Intracellular signalling pathways of okadaic acid leading to mitogenesis in Rat1 fibroblast overexpressing insulin receptors: Okadaic acid regulates Shc phosphorylation by mechanisms independent of insulin

Okadaic acid is an powerful inhibitor of serine/threonine protein phosphata ses 1 and 2A. Although it is known as a potent tumour promoter, the intrace llular mechanism by which okadaic acid mediates its mitogenic effect remain s to be clarified. We investigated the effect of okadaic acid on the activa tion of mitogenesis in Rat1 fibroblasts overexpressing insulin receptors. A s previously reported, insulin induced Shc phosphorylation, Shc.Grb2 associ ation, MAP kinase activation, and BrdU incorporation. Okadaic acid also sti mulated tyrosine phosphorylation of Shc and its subsequent association with Grb2 in a time- and dose-dependent manner without affecting tyrosine phosp horylation of insulin receptor beta-subunit and IRS. However, to a lesser e xtent, okadaic acid stimulated MAP kinase activity and BrdU incorporation. Interestingly, preincubation of okadaic acid potentiated insulin stimulatio n of tyrosine phosphorylation of Shc (213% of control), Shc.Grb2 associatio n (150%), MAP kinase activity (152%), and BrdU incorporation (148%). These results further confirmed the important role of Shc, but not IRS, in cell c ycle progression in Rat1 fibroblasts. Furthermore, serine/ threonine phosph orylation appears to be involved in the regulation of Shc tyrosine phosphor ylation leading to mitogenesis by mechanisms independent of insulin signall ing. (C) 1999 Elsevier Science Inc.