INSULIN STIMULATES THE PHOSPHORYLATION OF THE 66-KILODALTON AND 52-KILODALTON SHE ISOFORMS BY DISTINCT PATHWAYS

In contrast to the 52-kDa She isoform, insulin stimulation caused a qu antitative, time-dependent decrease in the SDS-PAGE mobility of 66-kDa She in both Chinese hamster ovary/IR cells and 3T3L1 adipocytes. Alka line phosphatase treatment and direct phosphoamino acid analysis demon strated that insulin stimulated an increase in serine phosphorylation of the 66-kDa isoform but not 52-kDa She, although the latter displaye d a marked increase in tyrosine phosphorylation. To identify the respo nsible kinase pathway, we compared the effects on 66-kDa She serine ph osphorylation by insulin, anisomycin, and osmotic shock, agents that s pecifically activate the ERK, JNK, or both pathways, respectively. Ins ulin and osmotic shock both stimulated a decrease in 66-kDa She mobili ty, whereas anisomycin had no effect. Furthermore, expression of a dom inant-interfering Ras mutant (N17Ras) prevented the insulin-stimulated , but not the osmotic shock-induced serine phosphorylation of 66-kDa S he. Consistent with a MEK-dependent pathway mediating 66-kDa She serin e phosphorylation, the specific MEK inhibitor (PD98059) and expression of a dominant-interfering MEK mutant partially inhibited both the ins ulin and osmotic shock-induced reduction in 66-kDa She mobility. In co ntrast, expression of the MAP kinase phosphatase (MKP-1) completely pr evented ERK activation but did not inhibit the serine phosphorylation of 66-kDa She. These data demonstrate that insulin stimulates the seri ne phosphorylation of tile 66-kDa She isoform through a MEK-dependent mechanism.