Hyperphosphorylation of Cytokeratins by Okadaic Acid Class 1\imor Promoters in Primary Human Keratinocytes1

Okadaic acid, dinophysistoxin-1 (35-methylokadaic acid), and calyculin A are potent tumor promoters on mouse skin (H. Fujiki, M. Suganuma, S. Nishiwaki, S. Yoshizawa, J. Yatsunami, R. Matsushima, H. Furuya, S. Okabe, S. Matsunaga, and T. Sugimura. In: R. D'Amato, T. J. Slaga, W. Farland, and C. Henry (eds.), Relevance of Animal Studies to the Evaluation of Human Cancer Risk, pp. 337-350. New York: John Wiley and Sons, Inc., 1992). These tumor promoters, which are also inhibitors of protein phosphatases 1 and 2A, induced hyperphosphorylation of ,17, 60,000, Mr 58,000, M, 56,000, A/r 52,000, MT 42,000, and Mr 27,000 pro teins in PHK 16-1 cells, human keratinocytes immortalized by human papillomavirus type 16. Except for the .17, 27,000 protein, these hyperphosphorylated proteins were identified to be cytokeratin peptides (CK) CK 5, CK 6, CK 7, CK 16, and CK 19, by anti-cytokeratin antibodies. CK 5 and CK 6 were more strongly phosphorylated than CK 16 and CK 19. The in vitro hyperphosphorylation of these cytokeratins was also found by incubation with an enzyme fraction containing a mixture of protein phosphatase 2A and protein kinases isolated from mouse brain and various concentrations of dinophysistoxin-1. Indirect immunofluorescence micros copy with anti-cytokeratin antibodies revealed that the hyperphosphorylated cytokeratins had retracted to the perinuclear area. The hyperphosphorylated ,17, 27,000 protein was identified as a heat shock protein, HSP27. Hyperphosphorylation of HSP27 and intermediate filaments, such as cytokeratins, is one of the early biochemical changes, or pleiotropic effects, in cells induced by the okadaic acid class of tumor promoters.