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

描述（由申请人提供）：磷酸肌肽3-激酶是增殖、运动、凋亡和囊泡运输的关键调节因子，在人类癌症中被组成性激活。IA类酶由一个调控亚基（p85）组成，它抑制一个不同的催化亚基（p110）。调节p110所需的p85的最小片段是一个卷曲卷曲的区域，即iSH2结构域，连接到单个SH2结构域（nSH2结构域）。分离的iSH2结构域足以结合p110，但抑制作用还需要nSH2结构域连接到iSH2结构域。nSH2结构域是如何发挥作用的？利用位点定向半胱氨酸诱变和EPR光谱，iSH2结构域已被证明是一个构象刚性卷曲线圈，由两个100 a长的反平行螺旋组成。这导致了一种新的模型，其中iSH2结构域结合p110，促进nSH2结构域和p110之间的额外抑制接触。该模型得到了最近的结构研究的支持，这些研究定义了远端iSH2结构域的一个区域，该区域与nSH2结构域密切接触。截断iSH2结构域使该区域消失，从而取消p85对p110的抑制作用。本研究验证了一个假设，即nSH2结构域的取向是p110活性的关键决定因素，并且这种取向是由nSH2结构域和iSH2结构域之间的特定接触维持的。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的结构，当被磷酸肽或致癌突变激活时，以及被丝氨酸自磷酸化抑制时。目的2使用EPR光谱研究p85/p110二聚体背景下nSH2和iSH2结构域的相对取向，并验证p85的不同激活输入（活化的Cdc42与BCR结构域的结合与磷酸化肽与SH2结构域的结合）通过类似机制发挥作用的假设。目的3将使用基于质谱的氧化足迹识别p85/p110的接触，确定这些接触如何被p85/p110的活化剂/抑制剂调节，并探索p85的三级组织。这些目标的完成将大大增加我们对p85/p110 PI 3-激酶的理解，并导致其在人类癌症中的激活机制的见解。

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.