Chakravarthy B R, Whitfield J F, Durkin J P
Institute for Biological Sciences, National Research Council of Canada, Ottawa.
Biochem J. 1994 Dec 15;304 ( Pt 3)(Pt 3):809-16. doi: 10.1042/bj3040809.
The activation of the multifunctional cell signalling enzymes, the protein kinase Cs (PKCs), is generally thought to result from the translocation of inactive cytosolic enzymes to activation sites in cell membranes. However, recent studies suggest that PKCs may also be stimulated in cells by processes independent of translocation. One possible mechanism is the modulation of the activity of PKCs already resident in membranes. A PKC assay that measures enzyme activity directly in isolated native membranes has revealed the presence of an activatable pool of PKCs resident in native membranes of various cells and tissues. In 3T3-L1 cells, some or all of this pool of membrane PKCs was stimulated within 10 min of exposing the cells to 10 ng/ml epidermal growth factor or 100 ng/ml fibroblast growth factor. Similar increases in PKC activity were observed in native membranes isolated from CTLL-2, WEHI-231 and S49 lymphoma cells that had been exposed to interleukin-2. These growth factors all stimulated membrane PKC activity without detectably translocating cytosolic enzymes to the membranes. In intact WEHI cells, low concentrations (5-10 microM) of a diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), or low concentrations (2-10 nM) of phorbol 12-myristate 13-acetate sufficed to activate PKCs already resident in membranes, but much higher concentrations (50-100 microM and 50-100 nM respectively) were needed to detectably stimulate the translocation of cytosolic PKCs. A phosphatidylcholine-specific phospholipase C also selectively stimulated membrane PKCs in WEHI cells at concentrations that were much less than those needed to induce the translocation of cytosolic enzymes. Furthermore, interleukin-2 and low concentrations of OAG both stimulated the phosphorylation of the 85 kDa PKC-selective substrate protein in intact WEHI cells in which translocation of PKCs was not evident. These results suggest that the membranes of some cells maintain a pool of activatable PKCs that respond to lower levels of extracellular stimuli than cytosolic PKCs, and that can be stimulated by signals which produce diacylglycerols through the hydrolysis of phospholipids other than polyphosphoinositides.
多功能细胞信号酶蛋白激酶C（PKCs）的激活通常被认为是由于无活性的胞质酶转位至细胞膜上的激活位点所致。然而，最近的研究表明，PKCs在细胞中也可能通过与转位无关的过程被刺激。一种可能的机制是对已经存在于膜中的PKCs活性进行调节。一种直接在分离的天然膜中测量酶活性的PKC检测方法揭示，在各种细胞和组织的天然膜中存在可被激活的PKC库。在3T3-L1细胞中，将细胞暴露于10 ng/ml表皮生长因子或100 ng/ml成纤维细胞生长因子后10分钟内，该膜PKC库中的部分或全部被刺激。在暴露于白细胞介素-2的CTLL-2、WEHI-231和S49淋巴瘤细胞分离的天然膜中也观察到PKC活性有类似增加。这些生长因子均刺激膜PKC活性，而未检测到胞质酶转位至膜上。在完整的WEHI细胞中，低浓度（5 - 10 microM）的二酰基甘油1-油酰基-2-乙酰基-sn-甘油（OAG）或低浓度（2 - 10 nM）的佛波酯12-肉豆蔻酸酯13-乙酸酯足以激活已经存在于膜中的PKCs，但需要更高得多的浓度（分别为50 - 100 microM和50 - 100 nM）才能检测到刺激胞质PKCs的转位。磷脂酰胆碱特异性磷脂酶C也以远低于诱导胞质酶转位所需的浓度选择性地刺激WEHI细胞中的膜PKCs。此外，白细胞介素-2和低浓度的OAG均刺激完整的WEHI细胞中85 kDa PKC选择性底物蛋白的磷酸化，而此时PKCs的转位并不明显。这些结果表明，某些细胞的膜维持着一个可被激活的PKC库，其对细胞外刺激水平的反应低于胞质PKCs，并且可被通过水解除多磷酸肌醇以外的磷脂产生二酰基甘油的信号所刺激。
