HYPERPHOSPHORYLATION OF KERATINS BY TREATMENT WITH OKADAIC ACID OF BALB MK-2 MOUSE KERATINOCYTES/

Protein hyper- or hypophosphorylation induced by okadaic acid (OA) tre atment was examined using quiescent cultures of the BALB/MK-2 mouse ep idermal keratinocytes. Treatment with OA enhanced the phosphorylation of five proteins with molecular weights of 65,000, 55,000, 50,000, 28, 000 and 15,000 (p65, p55, p50, p28, and p15, respectively) and decreas ed that of two proteins with molecular weights of 22,000 and 20,000 (p 22 and p20, respectively). The two major phosphorylated proteins, p65 and p55, were identified as type II and type I keratins, respectively, by immunoblotting and immunoprecipitation with keratin specific antib odies. Serine was the only phosphoamino acid residue in hydrolysates o f the P-32-labeled keratins purified from OA-treated cells. Two-dimens ional tryptic peptide maps of the phosphorylated keratins showed that the hyperphosphorylation was largely due to phosphorylation at several additional sites in both keratins. The hyperphosphorylation of kerati ns induced by OA treatment resulted in a drastic change in their solub ility. This change closely correlated with reorganization of the kerat in filament network, which finally collapsed into large perinuclear ag gregates. Concomitantly the cells changed from a typical epithelial sh ape to a round shape. Of several protein kinase inhibitors tested, onl y staurosporine interfered with this OA-induced morphological change a nd reorganization of the keratin network.