Characterization of PDK1 regulation and function in the insulin-stimulated PI3-kinase pathway

Three'-OH-phosphoinositide dependent protein kinase-1(PDK1) is a 65 kDa cytosolic protein involved in the PI 3-kinase signal transduction pathway. Whether and how PDK1 is regulated remains largely uncharacterized. In the present study, it was found that PDK1 undergoes insulin-stimulated and PI 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser 163 may provide a mechanism to positively regulate PDK1 activity and function in cells.; Insulin resistance is a major cause of Type II diabetes mellitus and is partly due to decreased insulin signaling through the PI 3-kinase pathway. However, the direct targets of insulin resistance are unknown. One mechanism of insulin resistance suggests that increased free fatty acids (FFA) stimulate Protein Kinase C (PKC) isozymes which phosphorylate and downregulate the insulin signaling pathway. Stimulating cells with a FFA led to increased phosphorylation of PDK1. In addition, overexpression of PKCtheta stimulates PDK1 phosphorylation in cells and in vitro. Also, FFA inhibit insulin-stimulated Akt phosphorylation by PDK1. Collectively, these data suggest that FFA-induced PKC phosphorylation of PDK1 may negatively regulate insulin-stimulated Akt phosphorylation.; Previously, our lab has characterized two constitutively active mutants of PDK1 (PDK1CA) which exhibit increased activity and phosphorylation of AktT308 in Chinese Hamster Ovary cells overexpressing the human insulin receptor. The use of PDK1CA provides us with an approach to study downstream signaling events such as the regulation of glycogen synthesis. Here we show that overexpression of PDK1CA induces phosphorylation of GSK3betaSer9, and leads to decreased phosphorylation of glycogen synthase in mouse hepatocytes. In addition, PDK1 CA is sufficient for activation of glycogen synthase in murine hepatocytes. Further characterization of this and other mechanisms of PDK1 signaling may contribute to our understanding of insulin signaling and resistance.