Diacylglycerol Kinase θ Binds to and Is Negatively Regulated by Active RhoA

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. To date, very little is known about the regulation of DGK activity. We have previously identified the DGKθ isotype, which is predominantly expressed in brain (Houssa, B., Schaap, D., van der Wal, J., Goto, K., Kondo, H., Yamakawa, A., Shibata, M., Takenawa, T., and Van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 10422–10428). We now report that DGKθ binds specifically to activated RhoA in transfected COS cells as well as in nontransfected neuronal N1E-115 cells. Binding is abolished by a point mutation (Y34N) in the effector loop of RhoA. DGKθ does not bind to inactive RhoA, nor to the other Rho-family GTPases, Rac or Cdc42. Like active RhoA, DGKθ localizes to the plasma membrane. Strikingly, the binding of activated RhoA to DGKθ completely inhibits DGK catalytic activity. Our results suggest that DGKθ is a downstream effector of RhoA and that its activity is negatively regulated by RhoA. Through accumulation of newly produced diacylglycerol, RhoA-mediated inhibition of DGKθ may lead to enhanced PKC activity in response to external stimuli. Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. To date, very little is known about the regulation of DGK activity. We have previously identified the DGKθ isotype, which is predominantly expressed in brain (Houssa, B., Schaap, D., van der Wal, J., Goto, K., Kondo, H., Yamakawa, A., Shibata, M., Takenawa, T., and Van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 10422–10428). We now report that DGKθ binds specifically to activated RhoA in transfected COS cells as well as in nontransfected neuronal N1E-115 cells. Binding is abolished by a point mutation (Y34N) in the effector loop of RhoA. DGKθ does not bind to inactive RhoA, nor to the other Rho-family GTPases, Rac or Cdc42. Like active RhoA, DGKθ localizes to the plasma membrane. Strikingly, the binding of activated RhoA to DGKθ completely inhibits DGK catalytic activity. Our results suggest that DGKθ is a downstream effector of RhoA and that its activity is negatively regulated by RhoA. Through accumulation of newly produced diacylglycerol, RhoA-mediated inhibition of DGKθ may lead to enhanced PKC activity in response to external stimuli. Diacylglycerol kinase (DGK) 1The abbreviations used are: DGK, diacylglycerol kinase; DAG, diacylglycerol; PKC, protein kinase C; PLD, phospholipase D; VSV, vesicular stomatitis virus; PBS, phosphate-buffered saline; GST, glutathione S-transferase; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GDPβS, guanyl-5′-yl thiophosphate; PA, phosphatidic acid 1The abbreviations used are: DGK, diacylglycerol kinase; DAG, diacylglycerol; PKC, protein kinase C; PLD, phospholipase D; VSV, vesicular stomatitis virus; PBS, phosphate-buffered saline; GST, glutathione S-transferase; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GDPβS, guanyl-5′-yl thiophosphate; PA, phosphatidic acid phosphorylates the second messenger diacylglycerol (DAG) to yield phosphatidic acid (1Kanoh H. Yamada K. Sakane F. Trends Biochem. Sci. 1990; 15: 47-50Abstract Full Text PDF PubMed Scopus (165) Google Scholar). Because DAG is a physiological activator of protein kinase C (PKC), DGK may act to attenuate PKC activation in response to external stimuli (1Kanoh H. Yamada K. Sakane F. Trends Biochem. Sci. 1990; 15: 47-50Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 2van Blitterswijk W.J. Schaap D. van der Bend R. Curr. Top. Membr. 1994; 40: 413-437Crossref Scopus (18) Google Scholar, 3Houssa B. van Blitterswijk W.J. Biochem. J. 1998; 331: 677-679Crossref PubMed Scopus (31) Google Scholar). To date, nine mammalian DGK isotypes have been cloned (excluding alternatively spliced variants), but surprisingly little is known about the regulation of these isotypes (for review, see Refs. 4van Blitterswijk W.J. Houssa B. Chem. Phys. Lipids. 1999; (in press)PubMed Google Scholarand 5Topham 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). Rho family GTPases (RhoA, Rac, and Cdc42) regulate vital cellular functions, particularly cytoskeletal reorganization and gene transcription (6Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 7Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (692) Google Scholar). These small GTPases regulate not only protein kinases (7Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (692) Google Scholar) but also lipid-metabolizing enzymes, such as phospholipase D (PLD) (8Bae C.D. Min D.S. Fleming I.N. Exton J.H. J. Biol. Chem. 1998; 273: 11596-11604Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and phosphatidylinositol 5- and 3-kinases (6Symons M. Trends Biochem. Sci. 1996; 21: 178-181Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 7Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (692) Google Scholar, 9Ren X.D. Schwartz M.A. Curr. Opin. Genet. Dev. 1998; 8: 63-67Crossref PubMed Scopus (78) Google Scholar). In addition, and interestingly enough, the Rac GTPase has been reported to form a functional complex with an unidentified DGK in vivo(10Tolias K.F. Couvillon A.D. Cantley L.C. Carpenter C.L. Mol. Cell. Biol. 1998; 18: 762-770Crossref PubMed Scopus (136) Google Scholar). We have recently cloned the cDNA of a new DGK isotype, termed DGKθ (11Houssa B. Schaap D. van der Wal J. Goto K. Kondo H. Yamakawa A. Shibata M. Takenawa T. van Blitterswijk W.J. J. Biol. Chem. 1997; 272: 10422-10428Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and have now considered the possibility that this DGKθ might be regulated by Rho GTPases. We report here that DGKθ specifically binds to active RhoA but not to Rac or Cdc42. Most strikingly, and unlike other RhoA effectors, DGKθ loses catalytic activity when it binds to RhoA. COS7-M6 and N1E-115 cells were grown in Dulbecco's modified Eagle's medium with 8% fetal calf serum and antibiotics. Cell transfections were all performed with pMT2 expression vectors using the DEAE-dextran method. Two days after transfection, cells were used for experiments. DGKθ cDNA was VSV-tagged at the 3′-end. Myc-tagged RhoA, Rac1, and Cdc42 constructs were described previously (12Gebbink M.F.B.G. Kranenburg O. Poland M. van Horck F.P.G. Houssa B. Moolenaar W.H. J. Cell Biol. 1997; 137: 1603-1613Crossref PubMed Scopus (140) Google Scholar). Point mutations were introduced into V14-RhoA cDNA by using QuickchangeTM site-directed mutagenesis kit from Stratagene and checked by DNA sequencing. P5D4 monoclonal antibody directed against VSV-tag was used for Western blotting and immunoprecipitation of VSV-tagged DGKθ. 9E10 monoclonal antibody directed against Myc-tag was used for Western blotting and immunoprecipitation of Myc-tagged small GTP binding proteins. Anti-DGKθ polyclonal antibody #101 was raised against a synthetic peptide corresponding to the stretch of amino acids 312 to 331 in the DGKθ primary sequence. For immunofluorescence, cells were fixed in 3.7% formaldehyde in PBS, permeabilized with PBS containing 0.1% Triton X-100, and subsequently blocked with 1% bovine serum albumin in PBS for 30 min. Cells were then stained with primary DGKθ antibody #101 and secondary goat-anti-rabbit antibody conjugated to Texas Red (Molecular Probes Inc.), each used at 1:100 dilution. After washing, cells were mounted with Vectashield, and fluorescence was analyzed on a Bio-Rad confocal microscope (MRC-600). Cells were disrupted by sonication in Hepes (50 mm, pH 8), sucrose (250 mm), supplemented with protease inhibitors. Cell debris and nuclei were removed by centrifugation (14,000 × g, 10 min). The supernatant was centrifuged at 100,000 × g for 1 h to separate cytosol (supernatant) from a pellet (particulate) fraction consisting of membranes and cytoskeleton. The pellet was resuspended in 1% Nonidet P-40 lysis buffer and centrifuged (100,000 × g, 1 h). The supernatant contains dissolved membranes, and the pellet contains the cytoskeleton. N1E-115 cells were lysed in a buffer containing 150 mm NaCl, 50 mmTris-HCl (pH 8.0), 5 mm MgCl2, 1% Nonidet P-40 and protease inhibitors, and incubated with purified GST-fusion protein (10 μg) at 4 °C. GST-fusion proteins were then collected with Glutathione-Sepharose beads. The beads were washed four times with lysis buffer and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-DGKθ polyclonal antibody #101. DGK activity assays were performed as described (11Houssa B. Schaap D. van der Wal J. Goto K. Kondo H. Yamakawa A. Shibata M. Takenawa T. van Blitterswijk W.J. J. Biol. Chem. 1997; 272: 10422-10428Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The enzymatic product, phosphatidic acid (PA), was separated by TLC using the solvent system chloroform/methanol/acetic acid (65:15:5, v/v/v). We first tested whether DGKθ can bind to Rho family members. To this end, DGKθ was VSV-epitope-tagged and transfected into COS7 cells together with expression vectors encoding various forms of Myc-epitope-tagged RhoA, Cdc42, or Rac1. Fig. 1A shows that DGKθ co-immunoprecipitates with (wild-type) RhoA but not with Rac or Cdc42. Wild-type RhoA, when expressed in COS7 cells, is partially active (GTP bound) as evidenced by its binding to a downstream effector, Rho kinase. 2O. Kranenburg, M. Poland, F. P. G. van Horck, and W. H. Moolenaar, manuscript in preparation. To assess how DGKθ binding depends on the activation state of RhoA, we used constitutively activated and inactive versions of RhoA. Fig. 1B shows that DGKθ co-immunoprecipitates with active V14-RhoA but not with inactive N19-RhoA. Furthermore, it is seen that DGKθ fails to co-precipitate with constitutively active Rac1 (V12-Rac) or Cdc42 (V12-Cdc42). This argues against DGKθ being the unidentified DGK that interacts with Rac (10Tolias K.F. Couvillon A.D. Cantley L.C. Carpenter C.L. Mol. Cell. Biol. 1998; 18: 762-770Crossref PubMed Scopus (136) Google Scholar). We next examined RhoA binding to endogenous DGKθ in nontransfected cells. DGKθ is predominantly expressed in brain and neuronal cell lines, such as N1E-115 neuroblastoma cells (11Houssa B. Schaap D. van der Wal J. Goto K. Kondo H. Yamakawa A. Shibata M. Takenawa T. van Blitterswijk W.J. J. Biol. Chem. 1997; 272: 10422-10428Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Purified GST-RhoA fusion proteins, immobilized on glutathione-Sepharose beads, were incubated with N1E-115 cell lysates, and RhoA-bound DGKθ was assayed by Western blotting using a polyclonal antibody against DGKθ. As shown in Fig. 1C, DGKθ is pulled down by GST-V14-RhoA but not with GST-N19-RhoA nor with free GST. The GTP dependence of the binding was confirmed in a similar experiment, using GST-wild-type RhoA fusion protein loaded with GTPγS versus GDPβS (data not shown). We conclude that DGKθ binds to RhoA in a GTP-dependent manner but not to other members of the Rho family, suggesting that DGKθ is a downstream effector of RhoA. RhoA target molecules bind to the effector-loop region of RhoA (13Wittinghofer A. Nasser N. Trends Biochem. Sci. 1996; 21: 488-491Abstract Full Text PDF PubMed Scopus (137) Google Scholar). Specific point mutations in this region have been shown to interfere with effector binding (8Bae C.D. Min D.S. Fleming I.N. Exton J.H. J. Biol. Chem. 1998; 273: 11596-11604Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 13Wittinghofer A. Nasser N. Trends Biochem. Sci. 1996; 21: 488-491Abstract Full Text PDF PubMed Scopus (137) Google Scholar, 14Sahai E. Alberts A.S. Treisman R. EMBO J. 1998; 17: 1350-1361Crossref PubMed Scopus (229) Google Scholar, 15Chang J.H. Pratt J.C. Sawasdikosol S. Kapeller R. Burakoff S.J. Mol. Cell. Biol. 1998; 18: 4986-4993Crossref PubMed Scopus (85) Google Scholar). For example, mutations in conserved amino acids Tyr-34 and Thr-37 are known to abolish binding and activation of PLD (8Bae C.D. Min D.S. Fleming I.N. Exton J.H. J. Biol. Chem. 1998; 273: 11596-11604Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and protein kinase N (14Sahai E. Alberts A.S. Treisman R. EMBO J. 1998; 17: 1350-1361Crossref PubMed Scopus (229) Google Scholar). We therefore mutated these residues in active V14-RhoA to yield V14/N34- and V14/A37-RhoA and tested whether these mutations affect the binding of DGKθ. Fig. 1D shows that the Y34N mutation disrupts the binding of V14-RhoA to DGKθ, whereas the T37A mutation has no effect. This suggests that the RhoA-DGKθ interaction is mediated by the RhoA effector loop, in which residue Tyr-34 is critical for binding. Activated RhoA is known to localize to the plasma membrane, whereas inactive GDP-bound RhoA is largely cytosolic (15Chang J.H. Pratt J.C. Sawasdikosol S. Kapeller R. Burakoff S.J. Mol. Cell. Biol. 1998; 18: 4986-4993Crossref PubMed Scopus (85) Google Scholar, 16Adamson P. Paterson H.F. Hall A. J. Cell Biol. 1992; 119: 617-727Crossref PubMed Scopus (327) Google Scholar, 17Kranenburg O. Poland M. Gebbink M. Oomen L. Moolenaar W.H. J. Cell Sci. 1997; 110: 2417-2427Crossref PubMed Google Scholar, 18Malcolm K.C. Elliott C.M. Exton J.H. J. Biol. Chem. 1996; 271: 13135-13139Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). We investigated DGKθ localization by immunofluorescence and cell fractionation studies. We used DGKθ-transfected COS7 cells because the level of endogenous DGKθ in N1E-115 cells was too low to allow proper detection. Fractionation of cell lysates revealed that the majority of DGKθ is present in the membrane fraction (Fig. 2A). The remainder is found in the cytosolic and the cytoskeleton fractions. Immunofluorescence analysis shows the presence of DGKθ at the cell periphery (Fig. 2B). In addition, DGKθ is present in the cytoplasm and the perinuclear region, as usual for an overexpression system. A similar subcellular distribution has been reported for overexpressed RhoA (17Kranenburg O. Poland M. Gebbink M. Oomen L. Moolenaar W.H. J. Cell Sci. 1997; 110: 2417-2427Crossref PubMed Google Scholar). The presence of DGKθ at the cell periphery supports a model in which DGKθ is regulated by RhoA-GTP at the inner side of the plasma membrane, where the DGK substrate DAG is generated following cell activation. We next investigated how activated RhoA may affect DGKθ activity. Epitope-tagged versions of DGKθ and V14-RhoA were co-expressed in COS7 cells, and DGKθ was immunoprecipitated either directly, using anti-VSV monoclonal antibody P5D4, or indirectly, through co-precipitation with RhoA using anti-myc monoclonal antibody 9E10. The catalytic activity of both pools of DGKθ was then tested in anin vitro kinase assay using 1,2-dioleoyl-sn-glycerol as a substrate. As shown in Fig. 3A, RhoA-bound DGKθ is completely inactive, whereas free DGKθ (precipitated with P5D4) is highly active. Likewise, when bound to active GST-V14-RhoA, endogenous DGKθ, pulled down from N1E-115 cell lysates, was completely inactive, in contrast to free DGKθ (Fig. 3B). From these results, we conclude that DGKθ is catalytically inactive when physically associated with active RhoA. In other words, RhoA is a negative regulator of DGKθ activity. Contrary to its inhibitory action on DGKθ, RhoA has been reported to stimulate the activity of PLD (8Bae C.D. Min D.S. Fleming I.N. Exton J.H. J. Biol. Chem. 1998; 273: 11596-11604Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 18Malcolm K.C. Elliott C.M. Exton J.H. J. Biol. Chem. 1996; 271: 13135-13139Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Both DGK and PLD generate PA, albeit from different lipid sources (DAG and phosphatidylcholine, respectively). How can one explain the significance of one PA-producing enzyme (PLD) being activated and the other (DGK) inactivated by RhoA? A likely possibility is that PLD and DGK and their respective PA products serve different cellular functions (19Hodgkin M.N. Pettitt T.R. Martin A. Michell R.H. Pemberton A.J. Wakelam M.J.O. Trends Biochem. Sci. 1998; 23: 200-204Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) and that these enzymes, their lipid substrates, and PA products may be located in distinct, spatially separated compartments within the cell. Consistent with this, the PA pools generated by PLD and DGK differ in fatty acid composition (19Hodgkin M.N. Pettitt T.R. Martin A. Michell R.H. Pemberton A.J. Wakelam M.J.O. Trends Biochem. Sci. 1998; 23: 200-204Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar,20van Blitterswijk W.J. Hilkmann H. de Widt J. van der Bend R.L. J. Biol. Chem. 1991; 266: 10337-10343Abstract Full Text PDF PubMed Google Scholar), and DGK in stimulated cells does not phosphorylate DAG generated by sequential PLD/PA phosphohydrolase activities (20van Blitterswijk W.J. Hilkmann H. de Widt J. van der Bend R.L. J. Biol. Chem. 1991; 266: 10337-10343Abstract Full Text PDF PubMed Google Scholar, 21van Dijk M.C.M. van Blitterswijk W.J. Biochim. Biophys. Acta. 1998; 1391: 273-279Crossref PubMed Scopus (8) Google Scholar) nor DAG that is randomly generated in the plasma membrane by exogenous phospholipase C (22van der Bend R.L. de Widt J. Hilkmann H. van Blitterswijk W.J. J. Biol. Chem. 1994; 269: 4098-4102Abstract Full Text PDF PubMed Google Scholar). An important implication of DGKθ inhibition is that RhoA should be able to regulate DAG levels and, hence, PKC activity. Interestingly, activated RhoA has recently been reported to associate with PKCαin vivo, important for membrane translocation and activation of PKCα (15Chang J.H. Pratt J.C. Sawasdikosol S. Kapeller R. Burakoff S.J. Mol. Cell. Biol. 1998; 18: 4986-4993Crossref PubMed Scopus (85) Google Scholar, 23Hippenstiel S. Kratz T. Krüll M. Seybold J. von Eichel-Streiber C. Suttorp N. Biochem. Biophys. Res. Commun. 1998; 245: 830-834Crossref PubMed Scopus (65) Google Scholar). Similar conclusions have been reached for activated Rho1 and Pkc1 in yeast (24Nonaka H. Tanaka K. Hirano H. Fujiwara T. Kohno H. Umikawa M. Mino A. Takai Y. EMBO J. 1995; 14: 5931-5938Crossref PubMed Scopus (304) Google Scholar, 25Kamada Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Combined with these data, our present results would suggest that RhoA may promote activation of PKC through a concerted positive action on PKC (at least PKCα) directly and a negative action on DGKθ. Whether RhoA proteins act in larger signaling complexes in association with both DGKθ and PKC, and whether other DGK isotypes are similarly regulated by Rho family members, remains to be investigated.