Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function

Incubation of cells with insulin leads to a transient rise in Tyr phosphory lation of insulin receptor substrate (IRS) proteins, accompanied by elevati on in their Ser(P)/Thr(P) content and their dissociation from the insulin r eceptor (IR), Wortmannin, a phosphatidylinositol 3-kinase inhibitor, select ively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissoci ation from IR, and the decrease in its Tyr(P) content following 60 min of i nsulin treatment. Four conserved phosphorylation sites within the phosphoty rosine binding/SAIN domains of IRS-1 and IRS-S served as in vitro substrate s for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylin ositol 3-kinase, Furthermore, PKB and IRS-1 formed stable complexes in vivo , and overexpression of PKB enhanced Ser phosphorylation of IRS-l. Overexpr ession of PKB did not affect the acute Tyr phosphorylation of IRS-l; howeve r, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced th e rate of Tyr dephosphorylation of IRS-I, while inclusion of vanadate rever sed this effect. These results implicate a wortmannin-sensitive Ser/Thr kin ase, different from PKB, as the kinase that phosphorylates IRS-I and acts a s the feedback control regulator that turns off insulin signals by inductin g the dissociation of IRS proteins from IR, In contrast, insulin-stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine bi nding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein- tyrosine phosphatases and enables it to maintain its Tyr-phosphorylated act ive conformation. These findings implicate PHB as a positive regulator of I RS-l functions.