The phorbol diester receptor present in the particulate fraction of rat brain was solubilized by divalent ion chelation in the absence of detergents. The soluble receptor was partially purified by (NH4)2SO4 precipitation, DEAE-cellulose, and gel filtration chromatography. The purified receptor required exogenous phospholipid for activity and displayed a Kd of7 nM for[H]phorbol 12,13-dibutyrate. Biologically active phorbol analogs inhibited binding, whereas inactive analogs did not. The Ca2+-dependent, phospholipid-sensitive protein Iinase C copurified with the phorbol receptor. Purified protein kinase C was activated directly by phorbol 12-myristate 13-acetate in the presence of phospholipid.The tumor-promoting phorbol diesters are agents that are noncarcinogenic but will induce tumor formation when administered after subthreshold doses ofa carcinogen (1). In an attempt to understand the mechanism of tumor promotion, the phorbol diesters have been studied in many systems and have been shown to induce a myriad ofin vitro functional and biochemical changes. These changes, which may be related to cocarcinogen activity, include morphological transformation (2), enhanced hexose transport (3), enhanced prostaglandin synthesis (4), altered phospholipid and protein synthesis (5, 6), loss of fibronectin (7), increased ornithine decarboxylase activity and polyamine production (8, 9), and altered rates ofDNA synthesis (10-13). The phorbols also induce secretion (14-15), superoxide production (16), and alteration of surface receptors (17)(18)(19) and influence differentiation of cells in vitro; most notably, all human myeloid leukemia cells can be induced to differentiate into macrophage-like cells (20)(21)(22). For induction of many of these responses, similar structure-function relationships were shown for a series of phorbol diester analogs, suggesting that each of these diverse responses was mediated via a common pathway.Further support for a common pathway has come from studies demonstrating high affinity, saturable, stereospecific receptors that mediate the activity of the phorbol diesters in many different cell types (23)(24)(25)(26)(27)(28). Although the binding properties of these receptors have been characterized extensively, their biochemical structure or possible mechanism of transduction is unknown.Recently, Castagna et aL were able to show direct activation of partially purified protein kinase C by active phorbol diester analogs (29). Based on these observations, they put forth the exciting hypothesis that protein kinase C may be a membrane target of the phorbol diesters. In this manuscript we present data that extends this hypothesis. The phorbol diester receptor of rat brain membranes, solubilized by divalent ion chelation in the absence of detergents, copurifies with protein kinase C.In the latter steps of purification, both activities require added phospholipid.MATERIALS Sprague-Dawley female weanling rats were a gift from P. M. Conn; Sephacryl S-200 was from Pharmacia (Uppsala, Sweden); DE 52 was...

Activation of cellular protein kinase C appears to be involved in the mechanism by which phorbol diesters induce differentiation of human myeloid leukemia cells . Protein kinase C is thought to be physiologically activated by diacylglycerol derived from receptor-mediated phosphatidylinositol hydrolysis. sn-1,2-diacylglycerols with short saturated acyl side chains (C4-C10) were synthesized and found to be potent activators of protein kinase C partially purified from HL-60 cells. These diacylglycerols were also competitive inhibitors of [3Hlphorbol dibutyrate binding to the soluble phorbol diester receptor. The most potent diacylglycerol, sn-1,2-dioctanoylglycerol, displaced >90% of[3H]phorbol dibutyrate from the phorbol diester receptor of intact HL-60 cells. Because of probable cellular metabolism of sn-1,2-dioctanoylglycerol, hourly doses were required to maintain persistent occupancy of the phorbol diester binding site. Treatment of HL-60 cells with either phorbol 12-myristate 13-acetate or sn-1,2-dioctanoylglycerol produced identical phosphoprotein changes. Finally, sn-1,2-dioctanoylglycerol induced differentiation of the HL-60 cells into cells with morphologic characteristics of macrophages. Substitution of the hydroxyl group at position 3 with a hydrogen, chloro, or sulfhydryl moiety inactivated sn-1,2-dioctanoylglycerol. These data strengthen the hypothesis that protein kinase C activation plays a role in macrophage differentiation.The phorbol diester tumor promoters are plant products that enhance tumor formation in two-stage models of skin carcinogenesis (1). Paradoxically, these same compounds will rapidly induce most myeloid leukemia cells (cell lines and fresh explants from patients) to cease proliferation and to differentiate into cells with the morphologic, antigenic, biochemical, and functional properties of macrophages (2, 3). The phorbol diesters exert these effects via a specific receptor (4, 5). Data from several laboratories indicate that this receptor is the Ca2+-phospholipid-dependent protein kinase, termed protein kinase C (6-9). We have recently presented data that suggest activation of protein kinase C is involved in differentiation of human promyelocytic leukemia cells (HL-60) induced by phorbol diesters (10).Protein kinase C is regulated in vitro by Ca2 + and diacylglycerol, and is presumably activated in vivo in response to receptor-mediated phosphatidylinositol turnover, which gives rise to diacylglycerol, and/or Ca2+ flux (11). In vitro, the phorbol diesters activate the kinase by interaction at the diacylglycerol site (12). In intact cells, the phorbol diesters bypass the receptor-phosphatidylinositol-mediated regulatory mechanisms and activate the kinase directly (11). Discrete and sustained changes in cellular phosphoproteins have been shown to occur during phorbol diester-induced differentiation of HL-60 cells, although evidence that these phosphorylation events are protein kinase C dependent or are involved in the process of differentiation has not been presented (13,14)...

As bstract. The phorbol diesters are the most potent inducers of differentiation of the promyelocytic leukemia cell line, HL-60. The soluble phorbol diester receptors prepared by all three methods copurified in a constant ratio with the Ca2-/ phospholipid-dependent protein kinase C through ammonium sulfate precipitation, DEAE ion exchange, and gel filtration chromatography. Partially purified protein kinase C was directly activated by the phorbol diesters even in the absence of exogenous Ca2+. The ability of a series of phorbol analogues to activate the kinase correlated with their known activity as inducers of cell differentiation. In addition, phorbol diester stimulation altered the phosphate acceptor substrate profile of protein kinase C, at least in part, by alteration of the Michaelis constant (Kin). These data suggest that protein kinase C is the phorbol diester receptor and that phorbol diesterinduced macrophage maturation of HL-60 cells may be mediated by activation of intracellular protein kinase C.