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Lck-Dependent Tyrosine Phosphorylation of Diacylglycerol Kinase Regulates Its Membrane Association in T Cells¹

Ernesto Merino^*, Antonia Ávila-Flores^*, Yasuhito Shirai, Ignacio Moraga^*, Naoaki Saito and Isabel Mérida^2,*

^* Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientificas, Madrid, Spain; and Biosignal Research Center, Kobe, Japan

TCR engagement triggers phospholipase C1 activation through the Lck-ZAP70-linker of activated T cell adaptor protein pathway. This leads to generation of diacylglycerol (DAG) and mobilization of intracellular Ca²⁺, both essential for TCR-dependent transcriptional responses. TCR ligation also elicits transient recruitment of DAG kinase (DGK) to the lymphocyte plasma membrane to phosphorylate DAG, facilitating termination of DAG-regulated signals. The precise mechanisms governing dynamic recruitment of DGK to the membrane have not been fully elucidated, although Ca²⁺ influx and tyrosine kinase activation were proposed to be required. We show that DGK is tyrosine phosphorylated, and identify tyrosine 335 (Y335), at the hinge between the atypical C1 domains and the catalytic region, as essential for membrane localization. Generation of an Ab that recognizes phosphorylated Y335 demonstrates Lck-dependent phosphorylation of endogenous DGK during TCR activation and shows that pY335DGK is a minor pool located exclusively at the plasma membrane. Our results identify Y335 as a residue critical for DGK function and suggest a mechanism by which Lck-dependent phosphorylation and Ca²⁺ elevation regulate DGK membrane localization. The concerted action of these two signals results in transient, receptor-regulated DGK relocalization to the site at which it exerts its function as a negative modulator of DAG-dependent signals.

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 This work was supported in part by Grants G03/79 from the Instituto de Salud Carlos III (Spanish Ministry of Health), BFU2004-01756 (Spanish Ministry of Education), and S-SAL-0311 from Comunidad de Madrid. E.M. is a Spanish Ministry of Science and Technology predoctoral fellow; A.Á.-F. is supported by the Juan de la Cierva programme from the Spanish Ministry of Science and Technology. The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (Consejo Superior de Investigaciones Cientificas) and by Pfizer.

² Address correspondence and reprint requests to Dr. Isabel Mérida, Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientificas, C/Darwin, 3, Universidad Autónoma de Madrid Campus de Cantoblanco, E-28049 Madrid, Spain. E-mail address: imerida{at}cnb.uam.es

³ Abbreviations used in this paper: PTK, protein tyrosine kinase; [Ca²⁺]_i, intracellular Ca²⁺; DAG, diacylglycerol; DGK, DAG kinase; HA, hemagglutinin; IP₃, inositol 1,4,5-triphosphate; KD, kinase dead; LAT, linker of activated T cell adaptor protein; MFI, mean fluorescence intensity; PA, phosphatidic acid; PKC, protein kinase C; PLC, phospholipase C1; pY, phosphotyrosine; SH, Src homology; SHP-1, SH region 2 domain-containing phosphatase-1; wt, wild type; Ras-GRP1, Ras guanyl nucleotide-releasing protein 1.