Revised Manuscript Received October 27, 1986 abstract: The Ca2+/phosphatidylserine-stimulated protein kinase C (PKC) appears to exist as interconvertible inactive, soluble and active, membrane-bound forms. Changes in the bimodal distribution of PKC induced by diacylglycerol or tumor-promoting phorbol esters have been proposed toregulate the activity of this kinase [Nishizuka, Y.(1984) Nature (London) 308, 693-698], A rapid microassay for assessment of protein kinase C translocation between cytosol and membranes was developed. This procedure, which relied on the selective digitonin-mediated release of cytoplasmic proteins, eliminated potential homogenization and fractionation artifacts. PKC activity toward histone HI was determined after limited trypsinolysis, which abolished the Ca2+/phospholipid requirement of the enzyme and prevented interference by inhibitory proteins. Complete translocation of PKC to the membrane fraction and subsequent down-regulation of the kinase in response to 12-O-tetradecanoylphorbol-13-acetate treatment of Swiss 3T3 cells could be demonstrated by this method. Platelet-derived growth factor, insulin-like growth factor 1, vasopressin, and prostaglandin F2a facilitated partial conversions of PKC to the membrane-bound form in quiescent 3T3 cells. e Ca2+-and phospholipid-stimulated enzyme protein ki-nase C (PKC) 1 plays a pivotal role in the regulation of cell metabolism and function in response to polyphosphoinositide turnover [for reviews, see Nishizuka (1983, 1984) and Berridge & Irvine (1984)]. A wide spectrum of hormones and other biologically activesubstances stimulates the phospholipase C catalyzed hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) into inositol 1, 4, 5-trisphosphate (an intracellular mo-bilizer of Ca2+) and diacylglycerol (an activator of PKC).

Diacylglycerol appears to enhance the affinity of PKC for both Ca2+ and phosphatidylserine (Kishimoto et al., 1980; Parker et al., 1984). Active tumor-promoting phorbol esters, notably 12-O-tetradecanoylphorbol-13-acetate (TPA), can substitute for diacylglycerol in the activation of PKC, and so far, PKC is the only phorbol ester receptor that has been identified (Nishizuka, 1983, 1984). However, other non-phorbol ester tumor promoters with no obvious diacylglycerol-like structure, such as teleocidin and mezerein, also activate PKC (Couturier et al., 1984; Miyake et al., 1984). Under resting conditions, PKC partitions with the cytosolic extract of cells that have been homogenized in the presence of calcium chelator (Katoh & Kuo, 1982). By contrast, when cultured cells are initially exposed to TPA, PKC is almost exclusively recovered with the particulate fraction upon cellular disruption (Kraft et al., 1982; Tapley & Murray, 1984; Wooten & Wrenn, 1984). Redistributionof PKCin TPA-treated cells apparently reflects the formation of a quaternary complex between the kinase, Ca2+, andthe membrane-asso-ciated phospholipid and phorbol ester (Wolf et al., 1985a; Ganong et al., 1986). Recent studies have implied that as-sociation of PKC with membranes, which is promoted by physiological levels of calcium and phosphatidylserine, is insufficient for full activation of PKC, but rather “primes” the kinase and renders it sensitive to the activators, diacylglycerol and phorbol esters (Wolf et al., 1985b; Ganong et al., 1986). PKC may be partially translocated to the membrane sub-fraction of a wide varietyof culturedcells in response to agents