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J. Biol. Chem., Vol. 282, Issue 48, 35396-35404, November 30, 2007

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Role of the Diacylglycerol Kinase -Conserved Domains in Membrane Targeting in Intact T Cells^*

From the Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Cantoblanco, E-28049 Madrid, Spain

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGK is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGK domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr³³⁵. This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.

Received for publication, March 9, 2007 , and in revised form, October 1, 2007.

^* This work was supported in part by Instituto de Salud Carlos III (Spanish Ministry of Health) Grants G03/79, Spanish Ministry of Education Grant BFU2004-01756, and Comunidad de Madrid Grant S-SAL-0311. 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 A Spanish Ministry of Science and Technology predoctoral fellow.

² Present address: St Jude Children's Research Hospital. Memphis, TN.

³ Present address: Burnham Institute for Medical Research. San Diego, CA.

⁴ To whom correspondence should be addressed: Dept. of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Científicas, Darwin 3, Cantoblanco, E-28049 Madrid, Spain. Tel.: 34-91/585-4665; Fax: 34-91/-372-0493; E-mail: imerida{at}cnb.uam.es.

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