Dynamics of Diacylglycerol Kinase ζ Translocation in Living T-cells

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 ζ (DGKζ) 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 DGKζ 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. DGKζ 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 DGKζ 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. 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 ζ (DGKζ) 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 DGKζ 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. DGKζ 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 DGKζ 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. diacylglycerol protein kinase C phosphatidic acid diacylglycerol kinase cysteine-rich domain myristoylated alanine-rich C kinase substrate T-cell receptor green fluorescent protein enhanced GFP phenylmethylsulfonyl fluoride C-terminal domain phosphate-buffered saline When agonists bind to their cellular receptors, an early signaling event is often the hydrolysis or modification of certain lipids present in cell membranes to produce second messengers. A well known signaling pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C enzymes to produce diacylglycerol (DAG)1 and soluble phosphatidylinositol 1,4,5-triposphate (1Payrastre B. Missy K. Giuriato S. Bodin S. Plantavid M. Gratacap M. Cell. Signal. 2001; 13: 377-387Crossref PubMed Scopus (189) Google Scholar). The DAG generated by this mechanism acts as a membrane recruitment signal and activator for a series of C1 domain-containing signaling proteins such as some PKC isoforms (2Ron D. Kazanietz M. FASEB J. 1999; 13: 1658-1676Crossref PubMed Scopus (552) Google Scholar), UNC-13 (3Nurrish S. Segalat L. Kaplan J. Neuron. 1999; 24: 231-242Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), RasGRP (4Ebinu J.O. Stang S.L. Teixeira C. Bottorff D.A. Hooton J. Blumberg P.M. Barry M. Bleakley R.C. Ostergaard H.L. Stone J.C. Blood. 2000; 95: 3199-3203Crossref PubMed Google Scholar), and chimerins (5Caloca M.J. Garcia-Bermejo M.L. Blumberg P.M. Lewin N.E. Kemmer E. Mischak H. Wang S. Nacro K. Bienfait B. Marquez V.E. Kazanietz M.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11854-11859Crossref PubMed Scopus (93) Google Scholar). These molecules are key regulators of a broad array of cellular functions including proliferation, differentiation, and/or apoptosis. The correct activation of DAG-regulated enzymes requires signal termination that is provided by the conversion of this lipid to phosphatidic acid (PA) in a reaction catalyzed by diacylglycerol kinase (DGK) (6Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). As the number of PA-binding proteins continues to grow (7Burger K. Demel R. Schmid S. de Kruijff B. Biochemistry. 2000; 39: 12485-12493Crossref PubMed Scopus (84) Google Scholar, 8Chalfant C. Kishikawa K. Mumby M. Kamibayashi C. Bielawska A. Hannun Y. J. Biol. Chem. 1999; 274: 20313-20317Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 9Dalton K. East J. Mall S. Oliver S. Starling A. Lee A. Biochem. J. 1998; 329: 637-646Crossref PubMed Scopus (43) Google Scholar, 10Frank C. Keilhack H. Opitz F. 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Biochem. 1999; 262: 291-298Crossref PubMed Scopus (23) Google Scholar, 18Rizzo M. Shome K. Watkins S. Romero G. J. Biol. Chem. 2000; 275: 23911-23918Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), a role for DGK-generated PA must also be considered. Because DGKs can remove a lipid messenger by creating another, the subcellular localization of these enzymes, together with their regulation in response to receptor stimulation, are anticipated to be vital for the correct onset of cellular responses. To date, nine DGK isoforms have been found in mammalian tissues, and similar genes are present in plants (19Katagiri T. Mizoguchi T. Shinozaki K. Plant Mol. Biol. 1996; 30: 647-653Crossref PubMed Scopus (45) Google Scholar), the nematodeCaenorhabditis elegans (3Nurrish S. Segalat L. Kaplan J. Neuron. 1999; 24: 231-242Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 20Miller K. Emerson M. Rand J. Neuron. 1999; 24: 323-333Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), and Drosophila melanogaster (21Masai I. Okazaki A. Hosoya T. Hotta Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11157-11161Crossref PubMed Scopus (136) Google Scholar). Structurally, the DGKs contain at least two N-terminal, zinc finger-like, cysteine-rich domains (CRD) and a conserved catalytic domain. Most mammalian DGKs present other distinct homology domains that have been used to classify them into five subclasses. The presence of different domains and the distinct tissue distribution of DGKs suggest very precise regulation and specific functions for these proteins in different cell types (22van Blitterswijk W.J. Houssa B. Cell. Signal. 2000; 12: 595-605Crossref PubMed Scopus (228) Google Scholar). Most DGKs are cytosolic enzymes, and translocation from the cytosol to other subcellular compartments appears to be a general mechanism for activation of this enzyme family. DGKζ, a type IV DGK, was originally cloned from human endothelial cell cDNA as well as from rat retina and brain (23Bunting M. Tang W. Zimmerman G. McIntyre T. Prescott S. J. Biol. Chem. 1996; 271: 10230-10236Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 24Goto K. Kondo H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11196-11201Crossref PubMed Scopus (148) Google Scholar). Not having the restricted expression pattern described for other isoforms, DGKζ appears much more ubiquitous, and DGKζ RNA is abundant in tissues such as skeletal muscle and lymphoid cells. There are two class IV DGK isoforms (DGKζ and ι) that are characterized by a domain homologous to the myristoylated alanine-rich protein kinase C substrate (MARCKS) protein phosphorylation domain, four C-terminal ankyrin repeats, and a PDZ-binding motif. Translocation of DGKζ from the cytosol to the nucleus is proposed to regulate cell growth by modulating nuclear DAG levels (25Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar). More recently, it was shown that this enzyme can interact with and regulate either integral or plasma membrane-associated proteins such as the long form leptin receptor (26Liu Z. Chang G. Leibowitz S. J. Biol. Chem. 2001; 276: 5900-5907Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), syntrophin (27Hogan A. Shepherd L. Chabot J. Quenneville S. Prescott S.M. Topham M. Gee S.H. J. Biol. Chem. 2001; 276: 26526-26533Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and RasGRP (28Topham M.K. Prescott S.M. J. Cell Biol. 2001; PubMed Scopus (99) Google Scholar). the signaling functions of DGKζ as a signal are well characterized (25Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar, M.K. Prescott S.M. J. Cell Biol. 2001; PubMed Scopus (99) Google Scholar), the by this enzyme is and to substrate Here, we the regulation of DGKζ in following the stimulation of an muscarinic type I receptor known to D. M. T. A. Nature. PubMed Scopus (99) Google M. M. A. J. 1996; Google Scholar). we the requirements of of the structural motifs for both enzymatic activity and translocation. DGKζ very from the cytosol to the plasma membrane in response to the of activity is not for the plasma phosphorylation of the domain is for enzyme activity for membrane The protein does not translocate following stimulation, that phospholipase DAG generation and PKC activation are not the signals for enzyme translocation. of the DGKζ C-terminal domain, four ankyrin and a PDZ-binding membrane translocation following that these domains a role in the regulation of membrane translocation. these demonstrate DGKζ translocation in living in response to and into the role of the protein domains specific to this DGK and by DGK was from and phenylmethylsulfonyl fluoride and from and from and was was from was from and was from from cDNA was a of K. of was with and the the cDNA was in the expression in the in the GFP C the was with and the was in To the ankyrin domains was and in was the To the of the was with as described (25Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar). two mutations in the homology in the first we and with and in the second the with To the the first conserved in of the two of the or with either in the type protein or in the The C-terminal domain including the four ankyrin and the PDZ-binding was generated by with two and The was in the and to be in the expression was in with and with to the and was into two for and a DAG activity The cell was generated by of the human muscarinic receptor in the Jurkat cell D. M. T. A. Nature. PubMed Scopus (99) Google Scholar). in with and by with of a at and a of to of growth and in an and for by five a The at for to remove and cell and the was and at for at The and the and by in and and and for at the for protein protein and an of protein of was by to and the expression of was with an and the The of was in and and by and The was and for protein. of protein from was with DAG as substrate in and into PA was by a in a to allowed to for at least at and in was by the with a the of with a confocal A was used for and a was used for series of and stimulation of the cells. to by of in at in a of and at it with of in was and with and in for the to cells. stimulation, with at a and series of by confocal To the subcellular localization and of DGKζ in we the cDNA of DGKζ to the C and this in cells. This is a of the Jurkat cell with the human muscarinic type receptor and has been used to examine the role of DAG T-cell D. M. T. A. Nature. PubMed Scopus (99) Google Scholar, M. M. A. J. 1996; Google Scholar). of this in and the expression of a protein of the with DGK activity in an in The was found in the of a cytosolic localization of the protein as to localization to membrane compartments of that the cytosolic very and to the plasma membrane following stimulation the of the protein was at the plasma it for at least stimulation membrane translocation enzyme activity. To a DGKζ was to This is found the that is conserved in DGK has been shown to be essential for enzymatic activity of this in the conserved of the D. melanogaster (21Masai I. Okazaki A. Hosoya T. Hotta Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11157-11161Crossref PubMed Scopus (136) Google Scholar). of this in human DGKζ has been described as essential for enzyme activity (25Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar). The of enzymatic activity by of this conserved is a well As expression of the a protein of the as the type with activity When by confocal videomicroscopy, the to the membrane with the as the type protein and IV DGKs domains with a role as regulators of enzyme activity and/or is a conserved the and the catalytic domain with a to the domain in This in the nucleus and the cytosol in a (25Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar). first the for PKC activity in enzyme translocation specific The of PKC activity by DGKζ that protein is activation generated in phosphorylation of the domain was or the the as the type protein of the mutations enzyme that the DGKζ domain does not in DAG phosphorylation in the subcellular localization of the following receptor stimulation was in living cells. The of to translocation of the enzyme to the membrane in response to receptor stimulation the other mutations that phosphorylation allowed protein translocation to the plasma membrane with similar to that of the type that protein phosphorylation of DGKζ at the domain is an for protein translocation from the cytosol to the of the domain is not the as the requires receptor stimulation for membrane translocation. DGKs have at least two of F. M. I. S. H. Biochem. J. 1996; PubMed Scopus Google of other DGK isoforms that both are and have enzymatic similar to of the intact To the role of DGKζ a was generated in the domain the two was This localization in intact DGKζ, was at the nucleus with cytosolic When stimulation of the in the localization of the enzyme To the role of the we generated two point in the first of is in zinc was with and These mutations in both the type protein and the domain proteins the as the type and in an in When the four cytosolic localization similar to that found for the type protein. of the to the plasma membrane in response to and cytosolic for the of the stimulation This that both in DGKζ are essential for PA and membrane in DGK ζ are essential for enzyme activity and translocation. and generated by of or to in the type or in the of protein expression and enzyme activity. of the was by a of an activity was in cell as described in the The a the of the to the of PA subcellular localization of the with of the different with for to type IV DGKs are to the Drosophila is from the a of the C-terminal ankyrin show that this domain is essential for enzyme in the retina (21Masai I. Okazaki A. Hosoya T. Hotta Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11157-11161Crossref PubMed Scopus (136) Google Scholar). Using DGKζ was found to interact with the leptin receptor the C-terminal domain the ankyrin (26Liu Z. Chang G. Leibowitz S. J. Biol. Chem. 2001; 276: 5900-5907Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This C-terminal of the enzyme also a PDZ-binding that with (27Hogan A. Shepherd L. Chabot J. Quenneville S. Prescott S.M. Topham M. Gee S.H. J. Biol. Chem. 2001; 276: 26526-26533Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). the of these domains functions to type IV two generated and used to enzyme translocation following receptor stimulation. the four of the PDZ-binding domain was by the C-terminal of the both the ankyrin and the PDZ-binding When both proteins of the and DAG in an in that these domains are not for enzyme activity of protein that the of the C-terminal domain or of the PDZ-binding protein translocation in response to in to a more also DGKζ translocation to the plasma we stimulation with was used as a of to the C. H. I. A. A. Nature. PubMed Scopus Google Scholar). to the plasma membrane at with the type not translocate to the membrane the The also to in response to stimulation. When the C-terminal was we rapid translocation to the was restricted to the of with the This shows in DGKζ, the C-terminal of the protein has a regulatory role that be by the signals The DGKζ C-terminal the PDZ-binding and the four ankyrin repeats, to the following stimulation. This the presence of some type of regulatory by the DGKζ C-terminal domain. The presence of ankyrin domains and a PDZ-binding the of a this domain. the of DGKζ expression in DGKζ translocation stimulation. As rapid translocation of DGKζ, was found at the membrane as as receptor stimulation When DGKζ was with the the domain, translocation of the enzyme was and cytosolic localization was found for the protein stimulation These point to a role for the DGKζ domain in the regulation of protein translocation. DGKζ is in lymphoid tissues, regulation of this enzyme following receptor stimulation has not been in Here, we used a Jurkat T-cell with a muscarinic type I this of DAG-regulated signals generated by the of a soluble Using this we show that DGKζ is in the cytosol of living T-cells and that it very to the plasma membrane in response to receptor stimulation. DGK isoforms have been shown to translocate to the membrane in response to and the and of this translocation was both the and the agonist this cell we M. I. J. Cell Biol. 2001; PubMed Scopus Google translocation of following of both muscarinic and this to the plasma membrane in response to and are Y. S. M. K. N. N. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (84) Google Scholar). at the plasma membrane in a in response to and in response to acid and Y. S. M. K. N. N. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Translocation to the these enzymes to their appears to be a general mechanism of DGK regulation as it is for other As we for the M. I. J. Cell Biol. 2001; PubMed Scopus Google Scholar), kinase activity is not for DGKζ translocation to the plasma activity of not for enzyme at the membrane M. I. J. Cell Biol. 2001; PubMed Scopus Google Scholar). Translocation of the type is a very rapid and a at the membrane for a of This the of regulation PA we that the of type I enzymes, is for this M. I. J. Cell Biol. 2001; PubMed Scopus Google Scholar). IV DGKs are not to this type of regulation as be of the of The of regulation for DGKζ be we that some agonists that translocation of the type enzyme a more translocation of the S. D. R. I. and A. DGKζ both in the cytosol and the cell in and translocation these two is proposed to be by phosphorylation of the domain (23Bunting M. Tang W. Zimmerman G. McIntyre T. Prescott S. J. Biol. Chem. 1996; 271: 10230-10236Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 24Goto K. Kondo H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11196-11201Crossref PubMed Scopus (148) Google Scholar, M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar). not nuclear localization in the cell or in the Jurkat T-cells at of the cell of the the expression of a with nuclear to of the nuclear localization in the domain (25Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Nature. 1998; 394: 697-700Crossref PubMed Scopus (250) Google Scholar). DGKζ appears to be a cytosolic enzyme in lymphoid and nuclear localization be with some specific in the nucleus of certain A role for DGKζ at the plasma membrane is by the that DGKζ has been found to be with proteins such as the leptin receptor, with cytosolic proteins that translocate to the plasma membrane such as or with plasma membrane such as (26Liu Z. Chang G. Leibowitz S. J. Biol. Chem. 2001; 276: 5900-5907Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, A. Shepherd L. Chabot J. Quenneville S. Prescott S.M. Topham M. Gee S.H. J. Biol. Chem. 2001; 276: 26526-26533Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, M.K. Prescott S.M. J. Cell Biol. 2001; PubMed Scopus (99) Google Scholar). DGKζ regulatory and we the structural requirements for lipid kinase activity of the The that the domains at the C-terminal of the protein are not in DAG phosphorylation and PDZ-binding are well known motifs M. C. C. J. Biol. 2001; PubMed Scopus Google Scholar, M. C. 2001; 24: PubMed Scopus Google it is not that their role is to the enzymatic activity of phosphorylation of is also in the regulation of protein with lipid membranes and/or A. D. S. 1998; PubMed Scopus Google Scholar, J. Cell Biol. 1998; PubMed Scopus Google Scholar). phosphorylation of this does not to be in PA by DGKζ, as of the in the domain a The DGKζ requirements for DAG phosphorylation to be an intact and the catalytic domain. The of including the ζ the for with G. Kazanietz M. Blumberg P. J. Cell. Full Text PDF PubMed Scopus Google of these conserved domains with DAG The that mutations produce an enzyme a structural in this domain that correct of the protein with the not with the be essential for DGKζ with membranes in the in with of the of DGKζ domain mutations translocation and enzyme activity This is the first report of DGKζ translocation in response to receptor in living cells. suggest that distinct are for DGKζ to from the cytosol to the plasma The generation of at the domain that phosphorylation of the in the domain with homology to is essential for translocation This domain has been found in proteins that the cytosol and the plasma membrane phosphorylation cytosolic localization by into the and A. D. S. 1998; PubMed Scopus Google Scholar, J. Cell Biol. 1998; PubMed Scopus Google Scholar). DGKζ, phosphorylation of the domain has the it is for membrane The domain is also found in proteins that interact with the and phosphorylation was shown to J. Cell Biol. 1998; PubMed Scopus Google Scholar). not the of modulating the in the DGKζ domain, we that this phosphorylation correct protein with the plasma membrane either by some or a A in domain are by that phosphorylation cytosolic and requires receptor stimulation for translocation. This the phosphorylation of in the DGKζ domain it is not to membrane localization of the the stimulation of T-cells the endogenous does not membrane localization of This that DGKζ is an enzyme in response to and not the of receptors. The DGKζ C-terminal the PDZ-binding and the four ankyrin repeats, is to to the following stimulation. the expression of the C-terminal domain of DGKζ translocation of the type protein in response to This us to a in membrane localization of DGKζ be by the of these domains with a to be characterized protein. this the activation of the type I muscarinic receptor the of isoforms with that the and membrane localization of other signaling molecules such as or S. M. J. Biochem. 2001; PubMed Scopus Google Scholar). a in cytosolic DGKζ in a with the C-terminal protein with the stimulation DGKζ with a phosphorylation of the domain enzyme to the The C-terminal domain of DGKζ is not for plasma membrane translocation is in turn essential for receptor of the C-terminal this the enzyme in the C-terminal domain is in response to The of signaling molecules to specific cellular is not by signals generated by receptor also by to the These a very specific enzymes and and the onset of different cellular responses. The of the of the DGKζ domains in enzyme activity and membrane translocation in living demonstrate that this enzyme is to regulation following receptor As we membrane localization of DGKζ requires intact and is DAG-regulated the C-terminal domain a for selectivity of DGKζ activation receptors. our to of the for and S. and M. for of the and C. for The cDNA from was a of K. The from A. with