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

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，在人类癌症中被组成性激活。IA类酶由抑制不同催化亚基（p110）的调节亚基（p85）组成。调节p110所需的p85的最小片段是一个卷曲螺旋区，iSH 2结构域，连接到一个单一的SH 2结构域（nSH 2结构域）。分离的iSH 2结构域足以与p110结合，但抑制作用还需要与iSH 2结构域连接的nSH 2结构域。nSH 2结构域如何发挥作用？使用定点半胱氨酸诱变和EPR光谱，已被证明是iSH 2结构域是一个构象刚性卷曲螺旋组成的两个100 A长的反平行螺旋。这导致了一种新的模型，其中iSH 2结构域结合p110，促进nSH 2结构域和p110之间的额外抑制性接触。该模型得到了最近的结构研究的支持，该研究定义了与nSH 2结构域密切接触的远端iSH 2结构域的区域。去除该区域的iSH 2结构域的截短废除p85对p110的抑制。目前的建议测试的假设，即nSH 2域的方向是一个关键的决定因素p110活性，这种方向是由特定的nSH 2域和iSH 2域之间的接触保持。目的1将使用NMR光谱来定义p85的nSH 2-iSH 2-cSH 2片段在其基础状态下，当被磷酸肽或致癌突变激活时，以及当被丝氨酸自磷酸化抑制时的结构。目的2使用EPR光谱检查的背景下的p85/p110二聚体的nSH 2和iSH 2结构域的相对取向，并测试的假设，不同的激活输入p85（激活Cdc 42的BCR结构域的结合与结合的磷酸肽的SH 2结构域）发挥其作用，通过类似的机制。目标3将使用基于质谱的氧化足迹来识别p85/p110接触，确定这些接触是如何被p85/p110的激活剂/抑制剂调节的，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.