Diacylglycerol-dependent binding recruits PKCtheta and RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes.
Diacylglycerol (DAG) signaling relies on the presence of conserved domain 1 (C1) in its target proteins. Phospholipase C-dependent generation of DAG after T cell receptor (TCR) triggering is essential for the correct immune response onset. Accordingly, two C1-containing proteins expressed in T lymphocytes, Ras guanyl nucleotide-releasing protein1 (RasGRP1) and protein kinase C (PKC), were shown to be fundamental for T-cell activation and proliferation. Although containing the same regulatory domain, they are proposed to relocate to distinct subcellular locations in response to TCR triggering. Here we studied intracellular localization of RasGRP1 and PKC C1 domains in living Jurkat T cells. The results demonstrate that, in the absence of significant primary sequence differences, the C1 domains of these proteins show specific localization within the cell and distinct responses to pharmacological stimulation and TCR triggering. These differences help explain the divergent localization and distinct functional roles of the full-length proteins, which contains them. The properties of these DAG-binding modules allow their characterization as functional markers that discriminate between DAG pools. Finally, we show that by binding to different diacylglycerol forms, overexpression of distinct C1 modules can attenuate DAG-dependent signals originating from the plasma or internal membranes. This is shown by analyzing the contribution of these two lipid pools to PLC-dependent Ras activation in response to TCR triggering.
Carrasco
Silvia
S
Department of Immunology and Oncology, National Center for Biotechnology, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, E-28049 Madrid, Spain.
Merida
Isabel
I
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D018106:CD28 Antigens / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D002470:Cell Survival; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism* / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019169:Jurkat Cells; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018631:ras Proteins / Q000378:metabolism
D018106:CD28 Antigens; D017252:CD3 Complex; D004268:DNA-Binding Proteins; D004075:Diglycerides; D020662:Guanine Nucleotide Exchange Factors; D007527:Isoenzymes; D008081:Liposomes; C491789:Rasgrp1 protein, rat; D011948:Receptors, Antigen, T-Cell; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate