P85/p110 PI3 Kinase--Structure, Function and Physiology

描述(申请人摘要)：磷脂酰肌醇3-激酶是关键 增殖、运动、凋亡和囊泡运输的调节者， 其活性和/或表达在人类癌症中升高。这项建议 重点关注IA类(p85/p110)PT 3-激酶，它受 受体酪氨酸激酶，并在完整细胞中产生第二信使PI 约3，4，5]P3。这些酶含有单独的调节性(P85)和催化 (P110)域。我们之前已经证明了p85/p110二聚体被激活 当磷酸酪氨酸肽与P85的SH2结构域结合时。我们还有 确定p85调节亚单位是p110的抑制因子 催化亚单位，而p85/p110异二聚体激活反映了 P110的去抑制作用。P85基因调控所需的最小片段 P110是一个假定的盘绕区域，即iSH2(InterSH2)结构域，连接在 其氨基末端为单一的SH2结构域(nSH2结构域)。隔离的nSH2 结构域不结合p110或影响其活性。隔离的iSH2域是 足以与p110的N端(残基1-108)结合，但这种结合 不影响p110活性。抑制作用仅见于nSH2-iSH2 片段(以下称为nSH2-iSH2)。因此，nSH2的存在 结构域调节iSH2结合对p110活性的影响。前三名 具体目的是研究nSH2-iSH2对p110的调控机制。目标1将 利用电子顺磁共振波谱和液质联用技术对样品进行了分析。 预测nSH2-iSH2的结构并识别经历 磷酸肽结合时的构象变化。AIM II将介绍 在nSH2-iSH2中的特定位置上的系留的化学蛋白水解酶，以便定位其 与p110的接触部位。AIM III将使用核磁共振波谱来解决 NSH2-iSH2的结构和p110的p85结合域。最后，目标四是 我们发现在大鼠腺癌细胞系中，一种亚型 P110催化亚单位(P110a)是表皮生长因子刺激的细胞骨架所必需的。 而另一个亚型(P110B)则不是。我们假设p110a 和p110b激活不同的下游效应器，这些下游效应器耦合 与细胞骨架不同，我们将确定下游组件 它们被p110a和p110b差异激活。成功完成 这些实验将极大地提高我们对信号的理解 由这种关键的和生理上重要的信号酶进行的转导。

DESCRIPTION (Applicant's abstract): Phosphoinositide 3-kinases are critical regulators of proliferation, motility, apoptosis and vesicular trafficking, whose activity and/or expression is elevated in human cancers. This proposal focuses on the Class IA (p85/p 110) PT 3-kinases, which are regulated by receptor tyrosine kinases, and in intact cells produce the second messenger PI about3,4,5]P3. These enzymes contain separate regulatory (p85) and catalytic (p110) domains. We have previously shown that p85/p110 dimers are activated when phosphotyrosine peptides bind to the SH2 domain of p85. We have also established that the p85 regulatory subunit is an inhibitor of the p110 catalytic subunit, and that activation of the p85/p110 heterodimer reflects disinhibition of p110. The minimal fragment of p85 required for regulation of p110 is a putative coiled-coil region, the iSH2 (inter-SH2) domain, linked at its amino terminus to a single SH2 domain (the nSH2 domain). Isolated nSH2 domains do not bind p110 or affect its activity. Isolated iSH2 domains are sufficient to bind to the N-terminus of p110 (residues 1-108), but this binding does not affect p110 activity. Inhibition is only seen with the nSH2-iSH2 fragment (hereafter referred to as nSH2-iSH2). Thus, the presence of the nSH2 domain modulates the effect of iSH2 binding on p110 activity. The first three specific aims examine the mechanism of p110 regulation by nSH2-iSH2. Aim 1 will use EPR spectroscopy and LC/MS analysis of deuterium exchange to test the predicted structure of nSH2-iSH2 and identify regions that undergo conformational changes upon phosphopeptide binding. Aim II will introduce tethered chemical proteases at specific sites in nSH2-iSH2, in order to map its sites of contact with p110. Aim III will use NMR spectroscopy to solve the structure of nSH2-iSH2 and the p85-binding domain of p110. Finally, Aim IV is motivated by our finding that in a rat adenocarcinoma cell line, one isoform of the p110 catalytic subunit (p1lOa) is required for EGF-stimulated cytoskeletal responses, whereas the other isoform (p1lOB) is not. We hypothesize that p110a and p110B activate different sets of downstream effectors, which couple differently to the cytoskeleton; we will identify the downstream components that are differentially activated by p1lOa and p1lOB. Successful completion of these experiments will substantially advance our understanding of signal transduction by this critical and physiologically important signaling enzyme.