| Abstract | 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. |
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