Cell-free system studies on the phosphorylation of the 17,000-20,000 dalton protein induced by phorbol ester in human leukemic cells and evidence for a similar event in virally transformed murine fibroblasts

Abstract

We have shown previously that a prominent early signal in the phorbol-12-myristate-13-acetate (PMA) effect on leukemic cells as well as on other malignant cells is a rapid and dramatic increase in the turnover of phosphate in a Mr 17,000 to 20,000 cytosolic protein and a moderate increase in turnover of phosphate in a Mr 27,000 protein, as detected in the intact cells by 2-dimensional gel electrophoresis. To further elucidate the mechanism of this phosphorylation event, we have examined the protein kinases which can reconstitute this event in a cell-free system. Activation of the endogenous Ca2+-activated phospholipid-dependent protein kinase (Ca-PL-PK) as well as addition of purified Ca-PL-PK to the cytosol of HL-60 leukemic cells resulted in enhanced phosphorylation of phosphoprotein Mr 27,000 (PP27) but did not affect the phosphorylation of phosphoprotein Mr 17,000 to 20,000 (PP17-20). In contrast, PP17-20 was heavily phosphorylated under cell-free conditions by the catalytic subunit of cAMP-dependent protein kinase (cAMP-PK). Exposure of intact cells to dibutyryl-cAMP resulted in increased phosphorylation of PP17-20. These conditions also enhanced the phosphorylation of PP27, showing that PP27 can act as a substrate for both Ca-PL-PK and cAMP-PK under cell-free conditions. Tryptic digest analysis of PP17-20 showed that one of four phosphopeptides is preferentially phosphorylated in PMA-induced PP17-20. An additional phosphopeptide was phosphorylated in cAMP-PK-catalyzed PP17-20. Thus, cAMP-PK alone mimics the effect of PMA on phosphorylation of PP17-20, but it introduces additional modifications. The precise role of this kinase in PMA-induced phosphorylation of PP17-20 remains to be clarified. We found further that enhanced phosphorylation of PP17-20 is also associated with malignant transformation of NIH/3T3 cells transformed by V-rasKi oncogene of Kirsten sarcoma virus. The tryptic phosphopeptide map of PP17-20 (phosphorylated in vivo) in the transformed cells was similar to that of PP17-20 in PMA-treated HL-60 cells but not to that induced by cAMP-PK, suggesting that the process activated by PMA which leads to phosphorylation of PP17-20 resembles an intrinsic cellular process which is enhanced in certain malignant cells.