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J. Biol. Chem., Vol. 280, Issue 29, 27329-27338, July 22, 2005

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A Novel Diacylglycerol-lactone Shows Marked Selectivity in Vitro among C1 Domains of Protein Kinase C (PKC) Isoforms and as Well as Selectivity for RasGRP Compared with PKC^*

Yongmei Pu, Nicholas A. Perry, Dazhi Yang, Nancy E. Lewin, Noemi Kedei, Derek C. Braun, Sung Hee Choi, Peter M. Blumberg¶, Susan H. Garfield||, James C. Stone**, Dehui Duan, and Victor E. Marquez

From the Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, ^|| Laboratory of Experimental Carcinogenesis, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, the ^**Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, and Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702

Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms , , , , and , with apparent K_i values ranging from 340 nM for PKC to 29 nM for PKC. When binding to isolated C1 domains of PKC and -, 130C037 showed good affinity (K_i = 1.78 nM) only for C1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of C1b. 130C037 bound intact RasGRP1 and RasGRP3 with K_i values of 3.5 and 3.8 nM, respectively, reflecting 8- and 90-fold selectivity relative to PKC and PKC. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED₅₀ = 286 nM), partial reduction of PKC (ED₅₀ > 10 µM), and no effect on PKC. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKC, and no translocation of PKC. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets.

Received for publication, December 16, 2004 , and in revised form, May 2, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^¶ To whom correspondence should be addressed: NCI, National Institutes of Health, Bldg. 37, Rm. 4048, 37 Convent Dr. MSC 4255, Bethesda, MD 20892-4255. Tel.: 301-496-3189; Fax: 301-496-8709; E-mail: blumberp{at}dc37a.nci.nih.gov.

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