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

描述（申请人摘要）：磷酸肌醇3-激酶是关键的 增殖、运动、凋亡和囊泡运输的调节剂， 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，其受 受体酪氨酸激酶，并在完整的细胞产生第二信使PI 约3、4、5]P3.这些酶含有单独的调节（p85）和催化（p85）酶。 （p110）结构域。我们以前已经证明，p85/p110二聚体被激活， 当磷酸酪氨酸肽与p85的SH 2结构域结合时。我们还 确定p85调节亚基是p110的抑制剂， p85/p110异二聚体的激活反映了 p110的去抑制。调节细胞凋亡所需的p85的最小片段， p110是一个假定的卷曲螺旋区，即iSH 2（inter-SH 2）结构域，连接在 其氨基末端连接到单个SH 2结构域（nSH 2结构域）。分离的nSH 2 结构域不结合p110或影响其活性。分离的iSH 2结构域是 足以与p110的N-末端（残基1-108）结合，但这种结合 不影响p110活性。仅在nSH 2-iSH 2中观察到抑制 片段（下文称为nSH 2-iSH 2）。因此，nSH 2的存在 结构域调节iSH 2结合对p110活性的影响。前三 具体目的是研究nSH 2-iSH 2调节p110的机制。目标1将 使用EPR光谱和LC/MS分析氘交换来测试 nSH 2-iSH 2的预测结构，并确定经历 磷酸肽结合后的构象变化。Aim II将介绍 在nSH 2-iSH 2中的特定位点处的拴系化学蛋白酶，以绘制其 与P110接触的部位。目标III将使用NMR光谱来解决 nSH 2-iSH 2的结构和p110的p85结合结构域。第四，目标是 我们发现在大鼠腺癌细胞系中， p110催化亚单位（p110 a）是EGF刺激的细胞骨架所必需的 反应，而另一种亚型（p110 B）不是。我们假设p110 a 和p110 B激活不同的下游效应子， 与细胞骨架不同;我们将识别下游组件 由p110 a和p110 b不同激活的细胞。成功完成 这些实验将大大推进我们对信号的理解 通过这种关键的和生理上重要的信号传导酶。

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.