Hyperphosphorylation of keratins by treatment with okadaic acid of BALB/MK-2 mouse keratinocytes.
Protein hyper- or hypophosphorylation induced by okadaic acid (OA) treatment was examined using quiescent cultures of the BALB/MK-2 mouse epidermal 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 decreased that of two proteins with molecular weights of 22,000 and 20,000 (p22 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 antibodies. Serine was the only phosphoamino acid residue in hydrolysates of the 32P-labeled keratins purified from OA-treated cells. Two-dimensional 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 keratins induced by OA treatment resulted in a drastic change in their solubility. This change closely correlated with reorganization of the keratin filament network, which finally collapsed into large perinuclear aggregates. Concomitantly the cells changed from a typical epithelial shape to a round shape. Of several protein kinase inhibitors tested, only staurosporine interfered with this OA-induced morphological change and reorganization of the keratin network.
Kasahara
K
K
Department of Cancer Cell Research, University of Tokyo, Japan.
Kartasova
T
T
Ren
X Q
XQ
Ikuta
T
T
Chida
K
K
Kuroki
T
T
D000818:Animals; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D004988:Ethers, Cyclic / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D007382:Intermediate Filaments / Q000378:metabolism / Q000648:ultrastructure; D015603:Keratinocytes / Q000378:metabolism*; D007633:Keratins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D019319:Okadaic Acid; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation
D004988:Ethers, Cyclic; D010750:Phosphoproteins; D019319:Okadaic Acid; D007633:Keratins; D010749:Phosphoprotein Phosphatases