[CANCER RESEARCH 48. 1-8. January I. I988| Special Lecture Protein Kinase C as the Receptor for the Phorbol Ester Tumor Promoters: Sixth Rhoads Memorial Award Lecture1 Peter M. Blumberg2 Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892 The focus of my research has been on understanding the ical potency, we predicted that a different derivative, phorbol initial events in phorbol ester action. The phorbol esters are 12,13-dibutyrate, would be optimized for specific binding activ natural products derived from Croton tiglium, the source of ity relative to nonspecific uptake due to lipophilicity (Fig. 1) croton oil, and from other plants of the family Euphorbiaceae (6). We therefore prepared radioactively labeled phorbol 12,13- (1). The phorbol esters initially became the object of intense dibutyrate; using this derivative, we and subsequently many research interest on the basis of their potent activity as mouse other laboratories were indeed able to demonstrate the existence skin tumor promoters (2). of specific phorbol ester receptors in a variety of cells and tissue The behavior of tumor promoters has been characterized in preparations (7). An alternative approach developed by Curt detail in the mouse skin system. Briefly, tumor promoters are Ashendel, in Dr. Boutwell's laboratory at the time, also proved compounds which by themselves are not carcinogenic or indeed successful. This approach used radioactive phorbol 12-myris mutagenic but which if administered chronically after exposure tate 13-acetate but reduced the nonspecific binding of the of an animal to a subeffective dose of a carcinogen are then able phorbol 12-myristate 13-acetate by means of washing with cold to lead to the rapid appearance of skin tumors. Whereas the acetone (8). action of the carcinogen is irreversible, as shown by the fact As we and others proceeded to characterize what we referred that one can delay the promotion treatment by up to 1 year to as the phorbol ester receptor, marked similarities began to after application of the carcinogen, referred to as an initiator, emerge with that of an enzymatic activity called protein kinase the action of the tumor promoters is reversible. Thus, if the C being characterized by Nishizuka et al. at Kobe University in chronic tumor promoter treatments precede rather than follow Japan (9, 10). In both cases, activity was highest in brain, high the treatment with the initiator, tumors do not arise. in lung and in neutrophils, and lower although present in other Although tumor promoting activity provided the initial mo tissues. The absolute amounts in brain were similar. In both tivation for studying the mechanism of the phorbol esters, as cases there was high conservation of activity over evolution. people began to examine the effects of the phorbol esters in Phorbol ester binding was demonstrated in nematodes, and different in vitro systems, it turned out that the phorbol esters had profound effects on a wide variety of biological systems (3- protein kinase C was found in earthworms. There was sensitiv ity to very low concentrations of calcium and, as will be de 5). These effects included (a) induction or inhibition of differ scribed later, evidence for association with specific phospho- entiation, (b) the partial mimicry in normal cells of the trans formed phenotype, (c) stimulation of secretory responses in a lipids. variety of cell types such as platelets and neutrophils, (d) It is now clear that the phorbol ester binding assays and the protein kinase C enzymatic assays were measuring different modulation of many membrane activities, and (e) generation of functional activities of the same protein: (a) Nishizuka's group active oxygen species. The range of these effects suggested early on that the phorbol esters might be functioning through stim demonstrated that under appropriate conditions with limiting ulation of some central regulatory pathway within cells. amounts of calcium and phospholipid, the required cofactors Two indirect arguments had suggested that the phorbol esters for protein kinase C, the phorbol esters were able to stimulate might bind to specific receptors: (a) the compounds had ex protein kinase C activity (11); (b) several laboratories, including traordinarily high potency, being active at nanomolar concen those of Dr. Niedel at Duke (12), Dr. Sando at the University trations; (h) small structural changes in the molecule led to of Virginia (13), Drs. Ashendel and Boutwell at McArdle Lab marked differences in activity. For example, elimination of the oratory (14), and our own group (15) demonstrated that protein hydroxyl group at position 20 of phorbol led to complete loss kinase C enzymatic activity and phorbol ester binding activity of activity, and methylation of the hydroxyl group at position copurified, ultimately to homogeneity. 4 led to a several hundred-fold loss in potency. Initial efforts to The identification of protein kinase C as the major target for demonstrate specific phorbol ester binding to cells were frus the phorbol esters was very exciting because it suggested that trated, however, because investigators used the biologically the phorbol esters were acting on one of the major signal most active phorbol ester, phorbol 12-myristate 13-acetate. The transduction mechanisms within cells. For a large class of myristate side chain makes the molecule highly lipophilic, and hormones and other cellular effectors, the initial action of the specific binding is therefore obscured by very high nonspecific hormones upon occupying their individual extracellular recep uptake. On the basis of structure-activity analysis which we had tors is to activate phospholipase C in what is now thought to carried out in vitro together with published data concerning the be a GTP binding protein mediated process (16, 17). Phospho lipophilicity of different phorbol esters as a function of biolog- lipase C degrades a specific phospholipid, phosphatidylinositol 4,5-bisphosphate, to generate two products, inositol 1,4,5-tris- Received 9/30/87; accepted 10/7/87. ' Presented at the 78th annual meeting of the American Association for Cancer phosphate and diacylglycerol. Nishizuka et al. demonstrated Research, May 22, 1987, in Atlanta, GA. that diacylglycerols, like the phorbol esters, are able to activate 1To whom requests for reprints should be addressed, at Laboratory of Cellular protein kinase C. Inositol 1,4,5-trisphosphate, in turn, activates Carcinogenesis and Tumor Promotion, Building 37, Room 3B2S, National Can cer Institute, Bethesda, MD 20892. the other arm of the signal transduction pathway, elevating 1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1988 American Association for Cancer Research. PIIORBOL ESTER TUMOR PROMOTERS ..OCICH^CH, CHjOH Fig. 1. Structure of phorbol 12.13-dibutyrate. intracellular calcium and thereby activating calcium dependent protein kinases. The demonstration that protein kinase C was the major receptor for the phorbol esters helped rationalize the broad range of biological activities that had been observed previously for the phorbol esters. In addition, because the phorbol esters are much better behaved as pharmacological agents than are 5 10 15 20 the diglycerides, it suggested that the phorbol esters could be [3H]PDBu BOUND, pmol/mg very useful probes for identifying the physiological systems in Fig. 2. Inhibition of |20-'H|phorbol 12,13-dibutyratc (|3H]PDBu) binding to which protein kinase C was involved. Finally, the detailed protein kinase C in the presence of phosphatidylserine and increasing concentra understanding of the interaction of phorbol esters with the tions of 1,2-diolein (from Ref. 29; reprinted with permission). regulatory domain on protein kinase C might permit the devel opment of agents which interfere with protein kinase C activity, was that diacylglycerol was simply functioning to perturb the thereby functioning as antipromoters or as inhibitors of phys lipid environment of the enzyme, thereby altering the phorbol iological effectors working through this pathway. ester binding affinity. The actual receptor is a complex between From the perspective of cancer researchers, the protein kinase protein kinase C and phospholipid, and, as described below, C pathway is important because, in addition to the phorbol binding affinity depends on the lipid component of the complex ester tumor promoters, a number of oncogenes and transform as well as on protein kinase C. ing gene products are thought to stimulate this pathway (18- There are three tests which must be met if diacylglycerols 20). sis and erb B are thought to enhance phosphatidylinositol actually inhibit the phorbol ester binding through a competitive turnover; ras has been shown to cause elevated levels of diacyl mechanism, (a) There is a specific relationship that must exist glycerol, and src and the polyoma middle T-antigen stimulate between the apparent binding affinity of phorbol 12,13-dibuty- phosphatidylinositol kinase, leading to elevated levels of sub rate for the receptor and the concentration of inhibitor present strate for phospholipase C. At the other end of the pathway, in the assay. This relationship in fact was found (29). (b) A protein kinase C is of interest because it phosphorylates a competitive inhibitor affects only the rate of association of a number of growth factor receptors such as the receptor for ligand with its receptor; it does not affect the rate of ligand epidermal growth factor (21). Secondly, protein kinase C mod release. For diacylglycerol this condition again was fulfilled ulates the activity of other signal transduction systems. For (30). Had the diacylglycerol been perturbing the lipid environ example, Grimm and Marks (22) and Mufson et al. (23) have ment, it most probably would have affected both the on and off shown that phorbol ester treatment blocks the elevation in rates for the binding of phorbol ester to the receptor, (c) A cyclic AMP in mouse skin induced by treatment with isopro- competitive inhibitor should bind with 1:1 stoichiometry.