Dynamics of diacylglycerol kinase zeta translocation in living T-cells. Study of the structural domain requirements for translocation and activity.
The diacylglycerol kinases (DGK) regulate diacylglycerol-based signals by phosphorylating this key lipid intermediate to phosphatidic acid. Here, we have investigated the spatial and temporal regulation of diacylglycerol kinase zeta (DGK zeta) in living Jurkat T-cells expressing a muscarinic type I receptor. Using real time confocal videomicroscopy, we show the rapid translocation of a green fluorescent protein-tagged enzyme from the cytosol to the plasma membrane following receptor stimulation. The generation of a panel of truncations, deletions, and point mutations of the enzyme allowed us to examine the requirements of the different structural motifs for both activity and receptor-regulated translocation. The data show that DGK zeta has strict requirements for intact zinc fingers and the conserved catalytic domain for full enzymatic activity. Protein kinase C-driven myristoylated alanine-rich C kinase substrate domain phosphorylation and intact zinc fingers are in turn essential for plasma membrane translocation. DGK zeta does not translocate to the membrane following stimulation of the endogenous T-cell receptor, and our data demonstrate that the specificity in terms of receptor response is provided by the regulatory motifs present at the C-terminal domain of the protein. This is the first report that shows in vivo DGK zeta translocation in response to agonist stimulation and establishes the role of the different domains in enzymatic activity and the selectivity of the response to receptors.
Santos
Teresa
T
Department of Immunology and Oncology, National Center for Biotechnology, Campus de Cantoblanco, Madrid 28049, Spain.
Carrasco
Silvia
S
Jones
David R
DR
Mérida
Isabel
I
Eguinoa
Alicia
A
D000818:Animals; D019556:COS Cells; D002217:Carbachol / Q000494:pharmacology; D019852:Diacylglycerol Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011487:Protein Conformation; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology*
D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D049452:Green Fluorescent Proteins; D002217:Carbachol; D019852:Diacylglycerol Kinase