PHORBOL ESTER INHIBITS THE PHOSPHORYLATION OF THE RETINOBLASTOMA PROTEIN WITHOUT SUPPRESSING CYCLIN D-ASSOCIATED KINASE IN VASCULAR SMOOTH-MUSCLE CELLS

To elucidate the role of protein kinase C in vascular smooth muscle ce ll proliferation, we examined the effects of phorbol 12-myristate 13-a cetate (PMA) on G(1) events in human arterial cells. About 15 h after G(0) cells were stimulated with fetal bovine serum and basic fibroblas t growth factor, [H-3]thymidine incorporation started. PMA (10 nM) inh ibited the incorporation over 90% when added earlier than 3 h after st imulation, but had no effect when added 12 h or later. PMA inhibited t he phosphorylation of the retinoblastoma protein (pRb), which normally began at about 9 h. PMA did not inhibit the gene expression of Cdk2, Cdk3, Cdk4, Cdk5, and cyclins G, C, and D, all of which began at 0-3 h . However, PMA reduced the expression of cyclins E and A, which usuall y began at 3-9 h and about 15 h, respectively. PMA inhibited the histo ne H1 kinase activity of Cdk2, which increased from about 9 h, whereas PMA did not inhibit the pRb kinase activities of cyclin D-associated kinase(s) and Cdk4, detectable from 0-3 h. These results suggested tha t the PMA-induced inhibition of pRb phosphorylation is not mediated by suppressing cyclin D-associated kinase(s) including Cdk4, but involve s the suppression of Cdk2 activity that results hom the reduced expres sion of cyclins E and A.