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.
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