Identification and characterization of regulatory mechanisms in the insulin receptor signal transduction pathway

Insulin resistance has been identified as a major causative factor in the pathogenesis of Type II diabetes. While initially believed to result from decreases in receptor or transporter expression or function, resistance to insulin is now thought mediated by changes in expression or function of intermediate protein(s) critical for transduction of the insulin signal. Activation of phosphatidylinositol-3 kinase (PI3-K) plays an essential role in insulin signaling. Protein kinase B (PKB/Akt) functions downstream of PI3-K and activation of this kinase requires phosphorylation at Thr308 and Ser 473. Phosphorylation of PKB at Thr308 is mediated by the newly identified 3′-phosphoinositide dependent protein kinase (PDK1). How PDK1 is activated and regulated in cells is currently unknown; however, subcellular localization and substrate-induced conformation changes have been suggested to play roles in regulating PDK1 function in cells. Our studies reveal that PDK1 undergoes autophosphorylation in vitro and in cells, and this phosphorylation may be mediated via intra- and intermolecular mechanisms. Using various biochemical techniques, we identified Ser 244 in the activation loop as a major site of mPDK1 phosphorylation in cells, and showed that phosphorylation at this site is mediated mainly through a trans-mechanism. We have also found that mPDK1 autophosphorylates at Ser399 and Thr516, and phosphorylation at these sites is mediated mainly through a cis-mechanism. Replacing Thr 516 of mPDK1 with glutamate, but not alanine, resulted in an increase in mPDK1 in vivo activity towards PKB to the same level as insulin stimulation. Similarly, we found that replacing Ala280 of mPDK1 with valine resulted in constitutive activation of PDK1 in cells. Deletion of the Pleckstrin homology domain (PH) of mPDK1 increases its autophosphorylation in vitro, but inhibits mPDK1-mediated PKB/Akt phosphorylation in cells, suggesting that this domain may play a duel role in regulating PDK1 and its function in vivo.; PI3-K is activated by binding with activated IRS1/2 following insulin stimulation. Alternatively, binding of activated p21Ras to the catalytic p110 subunit has been shown to activate PI3-K linking multiple pathways to the downstream activation of PKB/Akt. Insulin-mediated activation of p21Ras is negatively regulated by the 62 kDa protein d&barbelow;ownstream o&barbelow;f tyrosine k&barbelow;inases (p62dok), which is phosphorylated by activated insulin receptor in vitro and undergoes insulin-stimulated phosphorylation in cells. Given the role of phosphorylation in regulating signaling events, we hypothesized that phosphorylation of p62dok by the insulin receptor may play an important role in the regulation of Ras-mediated downstream signaling. Our data identified p62dok as a direct substrate for the insulin receptor in cells. In addition, insulin receptor-mediated phosphorylation of p62dok at Tyr362 and Tyr 398 was crucial for negatively regulated Ras downstream signaling to the PI-3K, but not the mitogen-activated protein kinase (MAPK) signaling pathways. Taken together, our results identify and characterize phosphorylation events in the insulin receptor signal transduction pathway which may function to regulate the insulin signal, and provides important information which may offer a better understanding of factors which lead to insulin resistance, the major causative factor in Type II diabetes.