Second-Messenger Induced Activation of PDK1 and PKB

描述(由申请人提供)：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(PI3K)的激活，激活的PI3K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)P3，它介导各种细胞生长和生存信号蛋白的膜移位，包括磷脂酰肌醇依赖激酶-1(PDKI)和蛋白激酶B(PKB，也称为Akt)。PDK1和PKB除了催化或激活区外，还含有一个Pleckstrin Homology(PH)区，它与第二信使结合，导致PDK1和PKB的磷酸化和激活。肿瘤抑制基因磷脂酰肌醇3-磷酸酶(PTEN，第10号染色体缺失的磷酸酶和张力蛋白同源物)下调PI3K对细胞生长和存活的刺激，已表明PTEN在多种人类癌症中发生突变。本研究的主要目的是(I)确定人PDK1和PKB的PH结构域介导的膜靶向性的结构基础；(Ii)确定PH结构域如何与膜结合的第二信使结合导致它们各自的激酶域的催化激活。结合高分辨率异核多维核磁共振方法、核Overhauser效应和核弛豫速率，将确定INS(1，3，4，5)P4结合对细菌表达的人PDK1和PKB的重组15N和13C同位素标记的PH结构域构建物的溶液结构和动力学的影响。此外，将PDK1和PKB的重组SN-同位素标记的PH结构域与其相应的细菌表达的重组未标记的激酶结构域进行剪接，以确定INS(1，3，4，5)P4与PH结构域结合对PH结构域相对于相应的激酶结构域的构象、动力学和位置的影响。最后，通过测量INS(1，3，4，5)P4与PH域的结合对与核苷酸、金属或蛋白质底物结合、构象变化和共价催化相关的平衡和激活自由能的影响，阐明了PDK1和PKB的激活模式。这种结构和机理的理解将有助于通过将底物类似物的结合自由能与磷脂第二信使的肌醇极性头基的类似物“连接”在有效和选择性抑制剂的合理设计中。

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.