被证明是一个构象上的刚性螺旋线圈，由200个A长的反平行螺旋组成。这导致了一个新模型，其中ISH2域结合了p110，从而促进了NSH2域和p110之间的其他抑制作用。该模型得到了最近定义远端ISH2结构域区域的结构研究的支持，该区域与NSH2域密切接触。去除该区域的ISH2结构域的截断消除了p110的p85抑制作用。本提案检验了以下假设：NSH2域的方向是p110活性的关键决定因素，并且通过NSH2域和ISH2域之间的特定接触来维持这种方向。 AIM 1将使用NMR光谱法来定义p85的NSH2-ish2-CSH2片段在其基础状态下，当被磷酸肽或致癌突变激活时，以及丝网膜自动磷酸化抑制时。 AIM 2使用EPR光谱检查在p85/p110二聚体的背景下检查NSH2和ISH2结构域的相对取向，并测试以下假设：不同的激活输入与p85（激活CDC42的结合与BCR结合了BCR领域与磷酸肽与SH2 DOMAINS的结合与结合的结合）的相似机制通过相似的机制而产生。 AIM 3将使用基于质谱的氧化脚打印来识别p85/p110触点，确定这些触点是如何通过p85/p110的激活剂/抑制剂调节的，并探索p85的第三级组织。这些目标的完成将大大增加我们对p85/p110 PI 3-激酶的理解，并导致对人类癌症激活的机械见解。