INSULIN INDUCTION OF PROTEIN-KINASE-C-ALPHA EXPRESSION IS INDEPENDENTOF INSULIN-RECEPTOR TYR(1162 1163) RESIDUES AND INVOLVES MITOGEN-ACTIVATED PROTEIN-KINASE KINASE-1 AND SUSTAINED ACTIVATION OF NUCLEAR P44(MAPK)/

We examined the effect of insulin on protein kinase C alpha (PKC alpha ) expression and the implication of the mitogen-activated protein kina se kinase 1 mitogen-activated protein kinase (MAPK) pathway in this ef fect. PKC alpha expression was measured by quantitative RT-PCR and Wes tern blotting using Chinese hamster ovary (CHO) cells overexpressing h uman insulin receptors of the wild type (CHO-R) or insulin receptors m utated at Tyr(1162/1163) autophosphorylation sites (CHO-Y2). In CHO-R cells, insulin caused a time- and concentration-dependent increase in PKC alpha messenger RNA, with a maximum at 6 h and 10(-8) M insulin. T his increase involved a transcriptional mechanism, as it was not due t o stabilization of PKC alpha messenger RNA and was associated with a s imilar increase in the immunoreactive PKC alpha level. Insulin inducti on of PKC alpha expression involved the MEK1-MAPK pathway, as it was 1 ) almost completely suppressed by the potent MEK1 inhibitor PD98059, 2 ) mimicked by the dominant-active MEK1 (S218D/S222D) mutant, and 3) as sociated with sustained MAPK activation. In CHO-Y2 cells in which the early phase of MAPK activation by insulin was lost and only the late a nd sustained phase of activation was observed, insulin signaling of PK C alpha expression was preserved and again involved the MEK1-MAPK path way. Moreover, we show that in both CHO-R and CHO-Y2 cells, insulin st imulation of PKC alpha gene expression was associated with prolonged a ctivation of nuclear p44(MAPK). These results indicate that induction of PKC alpha gene expression by insulin is independent of Tyr(1162/116 3) autophosphorylation sites and correlates with sustained activation of p44(MAPK) at the nuclear level.