Protein kinase C isozymes in progressively transformed rat embryo fibroblasts.
Protein kinase C isozymes in progressively transformed rat embryo fibroblasts.
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发表时间：
1994-11
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通讯作者：
L. Liao;K. Ramsay;S. Jaken
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作者：
L. Liao;K. Ramsay;S. Jaken
单个蛋白激酶C（PKC）亚型在大鼠胚胎成纤维细胞系（REF 52细胞）进行性转化中的作用进行了评价。用SV 40转染正常（REF A）细胞以产生部分转化的REF B细胞系。REF B细胞在形态上发生转化，但在软琼脂中仅具有有限的生长能力。在REF A和B细胞中，PKC-α和PKC-δ的信息和蛋白水平相似，表明SV 40大T的表达不直接影响PKC的量。然而，PKC-α定位受到影响。PKC-α与REF A细胞而非REF B细胞的局灶性接触相关，表明位置而非内容物的变化是REF 52细胞转化的早期事件。选择REF B细胞克隆在软琼脂中生长（REF C细胞）。在其中几个克隆中，PKC-δ的水平增加，但PKC-α或ε的水平没有增加，这表明PKC-δ含量的增加可能促进非贴壁生长。在其他研究中，我们已经确定PKC通过其调节结构域与其结合蛋白/底物相互作用（RD; L. Liao等人，Biochemistry，33：1229-1233，1994）。这些结果预测，RD可能通过与内源性野生型PKC竞争结合蛋白/底物相互作用来抑制PKC信号传导。RD的PKC-δ的过表达抑制了一个代表性REF C克隆在软琼脂中的生长，而RD的PKC-α的过表达促进了软琼脂中的生长。这些结果表明，RD表达可能是一个有用的方法显性负性PKC抑制剂与潜在的同工酶特异性。
The role of individual protein kinase C (PKC) isoforms in progressive transformation of a rat embryo fibroblast cell line (REF52 cells) has been evaluated. Normal (REF A) cells were transfected with SV40 to produce the partially transformed REF B cell line. REF B cells are morphologically transformed but have only limited capacity for growth in soft agar. Message and protein levels for PKC-alpha and PKC-delta were similar in REF A and B cells, indicating that expression of SV40 large T did not directly influence the amounts of PKCs. However, PKC-alpha localization was influenced. PKC-alpha was associated with focal contacts of REF A but not REF B cells, indicating that changes in location rather than content are an early event in REF52 cell transformation. Clones of REF B cells were selected for growth in soft agar (REF C cells). Levels of PKC-delta, but not PKC-alpha or epsilon, were increased in several of these clones, suggesting that increased PKC-delta content may facilitate anchorage-independent growth. In other studies, we have determined that PKCs interact with their binding proteins/substrates through their regulatory domains (RD; L. Liao et al., Biochemistry, 33: 1229-1233, 1994). These results predict that RDs could potentially inhibit PKC signaling by competing with endogenous wild-type PKCs for binding protein/substrate interactions. Overexpression of the RD of PKC-delta inhibited growth in soft agar of one representative REF C clone, whereas overexpression of the RD of PKC-alpha promoted growth in soft agar. These results suggest that RD expression may be a useful approach for dominant negative PKC inhibitors with potential isozyme specificity.