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J. Biol. Chem., Vol. 282, Issue 39, 28627-28638, September 28, 2007

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Identification of Acidic Amino Acid Residues in the Protein Kinase C V5 Domain That Contribute to Its Insensitivity to Diacylglycerol^*

From the Department of Laboratory Medicine, Center for Molecular Pathology, Malmö University Hospital, Lund University, SE-205 02 Malmö, Sweden

The protein kinase C (PKC) isoforms are maintained in an inactive and closed conformation by intramolecular interactions. Upon activation these are disrupted by activators, binding proteins and cellular membrane. We have seen that autophosphorylation of two sites in the C-terminal V5 domain is crucial to keep PKC insensitive to the activator diacylglycerol, which presumably is caused by a masking of the diacylglycerol-binding C1a domain. Here we demonstrate that the diacylglycerol sensitivity of the PKC isoforms also is suppressed by autophosphorylation of the V5 sites. To analyze conformational differences, a fusion protein ECFP-PKC-EYFP was expressed in cells and the FRET signal was analyzed. The analogous mutant with autophosphorylation sites exchanged for alanine gave rise to a substantially lower FRET signal than wild-type PKC indicating a conformational difference elicited by the mutations. Expression of the isolated PKC V5 domain led to increased diacylglycerol sensitivity of PKC. We identified acidic residues in the V5 domain that, when mutated to alanines or lysines, rendered PKC sensitive to diacylglycerol. Furthermore, mutation to glutamate of four lysines in a lysine-rich cluster in the C2 domain gave a similar effect. Simultaneous reversal of the charges of the acidic residues in the V5 and the lysines in the C2 domain gave rise to a PKC that was insensitive to diacylglycerol. We propose that these structures participate in an intramolecular interaction that maintains PKC in a closed conformation. The disruption of this interaction leads to an unmasking of the C1a domain and thereby increased diacylglycerol sensitivity of PKC.

Received for publication, March 15, 2007 , and in revised form, August 1, 2007.

^* This work was supported by grants from The Swedish Cancer Society, The Swedish Research Council, The Children's Cancer Foundation of Sweden, Malmö University Hospital Research Funds, and the Kock, Crafoord, Ollie, and Elof Ericsson, and Gunnar Nilsson Foundations. 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 To whom correspondence should be addressed: Lund University, Center for Molecular Pathology, Entrance 78, 3^rd floor, UMAS, SE-205 02 Malmö, Sweden. Tel.: 46-40-337404; E-mail: Christer.Larsson{at}med.lu.se.

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