COMMUNICATION
Diacylglycerol Kinase theta  Binds to and Is Negatively Regulated by Active RhoA*

Brahim Houssa, John de Widt, Onno Kranenburg, Wouter H. Moolenaar, and Wim J. van BlitterswijkDagger

From the Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

    ABSTRACT
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Abstract
Introduction
References

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 DGKtheta 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 DGKtheta 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. DGKtheta does not bind to inactive RhoA, nor to the other Rho-family GTPases, Rac or Cdc42. Like active RhoA, DGKtheta localizes to the plasma membrane. Strikingly, the binding of activated RhoA to DGKtheta completely inhibits DGK catalytic activity. Our results suggest that DGKtheta 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 DGKtheta may lead to enhanced PKC activity in response to external stimuli.

    INTRODUCTION
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Abstract
Introduction
References

Diacylglycerol kinase (DGK)1 phosphorylates the second messenger diacylglycerol (DAG) to yield phosphatidic acid (1). Because DAG is a physiological activator of protein kinase C (PKC), DGK may act to attenuate PKC activation in response to external stimuli (1-3). 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. 4 and 5).

Rho family GTPases (RhoA, Rac, and Cdc42) regulate vital cellular functions, particularly cytoskeletal reorganization and gene transcription (6, 7). These small GTPases regulate not only protein kinases (7) but also lipid-metabolizing enzymes, such as phospholipase D (PLD) (8) and phosphatidylinositol 5- and 3-kinases (6, 7, 9). In addition, and interestingly enough, the Rac GTPase has been reported to form a functional complex with an unidentified DGK in vivo (10).

We have recently cloned the cDNA of a new DGK isotype, termed DGKtheta (11), and have now considered the possibility that this DGKtheta might be regulated by Rho GTPases. We report here that DGKtheta specifically binds to active RhoA but not to Rac or Cdc42. Most strikingly, and unlike other RhoA effectors, DGKtheta loses catalytic activity when it binds to RhoA.

    EXPERIMENTAL PROCEDURES

Cells and Plasmids-- 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. DGKtheta cDNA was VSV-tagged at the 3'-end. Myc-tagged RhoA, Rac1, and Cdc42 constructs were described previously (12). Point mutations were introduced into V14-RhoA cDNA by using QuickchangeTM site-directed mutagenesis kit from Stratagene and checked by DNA sequencing.

Antibodies and Immunofluorescence-- P5D4 monoclonal antibody directed against VSV-tag was used for Western blotting and immunoprecipitation of VSV-tagged DGKtheta . 9E10 monoclonal antibody directed against Myc-tag was used for Western blotting and immunoprecipitation of Myc-tagged small GTP binding proteins. Anti-DGKtheta polyclonal antibody #101 was raised against a synthetic peptide corresponding to the stretch of amino acids 312 to 331 in the DGKtheta 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 DGKtheta 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).

Cell Fractionation-- 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.

GST-RhoA Binding and DGK Activity Assay-- N1E-115 cells were lysed in a buffer containing 150 mM NaCl, 50 mM Tris-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-DGKtheta polyclonal antibody #101.

DGK activity assays were performed as described (11). The enzymatic product, phosphatidic acid (PA), was separated by TLC using the solvent system chloroform/methanol/acetic acid (65:15:5, v/v/v).

    RESULTS AND DISCUSSION

We first tested whether DGKtheta can bind to Rho family members. To this end, DGKtheta 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 DGKtheta 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.2 To assess how DGKtheta binding depends on the activation state of RhoA, we used constitutively activated and inactive versions of RhoA. Fig. 1B shows that DGKtheta co-immunoprecipitates with active V14-RhoA but not with inactive N19-RhoA. Furthermore, it is seen that DGKtheta fails to co-precipitate with constitutively active Rac1 (V12-Rac) or Cdc42 (V12-Cdc42). This argues against DGKtheta being the unidentified DGK that interacts with Rac (10).


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Fig. 1.   Specific binding of DGKtheta to active RhoA in vivo. A, COS7 cells were co-transfected with DGKtheta (VSV-tagged) and either RhoA, Rac, or Cdc42 (wild-type; Myc-tagged). Cells were lysed (1% Nonidet P-40), and the small G proteins were immunoprecipitated with anti-Myc monoclonal antibody 9E10. Co-immunoprecipitated DGKtheta was visualized (upper panel) by Western blotting. B, same experiment but using the constitutively active mutants V14-RhoA, V12-Rac, and V12-Cdc42 and the inactive mutant N19-RhoA (all Myc-tagged). Lower panels show expression controls (total cell lysates). Positions of Myc-RhoA, Myc-Rac, and Myc-Cdc42 (small G proteins) and VSV-DGKtheta in the gel are indicated (arrowheads). C, endogenous DGKtheta from N1E-115 neuroblastoma cells binds to active RhoA. GST fusion proteins of active V14-RhoA or inactive N19-RhoA or GST alone were incubated with N1E-115 cell lysate and then collected through binding to glutathione-Sepharose beads. Bound DGKtheta was detected by Western blotting. D, RhoA effector loop mutation Y34N, but not T37A, abrogates binding to DGKtheta ; same experimental conditions as in panel A.

We next examined RhoA binding to endogenous DGKtheta in nontransfected cells. DGKtheta is predominantly expressed in brain and neuronal cell lines, such as N1E-115 neuroblastoma cells (11). Purified GST-RhoA fusion proteins, immobilized on glutathione-Sepharose beads, were incubated with N1E-115 cell lysates, and RhoA-bound DGKtheta was assayed by Western blotting using a polyclonal antibody against DGKtheta . As shown in Fig. 1C, DGKtheta 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 GTPgamma S versus GDPbeta S (data not shown).

We conclude that DGKtheta binds to RhoA in a GTP-dependent manner but not to other members of the Rho family, suggesting that DGKtheta is a downstream effector of RhoA. RhoA target molecules bind to the effector-loop region of RhoA (13). Specific point mutations in this region have been shown to interfere with effector binding (8, 13-15). For example, mutations in conserved amino acids Tyr-34 and Thr-37 are known to abolish binding and activation of PLD (8) and protein kinase N (14). 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 DGKtheta . Fig. 1D shows that the Y34N mutation disrupts the binding of V14-RhoA to DGKtheta , whereas the T37A mutation has no effect. This suggests that the RhoA-DGKtheta 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 (15-18). We investigated DGKtheta localization by immunofluorescence and cell fractionation studies. We used DGKtheta -transfected COS7 cells because the level of endogenous DGKtheta in N1E-115 cells was too low to allow proper detection. Fractionation of cell lysates revealed that the majority of DGKtheta 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 DGKtheta at the cell periphery (Fig. 2B). In addition, DGKtheta 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 (17). The presence of DGKtheta at the cell periphery supports a model in which DGKtheta is regulated by RhoA-GTP at the inner side of the plasma membrane, where the DGK substrate DAG is generated following cell activation.


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Fig. 2.   Subcellular localization of DGKtheta . COS7 cells were transfected with DGKtheta cDNA and maintained at 8% serum for 2 days. A, total cell lysate (Total) was fractionated into cytosol, particulate fraction (i.e. membranes plus cytoskeleton), membrane fraction, and cytoskeleton (see "Experimental Procedures"). Fractions were suspended/adjusted to equal volumes and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. B, immunofluorescence confocal microscopy using polyclonal antibody #101 against DGKtheta .

We next investigated how activated RhoA may affect DGKtheta activity. Epitope-tagged versions of DGKtheta and V14-RhoA were co-expressed in COS7 cells, and DGKtheta 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 DGKtheta was then tested in an in vitro kinase assay using 1,2-dioleoyl-sn-glycerol as a substrate. As shown in Fig. 3A, RhoA-bound DGKtheta is completely inactive, whereas free DGKtheta (precipitated with P5D4) is highly active. Likewise, when bound to active GST-V14-RhoA, endogenous DGKtheta , pulled down from N1E-115 cell lysates, was completely inactive, in contrast to free DGKtheta (Fig. 3B). From these results, we conclude that DGKtheta is catalytically inactive when physically associated with active RhoA. In other words, RhoA is a negative regulator of DGKtheta activity.


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Fig. 3.   DGKtheta is inactive when bound to RhoA. A, COS7 cells were transfected with VSV-tagged DGKtheta with (+) or without (-) Myc-tagged V14-RhoA cDNAs. Cells were lysed (1% Nonidet P-40), and DGKtheta was immunoprecipitated directly with P5D4 monoclonal antibody against the VSV-tag or indirectly via RhoA using 9E10 monoclonal antibody against the Myc-tag. One fourth of the immunoprecipitate was used for measuring DGK activity. PA, the product of DGK, was separated by TLC (lower panel; see "Experimental Procedures"). Co-precipitated DGKtheta was visualized by Western blotting (upper panel). B, endogenous DGKtheta from N1E-115 cells was recovered in precipitates, either by using GST-V14RhoA and glutathione beads (binding) or by anti DGKtheta antibody (I.P.). Half of the precipitates was analyzed by Western blotting using DGKtheta antibody #101 (upper panel), and the other half was used for DGK activity assay (lower panel). The histogram shows relative DGK activities, based on 32P-PA formation quantified after TLC separation, using a Fujix BAS 2000 TR phosphoimager. Inactive GST-N19-RhoA, free GST, and normal rabbit serum (NRS) were used as negative controls.

Contrary to its inhibitory action on DGKtheta , RhoA has been reported to stimulate the activity of PLD (8, 18). 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 (19) 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 (19, 20), and DGK in stimulated cells does not phosphorylate DAG generated by sequential PLD/PA phosphohydrolase activities (20, 21) nor DAG that is randomly generated in the plasma membrane by exogenous phospholipase C (22).

An important implication of DGKtheta 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 PKCalpha in vivo, important for membrane translocation and activation of PKCalpha (15, 23). Similar conclusions have been reached for activated Rho1 and Pkc1 in yeast (24, 25). 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 PKCalpha ) directly and a negative action on DGKtheta . Whether RhoA proteins act in larger signaling complexes in association with both DGKtheta and PKC, and whether other DGK isotypes are similarly regulated by Rho family members, remains to be investigated.

    FOOTNOTES

* This work was supported by a grant from the Netherlands Organization for Scientific Research (SON 330-210) and by the Dutch Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Fax: +31-20-5121989; E-mail: wblit{at}nki.nl.

2 O. Kranenburg, M. Poland, F. P. G. van Horck, and W. H. Moolenaar, manuscript in preparation.

    ABBREVIATIONS

The 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; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; GDPbeta S, guanyl-5'-yl thiophosphate; PA, phosphatidic acid.

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