Insulin stimulates PKC zeta-mediated phosphorylation of insulin receptor substrate-1 (IRS-1) - A self-attenuated mechanism to negatively regulate thefunction of IRS proteins

Incubation of rat hepatoma Fao cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins. This is followed by elevation in their P-Ser/Thr content, and their dissociation from the insulin receptor (IR), Wortmannin, a phosphatidylinositol 3-kinas e (PI3K) inhibitor, abolished the increase in the P-Ser/Thr content of IRS- 1, its dissociation from the IR, and the decrease in its P-Tyr content foll owing 60 min of insulin treatment, indicating that the Ser kinases that neg atively regulate IRS-l function are downstream effectors of PI3K. PKC zeta fulfills this criterion, being an insulin-activated downstream effector of PI3K, Overexpression of PKC zeta in Fao cells, by infection of the cells wi th adenovirus-based PKC zeta construct, had no effect on its own, but it ac celerated the rate of insulin-stimulated dissociation of IR IRS-1 complexes and the rate of Tyr dephosphorylation of IRS-1, The insulin-stimulated neg ative regulatory role of PKC zeta was specific and could not be mimic by in fecting Fao cells with adenoviral constructs encoding for PKC alpha, delta, or eta. Because the reduction in P-Tyr content of IRS-1 was accompanied by a reduced association of IRS-1 with p85, the regulatory subunit of PI3K, i t suggests that this negative regulatory process induced by PKC zeta has a built-in attenuation signal. Hence, insulin triggers a sequential cascade i n which PI3K-mediated activation of PKC zeta inhibits IRS-1 functions, redu ces complex formation between IRS-1 and PI3K, and inhibits further activati on of PKC zeta itself. These findings implicate PKC zeta as a key element i n a multistep negative feedback control mechanism of IRS-1 functions.