Second-Messenger Induced Activation of PDK1 and PKB

描述（申请人提供）：生长因子与受体酪氨酸激酶的结合事件导致磷脂酰肌醇3-激酶（PI 3 K）的活化，活化的PI 3 K产生膜结合的第二信使磷脂酰肌醇3，4-二磷酸[PI（3，4）P2]和PI（3，4，5）P3，介导多种细胞生长和存活信号蛋白的膜转位，包括磷酸肌醇依赖性激酶-1（PDKI）和蛋白激酶B（PKB，也称为Akt）。除了催化或激酶结构域之外，PDK 1和PKB还含有普列克底物蛋白同源（PH）结构域，其结合第二信使并导致PDK 1和PKB的磷酸化和活化。肿瘤抑制基因磷脂酰肌醇3-磷酸酶（PTEN，在染色体TEN上缺失的磷酸酶和TENSin同源物）下调细胞生长和存活的PI 3 K刺激，并且已经显示PTEN在多种人类癌症中突变。拟议研究的主要目标是（i）确定由人PDK 1和PKB的PH结构域介导的膜靶向特异性的结构基础，以及（ii）确定PH结构域与膜结合第二信使的结合如何导致其各自激酶结构域的催化活化。将使用高分辨率异相多维NMR方法、核Overhauser效应和核弛豫速率的组合来确定Ins（1，3，4，5）P4结合对细菌表达的重组15 N-和13 C-同位素标记的人PDK 1和PKB的PH结构域构建体的溶液结构和动力学的影响。此外，PDK 1和PKB的重组SN-同位素标记的PH结构域构建体将与其相应的细菌表达的重组未标记的激酶结构域剪接，以确定与PH结构域结合的Ins（1，3，4，5）P4对PH结构域相对于相应激酶结构域的构象、动力学和位置的影响。最后，PDK 1和PKB的激活模式将通过测量Ins（1，3，4，5）P4结合到PH结构域上对与核苷酸、金属或蛋白质底物、构象变化和共价催化结合相关的平衡和激活自由能的影响来阐明。这种结构和机制的理解将是有用的，在合理设计的有效和选择性的抑制剂，通过“链接”的自由能结合的底物类似物与类似物的肌醇极性头基团的磷脂第二信使。

DESCRIPTION (provided by applicant): Growth factor binding events to receptor tyrosine kinases result in activation of phosphatidylinositol 3-kinase (PI3K), and activated PI3K generates the membrane-bound second messengers phosphatidylinositol 3,4-diphosphate [PI(3,4)P2] and PI(3,4,5)P3, which mediate membrane translocation of a variety of cell growth and survival signaling proteins including the phosphoinositide-dependent kinase-1 (PDKI) and protein kinase B (PKB, also known as Akt). In addition to a catalytic or kinase domain, PDK1 and PKB also contain a pleckstrin homology (PH) domain, which binds to the second messenger and results in the phosphorylation and activation of both PDK1 and PKB. The tumor suppressor gene phosphatidylinositol 3-phosphatase (PTEN, Phosphatase and TENsin homologue deleted on chromosome TEN) down regulates PI3K stimulation of cell growth and survival, and it has been shown that PTEN is mutated in a variety of human cancers. The major objectives of the proposed research are (i) to determine the structural basis of specificity for membrane targeting mediated by the PH domains of human PDK1 and PKB and (ii) to determine how binding of the PH domains to the membrane-bound second messenger leads to the catalytic activation of their respective kinase domains. A combination of high-resolution heteronuclear multidimensional NMR methods, nuclear Overhauser effects, and nuclear relaxation rates will be used to determine the effects that Ins(1,3,4,5)P4 binding has on the solution structures and dynamics of the bacterially expressed recombinant 15N- and 13C-isotopically labeled PH domain constructs of both human PDK1 and PKB. In addition, the recombinant SN-isotopically labeled PH domain constructs of both PDK1 and PKB will be spliced with their corresponding bacterially expressed recombinant unlabeled kinase domains to determine the effects that Ins(1,3,4,5)P4 binding to the PH domain has on the conformations, dynamics, and position of the PH domain with respect to the corresponding kinase domain. Finally, the modes of activation of PDK1 and PKB will be elucidated by measuring the effects of Ins(1,3,4,5)P4 binding to the PH domains on the equilibrium and activation free energies associated with binding of nucleotide, metal, or protein substrates, conformational changes, and covalent catalysis. Such structural and mechanistic understanding will be useful in the rational design of potent and selective inhibitors by "linking" the free energies of binding of substrate analogs with analogs of the inositol polar head group of the phospholipid second messenger.