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

说明(申请人提供)：磷脂酰肌醇3-激酶是增殖、运动、凋亡和囊泡运输的关键调节因子，在人类癌症中被结构性激活。IA类酶由一个调节亚单位(P85)组成，它抑制一个独特的催化亚单位(P110)。调节p110所需的p85的最小片段是一个卷曲区域，即连接到单个SH2结构域(nSH2结构域)的iSH2结构域。分离的iSH2结构域足以与p110结合，但抑制另外需要连接到iSH2结构域的nSH2结构域。NSH2结构域是如何发挥作用的？利用半胱氨酸定点突变和EPR波谱分析表明，iSH2结构域是由两个100A长的反平行螺旋组成的刚性螺旋。这导致了一种新的模型，其中iSH2结构域与p110结合，促进了nSH2结构域和p110之间额外的抑制性接触。这一模型得到了最近的结构研究的支持，该研究定义了远端iSH2结构域的一个区域，该区域与nSH2结构域密切接触。删除该区域的iSH2结构域的截断取消了对p110的p85抑制。本提案检验了这样的假设，即nSH2结构域的取向是p110活性的关键决定因素，并且这一取向是通过nSH2结构域和iSH2结构域之间的特定接触来维持的。目的1将使用核磁共振波谱来确定p85的nSH2-iSH2-cSH2片段在其基础状态下，当被磷酸肽或致癌基因突变激活时，以及当被丝氨酸自动磷酸化抑制时的结构。目的2利用EPR波谱检测p85/p110二聚体中nSH2和iSH2结构域的相对取向，并检验P85的不同激活输入(活化的CDC42与bcr结构域的结合与磷酸肽与SH2结构域的结合)通过相似的机制发挥作用的假说。AIM 3将使用基于质谱学的氧化足迹技术来识别p85/p110接触，确定这些接触是如何被p85/p110的激活剂/抑制物调制的，并探索p85的第三级组织。这些目标的完成将极大地增加我们对p85/p110PI 3-激酶的理解，并导致对其在人类癌症中激活的机制的深入了解。

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 two 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.