Activation of messenger-independent protein kinases in wild-type and phorbol ester-resistant EL4 thymoma cells.

Phorbol esters, acting via activation of the protein kinase C family of protein serine/threonine kinases, are able to exert profound effects on various cellular functions. In this study, we used the EL4 thymoma cell line to study the potential role of "downstream" protein serine/threonine kinases in cellular responses to phorbol esters. In wild-type EL4 cells, addition of phorbol ester caused a rapid activation of kinase activity toward RRLSSLRA (S6P). This increased activity was maintained for at least 15 min but diminished to control levels by 60 min. Activation of a myelin basic protein (MBP) kinase was also seen in response to phorbol ester. In a variant EL4 cell line in which phorbol ester does not induce interleukin 2 transcription, phorbol ester failed to activate either the S6P kinase or MBP kinase. Partial purification of the activated S6P and MBP kinases from wild-type cells showed that they represent separate enzymes that are distinct from protein kinase C. Although the variant cells had reduced levels of protein kinase C as compared with the wild-type cells, the amount of membrane-bound enzyme increased in response to phorbol 12-myristate 13-acetate in both wild-type and variant cells. Treatment of intact cells with phorbol ester resulted in phosphorylation of some of the same protein substrates in both cell lines. Okadaic acid, a phosphatase inhibitor, increased S6P and MBP kinase activities in both wild-type and variant cells. Thus, phorbol ester failed to activate the S6P and MBP kinases in the variant cells even though these cells express activatable protein kinase C, S6P kinase, and MBP kinase. Two protein kinase inhibitors, staurosporine and H-7, inhibited the activity of all three kinases in vitro, while a peptide inhibitor (PKC 19-31) showed specificity for protein kinase C. In summary, these results suggest that activation of messenger-independent protein kinases may be critical for certain protein kinase C-dependent responses.