Effect of phorbol 12-myristate 13-acetate on protein kinase C and ras p21 phosphorylation

Abstract

The Ca{dollar}\sp{lcub}2+{rcub}{dollar}- and phospholipid-dependent protein kinase (protein kinase C) is an important regulatory enzyme involved in a variety of cellular functions such as proliferation, secretion and differentiation. Phosphatidylinositol turnover which results in the release of diacylglycerol (DG) in the cell membrane activates protein kinase C. Protein kinase C is the cellular receptor for the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA). PMA can substitute for DG and activate protein kinase C. Protein kinase C was purified to homogeneity from the 100,000 x g supernatant fluid of a rat brain homogenate by: DEAE-cellulose chromatography, Ultrogel AcA 34 gel filtration in the presence first of EGTA and then repeated in the presence of the enzyme activators, phosphatidylserine, 1,2-diolein and calcium. Two features of the enzyme were studied in detail. First, the biosynthesis and turnover of the enzyme using polyclonal antibodies elicited to the purified protein and second, the ability of the enzyme to catalyze the phosphorylation of the ras p21 protein. To study the biosynthesis and the effect of PMA on its own receptor we used a clonal line of rat pituitary tumor cells (GH{dollar}\sb3{dollar}). PMA accelerated the loss of ({dollar}\sp{lcub}35{rcub}{dollar}S) methionine-labeled protein kinase C in a time- and dose-dependent manner. PMA also induced the translocation on immunologically reactive protein kinase C from the cytosol to the membranes in a concentration-dependent manner. The results indicated that the turnover, but not the synthesis of protein kinase C is enhanced by membrane association. We then studied the relationship of PMA, the ras oncogene and protein kinase C to cellular transformation. We found that the ras oncogene protein (p21) encoded by the cKi-ras gene with exon 4B is phosphorylated in response to PMA in intact cells. In vitro, protein kinase C phosphorylated cKi-ras. Both in vitro and in intact cells the phosphorylation occurs on serine residues. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of the cKi-ras gene. Neither the GTP binding nor the GTPase activities of ras were affected by the phosphorylation of the serine residue in exon 4B.