RAPID PHOSPHORYLATION OF 28-KDA HEAT-SHOCK PROTEIN BY TREATMENT WITH OKADAIC ACID AND PHORBOL ESTER OF BALB MK-2 MOUSE KERATINOCYTES/

Protein phosphorylation by okadaic acid and 12-O-tetradecanoylphorbol- 13-acetate (TPA) was examined using quiescent cultures of BALB/MK-2, a cell line derived from mouse epidermal keratinocytes. Treatment with okadaic acid caused rapid phosphorylation of five proteins with molecu lar masses of 65, 55, 50, 28 and 15 kDa (p65, p55, p50, p28, p15, resp ectively) while TPA caused rapid phosphorylation of five proteins with molecular masses of 80, 70, 40, 34 and 28 kDa (p80, p70, p40, p34, p2 8, respectively). In the present study, we examined p28, a common targ et protein of okadaic acid and TPA. The phosphorylation of p28 increas ed depending on time of exposure and doses of okadaic acid and TPA. Co mbined treatment with okadaic acid and TPA resulted in an additive eff ect. Its position on two-dimensional gel electrophoresis suggested tha t p28 is the 28-kDa heat-shock protein (HSP28). This possibility was c onfirmed by migration of p28 with HSP28 and comparative peptide mappin g of the two proteins. The phosphoamino-acid residue of phosphorylated HSP28 was serine. In two-dimensional tryptic peptide maps, the same p eptides were phosphorylated after treatment with both okadaic acid and TPA.