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J. Biol. Chem., Vol. 279, Issue 27, 28603-28613, July 2, 2004
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Serves as an Upstream Suppressor of Rac1 and Lamellipodium Formation*
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From the
Departments of Biochemistry and ¶Neurosurgery, School of Medicine, and the Department of Liberal Arts and Sciences, School of Health Sciences, Sapporo Medical University, Chuo-ku, Sapporo 060-8556, Japan
Received for publication, December 22, 2003 , and in revised form, April 15, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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in regulating Rac1-governed cell morphology. We found that the expression of kinase-dead DGK, which acts as a dominant-negative mutant, and inhibition of endogenous DGK activity with R59949
[GenBank]
induced lamellipodium and membrane ruffle formation in NIH3T3 fibroblasts in the absence of growth factor stimulation. Reciprocally, lamellipodium formation induced by platelet-derived growth factor was significantly inhibited upon expression of constitutively active DGK. Moreover, the constitutively active DGK mutant suppressed integrin-mediated cell spreading. These effects are isoform-specific because, in the same experiments, none of the corresponding mutants of DGK
and DGK
, closely related isoforms, affected cell morphology. These results suggest that DGK specifically participates in the Rac1-mediated signaling pathway leading to cytoskeletal reorganization. In support of this, DGK co-localized with dominant-active Rac1 especially in lamellipodia. Moreover, we found that endogenous DGK was physically associated with cellular Rac1. Dominant-negative Rac1 expression blocked the lamellipodium formation induced by kinase-dead DGK, indicating that DGK acts upstream of Rac1. This model is supported by studies demonstrating that kinase-dead DGK selectively activated Rac1, but not Cdc42. Taken together, these results strongly suggest that DGK functions through its catalytic action as an upstream suppressor of Rac1 and, consequently, lamellipodium/ruffle formation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Mammalian DGK is known to exist as a large protein family consisting of nine isozymes classified into five subtypes according to their structural features (12–15). These subfamilies can be characterized by the presence of a variety of regulatory domains of known and/or predicted functions, clearly indicating their distinct functions and regulatory mechanisms. The type I DGKs, presently consisting of -, -, and -isozymes (16–21), contain two sets of Ca2+-binding EF-hand motifs at their N termini (22–24). The distinctive tissue- and cell-dependent expression patterns detected for these isozymes suggest that, even belonging to the same subfamily, each member exerts differentiated functions in particular types of cells (12–15). Moreover, we recently reported that the EF-hand motifs of the type I DGKs have properties distinct from each other with respect to affinities for Ca2+ and to Ca2+-induced conformational changes (24). Among the type I DGKs, DGK has recently been the subject of intensive investigations (25–28). However, the functions of the type I DGKs other than the -isozyme have not yet been defined, and it remains unknown to what extent the functions of the three type I DGKs are redundant or distinct.
Recently, we provided results suggesting that DGK negatively regulates macrophage differentiation of human leukemia HL-60 and U937 cells through its catalytic action (29). Moreover, we found that after phorbol ester treatment, DGK translocates from the cytoplasm to the cell periphery, resulting in its co-localization with F-actin. In addition, rat brain DGK was reported previously as a cytoskeleton-associated isozyme (21). However, the physiological implication of the cytoskeleton association of DGK remains to be further explored. In this work, to reveal cytoskeleton-related targets and regulators of DGK, we examined the effects of enzyme expression on cytoskeletal changes using NIH3T3 fibroblasts, whose F-actin-driven morphological changes are well characterized. Interestingly, a kinase-dead mutant of DGK enhanced lamellipodium/membrane ruffle formation in the cells in the absence of growth factor stimulation. In contrast, the corresponding mutants of DGK and DGK, closely related isoforms, failed to affect cell morphology. Several lines of evidence strongly suggest that DGK operates as a novel negative regulator of Rac1.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmid Constructs—The kinase-dead (DGK-G435D and DGK-G494D) and constitutively active (DGK-(
1–192) and DGK-(1–259)) versions of pig DGK and human DGK were generated as described previously (22, 29). The cDNA encoding DGK-(1–232) (constitutively active version) was generated from a rat DGK cDNA clone (19) by PCR and subcloned into pEGFP-C3 (BD Biosciences, Tokyo) at the SalI/BamHI sites. The kinase-dead version of DGK (DGK-G495D) was generated by replacing Gly495 with Asp using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
The human cdc42, rac1, and rhoA cDNAs were amplified by PCR using human whole brain cDNA (BD Biosciences) as template and then subcloned into the pGEX-6P-1 (Amersham Biosciences, Tokyo) or pECFP-C1 (BD Biosciences) expression vector. A dominant-active form of Rac1 (Rac1-G12V) was generated by replacing Gly12 with Val using the QuikChange site-directed mutagenesis kit. A dominant-negative mutant of Rac1 (Rac1-T17N) was generated by replacing Thr17 with Asn. The authenticity of the cDNA constructs was confirmed by DNA sequencing.
Fluorescence Microscopy—For immunofluorescence microscopy, NIH3T3 cells were grown on poly-L-lysine-coated glass coverslips and transiently transfected with expression plasmids containing cDNAs for DGK and Rho family small G-proteins N-terminally fused with green fluorescent protein (GFP), cyan fluorescent protein (CFP), or yellow fluorescent protein (YFP). After 24 h, cells were fixed with 3.7% formaldehyde. When F-actin staining was required, cells were permeabilized in phosphate-buffered saline containing 0.1% Triton X-100 and 1% bovine serum albumin. Coverslips were then incubated with phalloidin conjugated with rhodamine (Santa Cruz Biotechnology, Santa Cruz, CA) or with Alexa Fluor 594 (Molecular Probes, Inc, Eugene, OR). The coverslips were mounted using Vectashield (Vector Labs, Inc., Burlingame, CA). Cells were examined using a Zeiss LSM 510 inverted confocal laser scanning microscope.
For living cell imaging, NIH3T3 cells were transiently transfected with the expression plasmids encoding YFP-DGK and CFP-small G-proteins and observed in glass bottom chambers (Asahi Techno Glass, Tokyo). When fluorescence microscopy was carried out, the medium was changed to Dulbecco's modified Eagle's medium/nutrient mixture F-12 without phenol red and buffered with 15 mM HEPES (Invitrogen). After 24 h of transfection, cells were examined using the Zeiss LSM 510 inverted confocal laser scanning microscope.
To assess the extent of lamellipodium/membrane ruffle formation, cells were stained for F-actin using rhodamine-labeled phalloidin. Random fields of the cells were photographed, and >50 cells exhibiting fluorescence were examined. Cells exhibiting clearly discernible lamellipodia/membrane ruffles (in most cases, >10 µm in width) (see Figs. 1, 2, 3, 4, 6, and 8) were scored positive.
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forward primer, 5'-GATGAGCGAAGAACAATGGG-3' (nucleotides 32–51); mouse DGK reverse primer, 5'-CCTGAGGTCGCCCGGTC-3' (nucleotides 538–554); mouse DGK forward primer, 5'-CCATGACAAACCAGGAAAAATGG-3' (nucleotides 425–447); and mouse DGK reverse primer, 5'-CCTCGGGTCTTCCTCTTTCG-3' (nucleotides 868–887). PCR conditions were as follows: 94 °C for 2 min; 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 2 min; and 68 °C for 7 min. As a control, mouse glyceraldehyde-3-phosphate dehydrogenase mRNA was simultaneously amplified. PCR products amplified were separated by agarose gel electrophoresis and stained with ethidium bromide.
Co-immunoprecipitation Analysis—NIH3T3 cells (
1 x 107 cells/60-mm dish) were transiently transfected with p3xFLAG-CMV-DGK-WT together with pECFP alone, pECFP-RhoA-WT, pECFP-Rac1-WT, or pECFP-Cdc42-WT. Twenty-four hours after transfection, cells were washed once with phosphate-buffered saline and harvested in 500 µl of buffer A (50 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, Complete protease inhibitor mixture (Roche Applied Science, Tokyo), and phosphatase inhibitor mixture II (Sigma)). The mixture was centrifuged at 12,000 x g for 10 min at 4 °C to give cell lysates (input). Co-immunoprecipitation analysis using anti-GFP antibody was carried out as described previously (31). In some experiments, anti-DGK serum (2 µl) (20) was used to precipitate endogenous DGK.
Western Blot Analysis—Cell lysates (input) and immunoprecipitates were separated by SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Tokyo) and blocked with Block Ace (Dainippon Pharmaceutical, Tokyo). The membrane was incubated with anti-FLAG monoclonal antibody M2 (Sigma), anti-GFP monoclonal antibody (B-2, Santa Cruz Biotechnology), or anti-DGK serum in Block Ace for 1 h. The immunoreactive bands were visualized using peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and ECL (Amersham Biosciences). To measure the relative density of immunoreactive bands, images were scanned and analyzed by NIH Image software.
Pull-down Assay—Glutathione S-transferase (GST)-fused Rac1, which was bacterially expressed and coupled to glutathione-Sepharose beads (Amersham Biosciences), was loaded with either 1 mM GDPS (Sigma) or 100 µM GTPS (Sigma) at 30 °C for 2 h. COS-7 cells (1 x 107 cells/60-mm dish) expressing (FLAG)3-tagged DGK were lysed in 1 ml of buffer A. The mixture was centrifuged at 12,000 x g for 10 min at 4 °C to give cell lysates (input). Cell lysates (250 µl) were incubated with 5 µg of the GST fusion protein attached to glutathione-Sepharose beads for 45 min at 4 °C with constant rocking. The beads were washed four times with buffer A and once with buffer containing 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, and 5 mM MgCl2. The washed beads were then boiled in SDS sample buffer, and the extracts were analyzed by Western blotting using anti-FLAG and anti-GST (B-14, Santa Cruz Biotechnology) monoclonal antibodies.
Affinity Precipitation of Activated Rac1—NIH3T3 cells (1 x 107 cells/60-mm dish) were transiently cotransfected with pECFP-Rac1-WT and either p3xFLAG-CMV-DGK-G494D or p3xFLAG-CMV-DGK-(1–259). Twenty-four hours after transfection, cells were starved in Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum for 18–24 h and then harvested with 500 µl of buffer A containing 4 µg of GST-fused p21-binding domain (PBD) of p21-activated kinase (PAK), which binds GTP-bound Rac1 and Cdc42 specifically. The mixture was incubated on ice at 4 °C for 1 h and then centrifuged at 12,000 x g for 5 min at 4 °C to give cell lysates (input). Glutathione-Sepharose beads (10 µl) were added to the lysates and then further incubated at 4 °C for 1 h with constant rocking. The bead pellets were washed five times with 500 µl of buffer A and finally suspended in 40 µl of SDS sample buffer. The proteins extracted were analyzed by immunoblotting. The GTP-bound form of Rac1 thus recovered was detected by Western blotting using anti-GFP monoclonal antibody. In this case, GST-PAK-PBD present in the precipitates and DGK mutants in the lysates were detected with anti-GST and anti-FLAG monoclonal antibodies, respectively. When the effect of a DGK inhibitor (R59949
[GenBank]
) on the activation state of endogenous Rac1 was examined, non-transfected NIH3T3 cells were used. In this case, the GTP-bound form of Rac1 recovered was detected by Western blotting using anti-Rac1 monoclonal antibody (clone 23A8, Upstate Biotechnology, Inc., Lake Placid, NY).
DGK Activity Assay—The octyl glucoside mixed micellar assay of DGK activity was done as described previously (32). In brief, the assay mixture (50 µl) contained 50 mM MOPS (pH 7.4), 50 mM octyl glucoside, 1 mM dithiothreitol, 100 mM NaCl, 20 mM NaF, 10 mM MgCl2, 1 mM EGTA, 5 mM phosphatidylserine, 1.5 mM DAG, and 1 mM [-32P]ATP (10,000 cpm/nmol; ICN). The reaction was initiated by adding cell lysates (5 µg of protein) and continued for 5 min at 30 °C. Lipids were extracted from the mixture, and PA separated by thin-layer chromatography was scraped and counted using a liquid scintillation spectrophotometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Induces Lamellipodium/Membrane Ruffle Formation in NIH3T3 Fibroblasts—We first examined the intracellular localization of GFP-tagged DGK transiently expressed in NIH3T3 fibroblasts. We confirmed that DGK partly co-localized with F-actin at the cell periphery in quiescent NIH3T3 cells (Fig. 1, panels e and f). Interestingly, DGK was clearly accumulated at lamellipodia and membrane ruffles (Fig. 1, panels g–h') when the cells were stimulated with platelet-derived growth factor (PDGF), which is known to induce these F-actin-driven structures (33–35). On the other hand, DGK, which is known to be cytosolic (17, 22, 36), showed cytoplasmic localization and failed to show such marked accumulation at lamellipodia/ruffles (Fig. 1, panels i–l). The expression of GFP alone served as the control (Fig. 1, panels a–d). These results imply the potential involvement of DGK in the formation of lamellipodia/membrane ruffles.
To explore the functional relationship between DGK and the cytoskeleton reorganization, we attempted to examine the effects of expression of kinase-dead DGK-G494D or constitutively active DGK-(1–259) on the morphology of NIH3T3 fibroblasts. We confirmed that DGK-G494D possessed no detectable DGK catalytic activity in vitro (data not shown). The deletion of the N-terminal 259 residues, acting as an autoinhibitory site of type I DGK (22, 37), resulted in only a 2-fold increase in DGK activity when assayed in vitro (data not shown). However, this mutant showed constitutive localization at the cell periphery (Fig. 2A, panel j). Thus, we used this mutant as a constitutively active mutant. Indeed, our recent work showed that the effects of overexpression of this mutant in HL-60 cells are markedly stronger than those of wild-type DGK (29). We found that the expression of DGK-G494D markedly induced formation of lamellipodia/membrane ruffles in the absence of growth factor stimulation (Fig. 2A, panels g–i), indicating that the inactive mutant mimicked PDGF stimulation. In contrast, wild-type (WT) DGK and constitutively active DGK failed to enhance lamellipodium/ruffle formation (Fig. 2A, panels d–f and j–l). DGK-G494D co-localized with F-actin at the cell periphery, especially at lamellipodia/membrane ruffles. A quantitative analysis showed that lamellipodia/membrane ruffles were observed for 40% of the cells expressing kinase-dead DGK (Fig. 2B). On the other hand, kinase-dead DGK-G435D, in contrast to the corresponding DGK mutant, failed to induce the morphological changes (Fig. 2, A, panels m–o; and B). Moreover, although kinase-dead DGK-G495D constitutively localized at the cell periphery, this mutant did not affect the cell morphology (Fig. 2, A, panels p–r; and B). We also confirmed that the wild-type and constitutively active versions of DGK and DGK did not affect the morphology of the transfected cells (Fig. 2B). Throughout these experiments, the transfection efficiency of the various DGK mutants remained almost unchanged (30%) (data not shown), and Western blot analysis showed that the DGK mutants were expressed at comparable levels (Fig. 2C). Therefore, we consider that the differences in cell morphology are not caused by variable expression levels of the enzyme constructs.
Endogenous DGK Is Critically Involved in the Control of Lamellipodium Formation in NIH3T3 Cells—Since overexpressed kinase-dead DGK unexpectedly mimicked PDGF stimulation and altered the F-actin-driven cell morphology, we next attempted to see whether endogenous DGK indeed participates in the regulation of lamellipodium formation processes. For this purpose, we used R59949
[GenBank]
, which inhibits calcium-dependent DGK isoforms (type I: , , and ) (38). Indeed, 10 µM R59949
[GenBank]
significantly inhibited DGK activity (70% inhibition) in vitro (data not shown).
To examine the expression of endogenous type I DGK isoforms in NIH3T3 cells, we first performed RT-PCR analysis (30 cycles), revealing that NIH3T3 cells contain DGK mRNA (Fig. 3A). Western blot analysis of immunoprecipitates using anti-DGK antibody clearly showed that the cells indeed contain the isoform protein (Fig. 3B). Moreover, we confirmed that endogenous DGK was, at least in part, recovered in the cytoskeletal fraction (data not shown). DGK is known to exhibit brain-specific expression (19), and indeed, its mRNA was amplified using mouse brain cDNA as a template (Fig. 3A). However, the DGK mRNA was not detected in NIH3T3 cells by RT-PCR analysis (30 cycles). Moreover, no amplified products for the DGK mRNA were detected by even prolonged amplification (35 cycles) (data not shown). Although DGK mRNA was also detected by RT-PCR analysis (data not shown), the results given in Fig. 2 demonstrate that this isoform was not involved in the morphological changes. Therefore, if the morphological changes are induced by treating cells with R59949
[GenBank]
, the observed phenotype should be solely due to inhibition of the DGK activity.
As shown in Fig. 3 (C and D), lamellipodium/membrane ruffle formation was markedly enhanced by R59949
[GenBank]
(10 µM) treatment for 1 h in the absence of PDGF stimulation. Thus, the phenotype caused by the R59949
[GenBank]
treatment highly resembled that induced by the kinase-dead DGK expression (Fig. 2). These results strongly suggest that the inhibition of endogenous DGK activity by R59949
[GenBank]
mimics PDGF stimulation and that endogenous DGK is indeed involved in the negative control of lamellipodium/membrane ruffle formation.
To further substantiate the participation of endogenous DGK in the signaling pathway from physiological stimulations to cytoskeletal reorganization, we examined the effects of DGK expression on the lamellipodium/membrane ruffle formation induced by PDGF (33, 34, 39). If endogenous DGK needs to be in an inactive state to induce the formation of lamellipodia/ruffles, overexpression of constitutively active DGK followed by PDGF stimulation should inhibit the lamellipodium/ruffle formation. As shown in Fig. 4, the expression of DGK-(1–259) inhibited 70% of PDGF-induced lamellipodium/ruffle formation. On the other hand, the corresponding mutant of DGK, which is also expressed in NIH3T3 cells, did not give detectable effects. These results collectively suggest that DGK participates in the signaling pathway from PDGF receptor activation to cytoskeletal reorganization as represented by the formation of lamellipodia and membrane ruffles.
DGK Is Involved in the Control of Cell Spreading—In fibroblasts, Rac1 is known to regulate growth factor-stimulated lamellipodium/membrane ruffle formation, whereas two other closely related Rho family GTPases, Cdc42 and RhoA, regulate growth factor-induced filopodium and stress fiber formation, respectively (33, 34, 39, 40). Therefore, the effects of the DGK mutants on lamellipodium/ruffle formation imply that DGK participates in signal transduction events involving Rac1. To support this contention, we attempted to analyze another biological event, integrin-dependent cell spreading, in which the role of Rac1 is well defined (41, 42). NIH3T3 cells rapidly adhered to fibronectin-coated coverslips and spread considerably by 15 min after replating (Fig. 5, A, panels c and d; and B). This morphological change was markedly suppressed by the expression of DGK-(1–259) (Fig. 5, A, panels i and j; and B). However, DGK-WT and kinase-dead DGK failed to block cell spreading (Fig. 5, A, panels e–h; and B). In contrast to DGK-(1–259), the corresponding mutants of DGK and DGK failed to show detectable effects (Fig. 5, A, panels k–n; and B). We confirmed that adhesive properties in fibronectin-coated coverslips was not influenced by the expression of DGK-(1–259) (data not shown). These results further support that DGK, but not DGK and DGK, is involved in the cytoskeletal reorganization governed by Rac1.
DGK Is Co-localized and Associated with Rac1—We next tested whether DGK is indeed co-localized with Rac1 in NIH3T3 cells. In a subsequent experiment, CFP-tagged Rho family GTPases (Rac1, Cdc42, and RhoA) and YFP-tagged DGK were coexpressed in NIH3T3 fibroblasts, and their subcellular localization was investigated. CFP alone and CFP-RhoA, which were diffusely distributed in the cytoplasm, did not show significant co-localization with DGK (Fig. 6A, panels a–f). On the other hand, the fluorescence of YFP-DGK was virtually superimposed on that of CFP-Rac1 (Fig. 6A, panels g–i). Moreover, DGK co-localized especially at lamellipodia/ruffles induced by dominant-active Rac1 (Fig. 6B). Unfortunately, because immunofluorescence microscopy using anti-DGK antibody did not detect signals in NIH3T3 cells (data not shown), we could not confirm the co-localization of endogenous DGK and Rac1. CFP-Cdc42 also co-localized partly with YFP-DGK at the cell periphery (Fig. 6A, panels j–l). YFP-tagged DGK-(1–259) and DGK-G494D gave essentially the same intracellular localization pattern.
We next examined whether DGK physically interacts with Rac1 in vivo. To assess in vivo association between DGK and Rac1, we carried out co-immunoprecipitation analysis using the lysates of NIH3T3 cells coexpressing (FLAG)3-tagged DGK with CFP-tagged Cdc42, Rac1, or RhoA. When CFP-Rac1 was immunoprecipitated with anti-GFP antibody, DGK was clearly co-immunoprecipitated (Fig. 7A). Interactions of kinase-dead and constitutively active DGK mutants with Rac1 were also detected (data not shown). Although Cdc42 was, to a lesser extent, co-immunoprecipitated with the DGK isoform, RhoA failed to coprecipitate. We could not detect co-immunoprecipitation of DGK and Rac1 under the same experimental conditions (Fig. 7B), further demonstrating the specificity of the DGK-Rac1 interaction. To confirm that DGK is physiologically associated with Rac1, we investigated whether endogenous DGK and Rac1 could interact with each other in NIH3T3 cells. Consistent with the results obtained from the transfected cells, endogenous Rac1 was clearly co-immunoprecipitated with cellular DGK (Fig. 7C).
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. Similar to Rac1-WT, the dominant-active and dominant-negative mutants displayed comparable interaction with DGK (Fig. 7D), suggesting that the interaction is not significantly dependent on the activation state of the G-protein.
To substantiate the GTP-independent association between DGK and Rac1, we performed a pull-down assay. A bacterially expressed GST fusion protein of Rac1 was coupled to glutathione-agarose beads and loaded with either GTPS or GDPS. The beads were incubated with the lysates from COS-7 cells expressing DGK. In this experiment, we could not use highly purified DGK because most of the DGK expressed in Escherichia coli cells was recovered in inclusion bodies. (FLAG)3-tagged DGK recovered in the pull-down fractions was detected by Western blotting using anti-FLAG antibody. As shown in Fig. 7E, DGK bound to Rac1 in vitro, and this molecular interaction was found to be independent of GTP loading. Moreover, interactions of kinase-dead and constitutively active DGK mutants with Rac1 were also detected (data not shown). Taken together, the results reveal that DGK is a novel Rac1-associated enzyme and that the interaction does not depend on GTP.
DGK Operates as an Upstream Suppressor of Rac1—To analyze the hierarchical relationships between the actions of DGK and Rac1, we tested the effects of dominant-negative Rac1-T17N expression on kinase-dead DGK-G494D-induced lamellipodium formation. As shown in Fig. 2, the expression of kinase-dead DGK alone in NIH3T3 cells significantly induced lamellipodium formation. When inactive Rac1 was coexpressed with inactive DGK, they co-localized with each other, and lamellipodium formation was markedly suppressed (Fig. 8, A, panels a–c; and B). Reciprocally, we examined whether dominant-active Rac1-G12V expression overcame the effect of constitutively active DGK-(1–259), which, as shown in Fig. 4, was shown to suppress PDGF-induced lamellipodium formation. The expression of active Rac1 alone significantly induced lamellipodium formation in NIH3T3 cells (Fig. 8, A, panels d–f; and B). In contrast to the results of similar experiments using a combination of kinase-dead DGK and dominant-negative Rac1, the expression of constitutively active DGK failed to affect the lamellipodium formation induced by dominant-active Rac1, although the co-localization of DGK and Rac1 mutants was again clearly observed. These results thus suggest that DGK functions upstream of Rac1.
To confirm that DGK acts as an upstream regulator of Rac1, we examined whether DGK controls Rac1 activity. To assess this possibility, we performed affinity precipitation of GTP-bound (active) Rac1 using GST-PAK-PBD. When Rac1-WT and kinase-dead DGK were coexpressed, the level of GTP-bound Rac1 was markedly increased (Fig. 9, A and B). In contrast, coexpression of constitutively active DGK significantly reduced the active Rac1 level. On the other hand, the DGK mutants failed to affect Cdc42 activity. We next tested the effects of R59949
[GenBank]
on the activation state of cellular Rac1. Treatment with the DGK inhibitor clearly increased the level of GTP-bound Rac1 (2–3-fold increase in twice repeated experiments) (Fig. 9C), as observed for kinase-dead DGK expression, suggesting that endogenous DGK contributes, at least in part, to negative regulation of Rac1 activity.
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, as a reciprocal control, we next examined the effect of Rac1 expression on DGK activity. As shown in Fig. 9D, both dominant-negative and dominant-positive Rac1 mutants failed to affect DGK activity. Taken together, these results suggest that DGK functions upstream of Rac1 and further indicate that this isoform specifically and negatively regulates Rac1 activity. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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functions as a novel upstream suppressor of Rac1 and consequently of lamellipodium/membrane ruffle formation and cell spreading in NIH3T3 fibroblasts. This conclusion is based on the following observations. (a) DGK accumulated at lamellipodia/membrane ruffles induced by PDGF stimulation. (b) The expression of kinase-dead DGK induced lamellipodium/membrane ruffle formation. (c) The inhibition of endogenous DGK activity with R59949
[GenBank]
induced lamellipodium/membrane ruffle formation. (d) PDGF-induced lamellipodium/ruffle formation and integrin-mediated cell spreading were significantly inhibited by expression of constitutively active DGK. (e) DGK co-localized and interacted with Rac1. (f) Dominant-negative Rac1 expression blocked the lamellipodium/ruffle formation induced by kinase-dead DGK. (g) The expression of kinase-dead DGK and the inhibition of endogenous DGK activity with R59949
[GenBank]
activated Rac1, whereas constitutively active DGK inhibited Rac1 activity. In addition to cell spreading and lamellipodium/membrane ruffle formation, Rac1 plays crucial roles in diverse cellular events such as membrane trafficking, superoxide generation, transcriptional regulation, cell growth control, and development (33–35). Thus, DGK may potentially regulate a variety of cellular functions through controlling Rac1 activity.
Although the expression of constitutively active DGK significantly blocked PDGF-induced lamellipodium/ruffle formation, 30% of the PDGF-derived morphological changes still persisted (Fig. 4). This suggests that PDGF receptor-Rac1 signaling partly utilizes a distinct pathway(s) that is independent of DGK. In this respect, dominant-active Rac1 induced lamellipodium/ruffle formation in >80% of the G-protein-transfected cells, whereas only 30–40% of the kinase-dead DGK transfectants showed this phenotype (Fig. 2). This lesser potency of kinase-dead DGK compared with dominant-active Rac1 further suggests the critical but partial contribution of DGK to the signaling pathway leading to lamellipodium/membrane ruffle formation. The Rac1 activation mechanisms, which require participation of diverse factors and coordination with other signaling pathways, are undoubtedly complicated (33–35). The participation of DGK in the control of Rac1 as revealed in this work may represent one of the useful mechanisms for fine-tuning the spatial and temporal action of Rac1.
Constitutively active DGK, which reduced Rac1 activity (Fig. 9), inhibited integrin-mediated cell spreading (Fig. 5). However, kinase-dead DGK unexpectedly failed to accelerate spreading (Fig. 5), although this mutant activated Rac1 (Fig. 9). Moreover, the kinase-dead mutant did not enhance cell spreading even at earlier time points (data not shown). In this regard, we speculate that because both Rac1 and Cdc42 are necessary for integrin-mediated cell spreading (42), the activation of Rac1 alone is not sufficient to accelerate the morphological changes. In contrast, the inhibition of only one of these GTPases (Rac1 in this study) is considered to be sufficient to block cell spreading. Indeed, the expression of constitutively active DGK markedly inhibited integrin-dependent cell spreading (Fig. 5). On the other hand, kinase-dead DGK expression was sufficient to enhance lamellipodium/membrane ruffle formation (Fig. 2). This discrepancy can be explained by the fact that among the Rho family GTPases, only Rac1 contributes to lamellipodium/membrane ruffle formation (33, 34, 39, 40).
We demonstrated that DGK and Rac1 were co-immunoprecipitated. However, it is not clear at present whether DGK directly interacts with Rac1. It also remains unclear whether Rac1 activity is directly regulated by the catalytic action of DGK. Because no reports have been available to suggest a direct activation or inhibition of Rac1 by DAG or PA, it is likely that DGK indirectly regulates Rac1 activity via unknown mediators. In this regard, three possible effectors may be worthy of consideration. First, it has been reported that PKC is involved in lamellipodium/membrane ruffle formation (43, 44). Moreover, PKC plays a key role in the phosphorylation of the Rac1-specific guanine nucleotide exchange factor Tiam1 in Swiss 3T3 fibroblasts (45). An increased level of DAG, a PKC activator (2–6), caused by dominant-negative effects of kinase-dead DGK may enhance PKC activity and, subsequently, Rac1 activity. In this context, we found that DGK and several PKC isoforms transiently expressed in NIH3T3 cells were co-localized and associated with each other.2 Therefore, PKC may be one of the downstream effectors of DGK prior to the activation of Rac1. Interestingly, DGK was translocated from the cytoplasm to the cell periphery in the presence of phorbol ester via cysteine-rich regions homologous to the C1 domains of PKCs (46). This result further supports a functional correlation between DGK and PKCs.
Second, chimaerins, which are GTPase-activating proteins for Rac1, may serve as downstream effectors of DGK. Although chimaerins contain a C1 domain and bind to phorbol ester/DAG through this structure, this enzyme shows no significant changes in GTPase-activating activity even in the presence of phorbol ester/DAG (6, 47, 48). On the other hand, acidic phospholipids such as PA markedly increase chimaerin activity. It is thus possible that PA produced by DGK controls chimaerin activity and, subsequently, GTP-bound Rac1 levels. Again, phorbol ester/DAG-dependent translocation of chimaerins to the cell periphery (6, 47, 48) further supports a functional correlation between DGK and chimaerins.
Third, PA is known to stimulate phosphatidylinositol-4-phosphate 5-kinase activity (49, 50). In addition, it was reported that, in REF52 fibroblasts, phosphatidylinositol 4,5-bisphosphate functions as an attenuator of Vav, which is a guanine nucleotide exchange factor for Rac (35). Thus, DGK may negatively regulate Rac1 activity via phosphatidylinositol-4-phosphate 5-kinase. Tolias et al. (51) recently showed that unidentified DGK(s) interacts with phosphatidylinositol-4-phosphate 5-kinase and both GTP- and GDP-bound Rac1. In the present work, DGK also associated with both GTP- and GDP-bound Rac1 (Fig. 7). Therefore, although DGK
was recently reported to co-localize with Rac1 in myoblasts (52), the -isoform may also be one of the unidentified DGK(s) reported by Tolias et al. (51).
In addition to Rac1, Cdc42 partly co-localized and, to a lesser extent, interacted with DGK. It is thought that the functions of Cdc42 (filopodium formation) and Rac1 (lamellipodium/ruffle formation) are closely related and cross-talk with each other (33, 34, 39, 40). To date, many potential targets of Rac1 and Cdc42 have been identified, and several of these proteins have been shown to associate with both Rac1 and Cdc42 (33, 34, 39). Thus, it is not surprising that DGK associated with both Rac1 and Cdc42. Interestingly, overexpression of constitutively active DGK induced filopodium formation in NIH3T3 cells (Fig. 2).2 However, the induction mechanisms are still unclear. An analysis of Cdc42-related functions of DGK is an interesting target of future investigation.
We recently provided results suggesting that DGK negatively regulates macrophage differentiation of HL-60 and U937 cells through its catalytic action (29). Extensive reorganization of the cytoskeletal networks is closely linked to the acquisition of the macrophage-specific phenotypes (53). Thus, DGK may affect the cytoskeletal networks via controlling Rac1 activity during leukemia cell differentiation. Alternatively, it is possible that DGK indirectly regulates gene expression of important proteins for differentiation through affecting Rac1 activity.
Interestingly, DGK is abundantly expressed in neuronal cells such as retina (20) and Purkinje cells (21). During development of the nervous system, growth cones guide advancing neurites to their appropriate targets in a process called neuronal pathfinding. Lamellar membrane protrusions controlled by Rac1 are critical in regulating growth cone steering and neuronal pathfinding (39, 54, 55). The abundant expression of DGK in neuronal cells suggests that this isoform actively participates in synaptic plasticity through timely regulation of lamellipodium formation in growth cones. Further spatial and temporal analyses of the cytoskeletal reorganization regulated by DGK will provide new insight into growth cone-mediated neurite guidance.
In this work, we have provided results strongly suggesting that DGK is associated with Rac1, serving as a negative regulator of this Rho family GTPase. However, DGK and DGK failed to affect the Rac1-mediated morphological changes. Therefore, although -, -, and -isoforms of DGK share a highly similar structure with each other, these isozymes have their own unique functions. Interestingly, it was recently reported that DGK interacts with and negatively regulates Ras guanyl nucleotide-releasing protein (56). In addition to co-localization with Rac1 (52), DGK was also found to bind to and regulate the Ras-guanyl nucleotide exchange factor (57). Moreover, RhoA binds to and negatively regulates DGK
(58). Thus, DGKs, including the isoforms not yet analyzed, may, at least in part, act in concert with small GTPases. The present results also imply that phosphorylation of DAG by DGK occurring in a certain cellular microenvironment plays key roles in regulating basic cellular functions and that each of the DGK members possesses non-redundant functions despite structural similarities.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Dept. of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan. Tel.: 81-11-611-2111; Fax: 81-11-622-1918; E-mail: sakane{at}sapmed.ac.jp.
1 The abbreviations used are: DGK, diacylglycerol kinase; DAG, diacylglycerol; PA, phosphatidic acid; PKC, protein kinase C; GFP, green fluorescent protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; RT, reverse transcription; GST, glutathione S-transferase; GDPS, guanosine 5'-(-thio)diphosphate; GTPS, guanosine 5'-(-thio)triphosphate; PBD, p21-binding domain; PAK, p21-activated kinase; MOPS, 3-(N-morpholino)propanesulfonic acid; PDGF, platelet-derived growth factor; WT, wild-type.
2 M. Kai, S. Tsushima, F. Sakane, and H. Kanoh, unpublished data.
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