INACTIVE MEMBRANE-PROTEIN KINASE CS - A POSSIBLE TARGET FOR RECEPTOR SIGNALING

The activation of the multifunctional cell signalling enzymes, the pro tein kinase Cs (PKCs), is generally thought to result from the translo cation of inactive cytosolic enzymes to activation sites in cell membr anes. However, recent studies suggest that PKCs may also be stimulated in cells by processes independent of translocation, One possible mech anism is the modulation of the activity of PKCs already resident in me mbranes. A PKC assay that measures enzyme activity directly in isolate d native membranes has revealed the presence of an activatable pool of PKCs resident in native membranes of various cells and tissues. In 3T 3-L1 cells, some or all of this pool of membrane PKCs was stimulated w ithin 10 min of exposing the cells to 10 ng/ml epidermal growth factor or 100 ng/ml fibroblast growth factor. Similar increases in PKC activ ity were observed in native membranes isolated from CTLL-2, WEHI-231 a nd S49 lymphoma cells that had been exposed to interleukin-2. These gr owth factors all stimulated membrane PKC activity without detectably t ranslocating cytosolic enzymes to the membranes. In intact WEHI cells, low concentrations (5-10 mu M) of a diacylglycerol, 1-oleoyl-2-acetyl -sn-glycerol (GAG), or low concentrations (2-10 nM) of phorbol 12-myri state 13-acetate sufficed to activate PKCs already resident in membran es, but much higher concentrations (50-100 mu M and 50-100 nM respecti vely) were needed to detectably stimulate the translocation of cytosol ic PKCs. A phosphatidylcholine-specific phospholipase C also selective ly stimulated membrane PKCs in WEHI cells at concentrations that were much less than those needed to induce the translocation of cytosolic e nzymes. Furthermore, interleukin-2 and low concentrations of OAG both stimulated the phosphorylation of the 85 kDa PKC-selective substrate p rotein in intact WEHI cells in which translocation of PKCs was not evi dent. These results suggest that the membranes of some cells maintain a pool of activatable PKCs that respond to lower levels of extracellul ar stimuli than cytosolic PKCs, and that can be stimulated by signals which produce diacylglycerols through the hydrolysis of phospholipids other than polyphosphoinositides.