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J. Biol. Chem., Vol. 283, Issue 16, 10543-10549, April 18, 2008

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Characterization of the Interaction of Phorbol Esters with the C1 Domain of MRCK (Myotonic Dystrophy Kinase-related Cdc42 Binding Kinase) /β^*

Sung Hee Choi, Gabriella Czifra, Noemi Kedei, Nancy E. Lewin, Jozsef Lazar, Yongmei Pu, Victor E. Marquez, and Peter M. Blumberg¹

From the Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 and Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21701

C1 domains mediate the recognition and subsequent signaling response to diacylglycerol and phorbol esters by protein kinase C (PKC) and by several other families of signal-transducing proteins such as the chimerins or RasGRP. MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase), a member of the dystrophia myotonica protein kinase family that functions downstream of Cdc42, contains a C1 domain with substantial homology to that of the diacylglycerol/phorbol ester-responsive C1 domains and has been reported to bind phorbol ester. We have characterized here the interaction of the C1 domains of the two MRCK isoforms and β with phorbol ester. The MRCK C1 domains bind [20-³H]phorbol 12,13-dibutyrate with K_d values of 10 and 17 nM, respectively, reflecting 60–90-fold weaker affinity compared with the protein kinase C C1b domain. In contrast to binding by the C1b domain of PKC, the binding by the C1 domains of MRCK and β was fully dependent on the presence of phosphatidylserine. Comparison of ligand binding selectivity showed resemblance to that by the C1b domain of PKC and marked contrast to that of the C1b domain of PKC. In intact cells, as in the binding assays, the MRCK C1 domains required 50–100-fold higher concentrations of phorbol ester for induction of membrane translocation. We conclude that additional structural elements within the MRCK structure are necessary if the C1 domains of MRCK are to respond to phorbol ester at concentrations comparable with those that modulate PKC.

Received for publication, September 6, 2007 , and in revised form, February 7, 2008.

^* This work was supported by the Intramural Research Program of the Center for Cancer Research, NCI, National Institutes of Health. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Laboratory of Cancer Biology and Genetics, NCI, National Institutes of Health, Bldg. 37, Rm. 4048B, 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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