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¹ Department of Biology, Gallaudet University, Washington, District of Columbia; Laboratories of ² Cellular Carcinogenesis and Tumor Promotion and ³ Experimental Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland; ⁴ Departments of Internal Medicine and Medicinal Chemistry and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan

Requests for reprints: Peter M. Blumberg, National Cancer Institute, NIH, Building 37, Room 4048, 37 Convent Drive, MSC 4255, Bethesda, MD 20892-4255. Phone: 301-496-3189; Fax: 301-496-8709. E-mail: blumberp{at}dc37a.nci.nih.gov

The diacylglycerol signaling pathway, involving protein kinase C (PKC) and RasGRP, is a promising therapeutic target for both cancer and other indications. The phorbol esters, ultrapotent diacylglycerol analogues, bind to and activate PKC and RasGRP. Here, using fluorescent phorbol esters and complementary fluorescent PKC and RasGRP constructs, we determined the localization of the phorbol ester as a function of time after addition and how the resultant PKC or RasGRP3 translocation related to ligand localization. For these studies, we prepared fluorescently labeled phorbol esters of varying lipophilicities based on the BODIPY FL (green) or BODIPY 581/591 (red) fluorophores, and by using fusion constructs of green fluorescent protein or DsRed with PKC isoforms or RasGRP3 expressed in Chinese hamster ovary cells, we simultaneously compared the kinetics and pattern of localization of PKC or RasGRP3 with that of the fluorescent red or green phorbol esters. Binding assays showed that the fluorescent derivatives were potent ligands. Uptake followed a one-compartment pharmacokinetic model with a half-time of minutes to hours, depending on the ligand, and all of the fluorescent phorbol esters localized primarily to intracellular membranes, with little plasma membrane localization. The fluorescent phorbol esters induced translocation of and generally colocalized with PKC or RasGRP3. However, PKC and, initially, PKC, translocated to the plasma membrane, in which little phorbol ester accumulated. The findings argue that the rate of uptake of phorbol esters influences the subsequent pattern of PKC translocation, and that the specificity for PKC translocation is dominated by factors other than the localization of the ligand.

Key Words: phorbol ester • protein kinase C • green fluorescent protein • subcellular localization, kinetics • uptake

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Received 8/ 4/04; revised 10/15/04; accepted 11/ 8/04.

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