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J. Biol. Chem., Vol. 281, Issue 44, 33773-33788, November 3, 2006

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Effects on Ligand Interaction and Membrane Translocation of the Positively Charged Arginine Residues Situated along the C1 Domain Binding Cleft in the Atypical Protein Kinase C Isoforms^*

Yongmei Pu¹, Megan L. Peach¹, Susan H. Garfield^¶, Stephen Wincovitch^¶, Victor E. Marquez^||, and Peter M. Blumberg²

From the Laboratory of Cellular Carcinogenesis and Tumor Promotion and ^¶Laboratory of Experimental Carcinogenesis, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the Basic Research Program, SAIC-Frederick, Inc. and ^||Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, Frederick, Maryland 21702

The C1 domain zinc finger structure is highly conserved among the protein kinase C (PKC) superfamily members. As the interaction site for the second messenger sn-1,2-diacylglycerol (DAG) and for the phorbol esters, the C1 domain has been an important target for developing selective ligands for different PKC isoforms. However, the C1 domains of the atypical PKC members are DAG/phorbol ester-insensitive. Compared with the DAG/phorbol ester-sensitive C1 domains, the rim of the binding cleft of the atypical PKC C1 domains possesses four additional positively charged arginine residues (at positions 7, 10, 11, and 20). In this study, we showed that mutation to arginines of the four corresponding sites in the C1b domain of PKC abolished its high potency for phorbol 12,13-dibutyrate in vitro, with only marginal remaining activity for phorbol 12-myristate 13-acetate in vivo. We also demonstrated both in vitro and in vivo that the loss of potency to ligands was cumulative with the introduction of the arginine residues along the rim of the binding cavity rather than the consequence of loss of a single, specific residue. Computer modeling reveals that these arginine residues reduce access of ligands to the binding cleft and change the electrostatic profile of the C1 domain surface, whereas the basic structure of the binding cleft is still maintained. Finally, mutation of the four arginine residues of the atypical PKC C1 domains to the corresponding residues in the C1b domain conferred response to phorbol ester. We speculate that the arginine residues of the C1 domain of atypical PKCs may provide an opportunity for the design of ligands selective for the atypical PKCs.

Received for publication, July 10, 2006 , and in revised form, August 24, 2006.

^* This work has been supported in part by NCI, National Institutes of Health (NIH), under Contract N01-CO-12400 and by the NIH Intramural Research Program, NCI, Center for Cancer Research. 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.

¹ These authors contributed equally to this work.

² 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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