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J. Biol. Chem., Vol. 282, Issue 29, 21467-21476, July 20, 2007

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Mechanism of Diacylglycerol-induced Membrane Targeting and Activation of Protein Kinase C^*

Heather R. Melowic¹, Robert V. Stahelin¹², Nichole R. Blatner¹, Wen Tian, Keitaro Hayashi, Amnon Altman, and Wonhwa Cho³

From the Department of Chemistry, University of Illinois, Chicago, Illinois 60607 and Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037

Protein kinase C (PKC) is a novel PKC that plays a key role in T lymphocyte activation. PKC has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC. PKC showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKC had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKC. Furthermore, the Ca²⁺-independent C2 domain of PKC has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKC. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr⁹⁰ of the C2 domain enhances the affinity of PKC for model and cell membranes. Collectively, these results show that PKC has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties.

Received for publication, January 4, 2007 , and in revised form, May 31, 2007.

^* This work was supported by National Institutes of Health Grants GM76581 (to W. C.) and CA35299 (to A. A.). 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 Fig. S1.

¹ These authors equally contributed to this work.

² Current address: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine-South Bend and Department of Chemistry and Biochemistry and The Walther Center for Cancer Research, University of Notre Dame, South Bend, IN 46617.

³ To whom correspondence should be addressed: Dept. of Chemistry (M/C 111), University of Illinois at Chicago, 845 West Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-2183; E-mail: wcho{at}uic.edu.

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