Feuerstein N, Nishikawa M, Cooper H L
Cancer Res. 1985 Jul;45(7):3243-51.
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.
我们先前已经表明，佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）对白血病细胞以及其他恶性细胞作用的一个显著早期信号，是通过二维凝胶电泳在完整细胞中检测到的，分子量为17,000至20,000的胞质蛋白中磷酸盐周转迅速且显著增加，以及分子量为27,000的蛋白中磷酸盐周转适度增加。为了进一步阐明这种磷酸化事件的机制，我们研究了能够在无细胞系统中重建此事件的蛋白激酶。内源性Ca²⁺激活的磷脂依赖性蛋白激酶（Ca-PL-PK）的激活以及向HL-60白血病细胞胞质溶胶中添加纯化的Ca-PL-PK，导致分子量为27,000的磷蛋白（PP27）磷酸化增强，但不影响分子量为17,000至20,000的磷蛋白（PP17-20）的磷酸化。相反，在无细胞条件下，PP17-20被cAMP依赖性蛋白激酶（cAMP-PK）的催化亚基大量磷酸化。完整细胞暴露于二丁酰-cAMP导致PP17-20的磷酸化增加。这些条件也增强了PP27的磷酸化，表明在无细胞条件下PP27可以作为Ca-PL-PK和cAMP-PK的底物。对PP17-20的胰蛋白酶消化分析表明，在PMA诱导的PP17-20中，四个磷酸肽之一优先被磷酸化。在cAMP-PK催化的PP17-20中，另一个磷酸肽被磷酸化。因此，单独的cAMP-PK模拟了PMA对PP17-20磷酸化的作用，但它引入了额外的修饰。这种激酶在PMA诱导的PP17-20磷酸化中的精确作用仍有待阐明。我们进一步发现，PP17-20磷酸化增强也与由柯斯顿肉瘤病毒的V-rasKi癌基因转化的NIH/3T3细胞的恶性转化有关。转化细胞中（体内磷酸化的）PP17-20的胰蛋白酶磷酸肽图谱与PMA处理的HL-60细胞中PP17-20的图谱相似，但与cAMP-PK诱导的图谱不同，这表明由PMA激活导致PP17-20磷酸化的过程类似于某些恶性细胞中增强的内在细胞过程。
