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Mol Cell Biol, February 1998, p. 762-770, Vol. 18, No. 2
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Characterization of a Rac1- and RhoGDI-Associated Lipid Kinase Signaling Complex

Kimberley F. Tolias,1,2,* Anthony D. Couvillon,1 Lewis C. Cantley,1,2 and Christopher L. Carpenter1,3

Division of Signal Transduction, Beth Israel Deaconess Medical Center,1 and Departments of Cell Biology2 and Medicine,3 Harvard Medical School, Boston, Massachusetts

Received 11 August 1997/Returned for modification 27 September 1997/Accepted 4 November 1997

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rac1, RhoA, and Cdc42 are members of the Rho subfamily of Ras-related small GTP-binding proteins. Rho family GTPases are best known for their ability to regulate actin cytoskeletal remodeling in response to extracellular signals, leading to changes in cell morphology, adhesion, and motility (21). In Swiss 3T3 cells, Rac1 induces the formation of lamellipodia and membrane ruffles by mediating actin polymerization and focal complex assembly at the plasma membrane (55). RhoA, in contrast, regulates the assembly of actin stress fibers and focal adhesions (54), whereas Cdc42 controls the formation of filopodia and associated focal complexes (37, 47). In addition to regulating the actin cytoskeleton, Rho family members activate gene transcription (14, 25, 44), are required for G1 phase progression (48), and are transforming (33, 51, 52). Rac and Rho also appear to be involved in exocytosis and endocytosis (40, 42, 49, 50). Rac has an additional role in superoxide production. In fibroblasts, the pathway is not well characterized, but in neutrophils, Rac is required for the activation of the NADPH oxidase enzyme complex (1, 2, 28, 61, 62).

Conventionally, G proteins are thought to signal when bound to GTP. Exchange of GDP for GTP causes the effector domain of the G protein to undergo a conformational change that allows effector molecules to bind (8). Interestingly, activation of the NADPH oxidase also requires the C terminus of Rac, but no effectors have yet been shown to bind to this region (15, 30, 31, 38). The nucleotide state of Rho GTPases is regulated in response to extracellular signals by three different classes of proteins. Guanine nucleotide exchange factors catalyze the exchange of GDP for GTP, GTPase-activating proteins (GAPs) accelerate the intrinsic GTPase activity, and guanine nucleotide dissociation inhibitors (GDIs) stabilize the bound nucleotide by inhibiting nucleotide dissociation and GAP activity. In addition, RhoGDI controls membrane localization of the GTPases (6, 11, 39).

Although RhoGDI is predominantly thought to be a negative regulator of Rho family G proteins, recent work suggests a potential positive role for RhoGDI in Rho family signaling. A Rac-RhoGDI complex was required for secretion in permeabilized mast cells (49). Wild-type Rac alone had no effect in the assay and RhoGDI inhibited exocytosis (49). RhoGDI was also found to associate with a protein complex containing ezrin, radixin, and moesin (ERM) proteins and their membrane binding partner, CD44 (26). The interaction between ERM proteins and CD44, which appears to be regulated by Rho, is important for cross-linking the plasma membrane with actin filaments (26, 36). The role of RhoGDI in these systems could be explained by a requirement for shuttling G proteins to the appropriate membrane locations for signaling.

Much recent effort has focused on identifying downstream targets of Rac, Rho, and Cdc42, and as result, many candidate molecules have been described. Targets of Rho family members include serine/threonine and tyrosine kinases, a protein phosphatase, and adapter molecules (reviewed in reference 64). The majority of these Rho family effector proteins bind preferentially to the GTP-bound form of the GTPase. We, and others, find that phosphoinositide kinases are targets of Rho GTPases. We demonstrated that a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase interacts with Rac1 in a GTP-independent manner (65). This connection between Rac and a PtdInsP 5-kinase was further supported by Hartwig et al. (23), who showed that a constitutively activated RacV12 mutant mimicked thrombin stimulation in permeabilized platelets by increasing PtdIns-4,5-P2 levels, which led to actin filament uncapping. Rho has also been linked to a PtdInsP 5-kinase. Posttranslationally modified Rho-GTP stimulated PtdInsP 5-kinase activity in fibroblast lysate (12), and Rho was shown to associate with a type I PtdInsP 5-kinase in a GTP-independent manner (53). In addition, phosphatidylinositol (PI) 3-kinase may be a target of Rho family members since it binds to Cdc42 and Rac1 in a GTP-dependent manner (7, 65, 67).

The finding that lipid kinases are targets of Rho family GTPases is intriguing given that phospholipids, and in particular PI-4,5-P2, have also been implicated in a number of Rho family functions. PI-4,5-P2 binds to, and regulates, several actin regulatory proteins, including gelsolin, profilin, alpha -actinin, and capZ (reviewed in reference 60). PI-4,5-P2 binding to gelsolin leads to actin uncapping and allows further polymerization (23). PI-4,5-P2 also enhances the interaction of ERM proteins with CD44, which anchors actin filaments to the plasma membrane (26). In addition, PI-4,5-P2, produced by a type I PtdInsP 5-kinase, is required for secretion in permeabilized PC12 cells (24).

The Rac-associated type I PtdInsP 5-kinase is stimulated by phosphatidic acid (PA), in contrast to the type II PtdInsP 5-kinases (29, 65). It is not clear, however, how PA might function to activate PtdInsP 5-kinases in vivo. Small GTP-binding proteins can act as foci for the formation of multienzyme complexes, such as Ras activation of Raf (46). We therefore investigated whether Rac, in addition to binding to a PtdInsP 5-kinase, might also associate with a diacylglycerol (G) kinase (DGK) and thus form a multienzyme complex that could synthesize PA and activate the PtdInsP 5-kinase and PI-4,5-P2 production. We now present evidence of such a multienzyme complex. Additionally, we have mapped the region of Rac sufficient for the interaction and found that phospholipids potentiate complex formation. Since the association of Rac with the PtdInsP 5-kinase and DGK is GTP independent, and the majority of GDP-bound Rac in cells is present in a complex with RhoGDI (13), we investigated whether the lipid kinases that associate with Rac also associate with RhoGDI. We found that RhoGDI bound to both the DGK and the PtdInsP 5-kinase in vitro and in vivo. This finding supports the idea that RhoGDI could have positive signaling functions.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. The peptides corresponding to amino acids 166 to 188 of Rac1 (KTVFDEAIRAVLCPPPVKKRKRK), Cdc42a (KNVFDEAILAALEPPETQPKRK), and Cdc42b (KNVFDEAILAALEPPEPKKSRR), residues 168 to 190 of RhoA (REVFEMATRAALQARRGKKSG), and residues 130 to 148 of Rac1 (KEKKLTPITYPQGLAMAKE) were synthesized by the protein facility of Tufts Medical School. Rac1 and RhoGDI antibodies were obtained from Santa Cruz Biotechnology Inc. Frozen rat brains were obtained from Pel Freez Biologicals. [gamma -32P]ATP was purchased from Dupont NEN. All other reagents were purchased from Sigma Chemical Co.

Plasmids. A Rac C-terminal construct (RacCT), containing amino acid residues 165 to 192, was generated by digesting human Rac1 cDNA in pGEX-2T with StuI and EcoRI and then subcloning the fragment back into the pGEX-2T vector. pGEX-2T plasmids encoding various alleles of Rac1, RhoA, and Cdc42, and the Rac-Rho chimeras were generously provided by Alan Hall, University College London, and Larry Feig, Tufts University. RhoGDI cDNA was a gift from Bing Lim, Harvard University. Rac, Rho, and Cdc42 alleles subcloned in the mammalian expression vector pEBG were kindly provided by Margaret Chou, University of Pennsylvania.

Preparation of recombinant proteins. Glutathione S-transferase (GST) and the GST fusion proteins were prepared as previously described (59), with minor modifications. Briefly, bacteria were sonicated on ice in 50 mM Tris (pH 7.5)-150 mM NaCl-5 mM MgCl2-5 mM dithiothreitol (DTT)-4 µg each of leupeptin and pepstatin per ml-200 µM 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). After the addition of Triton X-100 to a final concentration of 1%, the bacteria were centrifuged for 10 min at 15,000 rpm. Supernatants were incubated with glutathione (GSH)-agarose beads for 2 h at 4°C. Beads were washed twice with lysis buffer A (50 mM Tris [pH 7.5], 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40 [NP-40]), once with 1 M NaCl in 50 mM Tris (pH 7.5), and twice with TNM (50 mM Tris [pH 7.5], 50 mM NaCl, 5 mM MgCl2). Following the washes, the beads were incubated with 2 mg of bovine serum albumin per ml for 30 min at 4°C. The fusion proteins were stored at -80°C in storage buffer (50 mM HEPES [pH 7.0], 150 mM NaCl, 5 mM MgCl2, 5 mM DTT, 50% glycerol). The proteins were determined to be active based on their ability to bind [3H]GTP and were quantified both by nucleotide loading and by comparison to bovine serum albumin standards on sodium dodecyl sulfate-polyacrylamide gels that were stained with Coomassie blue. G proteins were loaded with nucleotide as described previously (65).

Lipid kinase in vitro binding assays. Rat brains were homogenized with a Dounce homogenizer in lysis buffer B (50 mM Tris [pH 7.5], 50 mM NaCl, 5 mM MgCl2, 1% NP-40, 10% glycerol, 1 mM DTT, 4 µg each of leupeptin and pepstatin per ml, 200 µM AEBSF) and then centrifuged for 15 min at 15,000 rpm. GST and GST fusion proteins (10 µg of each) were incubated with cleared homogenate (3 mg/ml) for 45 min at 4°C with constant rocking. The beads were washed twice with lysis buffer B and twice with TNM and then assayed for lipid kinase activities as described below.

For peptide competition experiments, rat brain homogenate diluted to 0.4 mg/ml in lysis buffer A was used to prevent protein precipitation at high peptide concentrations. Peptides were preincubated with homogenate for 30 min at 4°C. GST-Rac or GST-RhoGDI (10 µg of each) was added to the homogenate, and the in vitro binding experiment was conducted as described above. To determine the effect of peptides on the association between Rac and RhoGDI, GST-RhoGDI bound to GSH-agarose was incubated for 1 h with Spodoptera frugiperda SF9 cell lysate from cells expressing Rac1 (10). The beads were washed and incubated for 30 min with 1 mM Rac peptides. The beads were again washed and suspended in sodium dodecyl sulfate sample buffer, and the associated proteins were analyzed by Western blotting.

Kinase assays. Lipid kinase assays were performed in 50 µl, containing 50 mM Tris (pH 7.5), 30 mM NaCl, 12 mM MgCl2, 80 µM PtdInsP, 500 µM DG, 50 µM [gamma -32P]ATP (10 µCi/assay), and 1 mM deoxycholate (DOC), unless otherwise noted. Reactions were stopped after 10 min by adding 80 µl of 1 N HCl and then 160 µl of chloroform-methanol (1:1). Lipids were separated by thin-layer chromatography in chloroform-methanol-water-ammonium hydroxide (60:48:11:1.8). Phosphorylated lipids were visualized by autoradiography and quantified by using a Bio-Rad Molecular Analyst. The DGK inhibitors R59949 and R59002 (dissolved in dimethyl sulfoxide), or dimethyl sulfoxide only, were added to the proteins 15 min prior to the kinase assay, in experiments in which they were used.

Cell culture and transfections. Cos-7 cells and 293 cells were maintained in Dulbecco's modified Eagle medium containing 10% fetal calf serum and 10% heat-inactivated fetal calf serum, respectively. Cos-7 cells were transfected by the DEAE-dextran method, using 5 µg of RacV12-pEBG per 10-cm-diameter plate. Cells were harvested 48 h after transfection in buffer A plus 1 mM DTT, 4 µg each of leupeptin and pepstatin per ml, and 200 µM AEBSF. GSH-agarose beads were incubated with cell lysates for 2 h at 4°C and then washed as described for the bacterially expressed G proteins. 293 cells were transfected essentially as above, but Lipofectamine was used.

Effects of phospholipids on lipid kinase binding to Rac. In vitro binding experiments were conducted with GST-RacV12 produced in Cos-7 cells as described above. GST-RacV12 was preincubated with 100 µM lipid for 30 min at room temperature in buffer C (50 mM Tris [pH 7.5], 150 mM NaCl, 0.5 mM MgCl2, 0.02% NP-40, 1 mM DTT, 4 µg each of leupeptin and pepstatin per ml, 200 µM AEBSF); 300 µl of rat brain homogenate in buffer C was then added to the Rac-lipid mixture and incubated for an additional 30 min. Beads were washed twice with lysis buffer B and twice with TNM and then assayed for lipid kinase activities. The effects of phosphatidylserine (PS), PA, PtdInsP, and PI-4,5-P2 were determined on partially purified lipid kinases, and no effects on activity were found at concentrations up to 25 µM.

Immunoprecipitations. Rat brain homogenate prepared in lysis buffer B was incubated at 4°C for 90 min with 2 µg of polyclonal Rac1 antiserum, 1 µg of RhoGDI antiserum, or nonimmune serum followed by a 90-min incubation with protein A-Sepharose beads. The beads were washed twice with lysis buffer B and twice with TNM and then assayed for lipid kinase activities as described above.

Column chromatography. Two to ten rat brains were homogenized in 20 volumes of homogenization buffer (25 mM HEPES [pH 7.5], 25 mM NaCl, 5 mM MgCl2, 4 µg each of leupeptin and pepstatin per ml, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride). Triton X-100 was added to a final concentration of 1%, and the homogenate was stirred at 4°C for 15 min. After centrifugation at 100,000 × g for 45 min, the supernatant was incubated with 20 to 250 µg of GST-Rac1 beads for 45 min at 4°C. The beads were washed twice with homogenization buffer plus 1% Triton X-100 and twice in homogenization buffer plus 0.1% Triton X-100. The Rac1 beads were poured into a column, and the DGK-PtdInsP 5-kinase complex was eluted with a linear gradient of NaCl (0 to 2 M). Enzyme activity was assayed as described, and the active fractions were pooled, diluted to 100 mM NaCl with homogenization buffer plus 0.1% Triton X-100, and loaded onto a Fast Flow heparin-agarose column (Pharmacia). Proteins were eluted with a linear NaCl gradient (0.25 to 0.8 M), and fractions were assayed for DGK and PtdInsP 5-kinase activities. The active fractions from the heparin column were desalted on a Sephadex G-25 column, applied to a Mono-Q column, and eluted with a linear NaCl gradient (0 to 0.5 M).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

A DGK associates with Rac1. We have previously described the interaction of Rac1 with two phosphoinositide kinases. PI 3-kinase associates with Rac in a GTP-dependent manner, whereas a type I PtdInsP 5-kinase binds to Rac in a GTP-independent manner (65). The Rac-associated PtdInsP 5-kinase is activated by PA. Since other small G proteins are known to form signaling complexes, we investigated whether Rac might also bind to a DGK, which could synthesize PA and activate the PtdInsP 5-kinase. Bacterially expressed GST fusion proteins of Rac1, RhoA, and Cdc42 bound to GSH-agarose beads were loaded with either GTPgamma S or GDPbeta S and then incubated with rat brain homogenate. After washing, the beads were assayed simultaneously for DGK and PtdInsP 5-kinase activities. We found that Rac specifically associated with a DGK (Fig. 1A). As with the PtdInsP 5-kinase, this association did not depend on GTP. GST-Rac associated with 32-fold more DGK activity than GST alone (Fig. 1A, lanes 2 and 3). A smaller amount of DGK activity (fivefold over GST) also associated with the GST fusion proteins of RhoA and Cdc42 (Fig. 1A, lanes 4 to 7). No lipid kinase activities were associated with GST-Rac that had not been exposed to homogenate (Fig. 1A, lanes 8 and 9).


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  		FIG. 1.   Association of Rac1 with a DGK. (A) GST and GST fusion proteins of Rac1, RhoA, and Cdc42 bound to GSH beads were loaded with GTPgamma S or GDPbeta S and then incubated with rat brain homogenate. The beads were washed and assayed simultaneously for DGK and PtdInsP 5-kinase activity. GST-Rac which had not been exposed to lysate was also assayed as a negative control (lanes 8 and 9). (B) Rac1 was immunoprecipitated from rat brain homogenate, washed, and assayed for DGK and PtdInsP 5-kinase activities. An immunoprecipitation using nonimmune (NI) serum was done as a negative control. The migration positions of the lipid standards in both experiments are indicated. The data are representative of five experiments.

We previously determined that the association between Rac and the type I PtdInsP 5-kinase occurred in vivo, based on the presence of the activity in Rac immunoprecipitates. To determine whether Rac also associates with the DGK in vivo, we immunoprecipitated Rac from rat brain homogenate and assayed it for lipid kinase activities. As shown in Fig. 1B, we detected both DGK and PtdInsP 5-kinase activity in Rac1 immunoprecipitates. In contrast, no lipid kinase activity was found in the immunoprecipitate obtained by using nonimmune serum.

Characteristics of the Rac-associated DGK. To further characterize and identify the DGK associated with Rac, we determined its apparent Km (Kmapp) for ATP and DG, its substrate specificity, and the effect of detergents, inhibitors, and calcium on its activity (Table 1). In the presence of 75 mM octylglucoside, the Rac-associated DGK had a Kmapp for ATP of 120 µM and a Kmapp for DG of 240 µM (0.32 mol%). At a DG concentration of 500 µM, the DGK was equally active in 1 mM DOC or 75 mM octylglucoside. While the Rac-associated DGK did not display selectivity for long-chain DGs (1,2-dioleoyl-sn-glycerol and 1-stearoyl-2-arachidonyl-sn-glycerol were equally good substrates), 1,2-diacylglycerol was strongly preferred over 1,3-diacylglycerol. The Rac-associated DGK also phosphorylated 2-monoacylglycerol. The DGK inhibitors R59002 and R59949 had a partial effect on the Rac-associated DGK activity (10% ± 7% and 30% ± 4% inhibition for 59002 and 59949, respectively). Some DGKs contain EF hand motifs and are stimulated by calcium (18). However, the activity of the Rac-associated DGK was unaffected by CaCl2. These experiments did not allow us to conclusively identify the DGK isoform that associates with Rac.

                              
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  		TABLE 1.   Characteristics of Rac-associated DGK

The PtdInsP 5-kinase and the DGK bind to the C terminus of Rac. Most Rho family GTPase target proteins require the effector domain for binding (15). Since the association of both the PtdInsP 5-kinase and the DGK is independent of GTP binding, we were interested in determining the region of Rac that binds to these lipid kinases. We used chimeric proteins, peptides, and a C-terminal Rac construct to identify this region.

Rac-Rho chimeras and Rac point mutants were expressed in bacteria as GST fusion proteins and purified with GSH beads. The proteins were incubated with rat brain homogenate, washed, and assayed for associated lipid kinase activities. We found that the two chimeras containing the C-terminal region of Rac, Rho73Rac and Rac73Rho143Rac, associated with PtdInsP 5-kinase and DGK activity at levels comparable to those of wild-type Rac and constitutively activated Rac (RacV12) (Fig. 2). In contrast, chimeras containing the C terminus of RhoA, Rac73Rho, Rac143Rho, and Rac178Rho, associated with significantly less PtdInsP 5-kinase and DGK activity (Fig. 2). These results indicate that the C terminus of Rac is necessary for binding to both the PtdInsP 5-kinase and DGK. Interestingly, we also detected reduced lipid kinase activity associated with the effector mutant RacA38, which suggests that other regions of Rac also participate in binding to the lipid kinases.


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  		FIG. 2.   The C terminus of Rac is necessary for binding to the DGK and the PtdInsP 5-kinase. GST and GST fusion proteins of Rho family members, Rac point mutants, and Rac-Rho chimeras bound to GSH beads were incubated with rat brain homogenate, washed, and assayed for lipid kinase activities. The data are representative of 12 experiments.

To confirm the finding that the C-terminal region of Rac binds to the PtdInsP 5-kinase and DGK, we synthesized peptides corresponding to the C termini of Rac1, RhoA, and Cdc42 (both splice variants) and used these peptides in competition experiments. Peptides were preincubated with rat brain homogenate for 30 min. GST-Rac bound to GSH beads was then added to the homogenate and incubated for an hour. After the incubation, the beads were washed and assayed for lipid kinase activities. We found that the Rac C-terminal peptide effectively competed with GST-Rac for binding to both the DGK (Fig. 3A) and the PtdInsP 5-kinase (Fig. 3B), with an apparent Ki of approximately 30 µM for both. In contrast, the control peptides corresponding to the C termini of Rho and Cdc42a did not compete (Fig. 3). At high concentrations, a peptide based on the C terminus of an alternatively spliced form of Cdc42, Cdc42b, partially competed for binding to both the PtdInsP 5-kinase and the DGK (60% of the kinase activities remained, compared to 5% with the Rac C-terminal peptide). The Cdc42b C-terminal peptide may have competed more efficiently than the other two control peptides because its sequence more closely resembles that of the Rac C-terminal peptide. The results from the peptide competition experiments confirm that the C terminus of Rac is necessary for binding to the PtdInsP 5-kinase and the DGK.


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  		FIG. 3.   A peptide corresponding to the C terminus of Rac competes with GST-Rac for binding to the lipid kinases. Increasing concentrations of a peptide corresponding to the C terminus of Rac (residues 166 to 188) were preincubated with rat brain homogenate for 30 min. GST-Rac beads were added to the homogenate and incubated for an hour. The beads were then washed and assayed for DGK activity (A) and PtdInsP 5-kinase activity (B). Control peptides corresponding to the C termini of RhoA and Cdc42 (splice variants a and b) were also used in the experiment at 1 mM. The activity of the lipid kinases associated with GST-Rac in the absence of peptide was taken as 100%. Data shown are the mean ± standard error of the mean of 11 experiments.

To determine whether the C terminus of Rac is sufficient for lipid kinase binding, we made a C-terminal GST-Rac construct containing only the last 27 amino acids (RacCT). This construct was expressed in bacteria, purified with GSH-agarose beads, and tested for its ability to associate with the lipid kinases in rat brain homogenate. We found that RacCT bound to both the PtdInsP 5-kinase and the DGK, indicating that the C terminus of Rac is sufficient for lipid kinase binding (Fig. 4). The affinity, however, appeared to be lower, particularly for the PtdInsP 5-kinase. The amount of PtdInsP 5-kinase activity associated with RacCT was diminished threefold compared to full-length Rac (Fig. 4). Since we cannot distinguish activity from protein binding, we are not certain whether this reduction of associated activity indicates that other regions of Rac are necessary for high-affinity PtdInsP 5-kinase binding or for stimulation of bound enzyme. Residues 1 to 73 of Rac may be involved, given that the Rho73Rac chimera and RacA38 mutant (Fig. 2) associate with less PtdInsP 5-kinase activity than wild-type Rac.


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  		FIG. 4.   The C terminus of Rac is sufficient for binding to PtdInsP 5-kinase and DGK. GST, GST-Rac, and a deletion mutant (RacCT) which contained only the C-terminal 27 amino acid residues of Rac fused to GST (residues 165 to 192) were incubated with rat brain homogenate, washed, and assayed for lipid kinase activities. The migration positions of the lipid standards are indicated. The results shown are representative of three experiments.

Evidence for a complex between the PtdInsP 5-kinase and the DGK. Since DGK and PtdInsP 5-kinase bind to the same region of Rac, we investigated whether the lipid kinases interact with Rac individually or as a complex. We incubated GST-Rac bound to GSH beads with brain homogenate, washed the beads, and packed them in a column. The column was then eluted with a NaCl gradient. We found that the DGK and the PtdInsP 5-kinase activities eluted in the same fractions, at a NaCl concentration of about 250 mM. Separation of the lipid kinase activities eluted from Rac on a heparin column also showed that the kinases eluted in identical fractions (Fig. 5A). We consistently recovered 200 to 300% of the PtdInsP 5-kinase activity and 50% of the DGK from the heparin column, suggesting that the majority of the PtdInsP 5-kinase and the DGK are present as a complex. The activities also eluted together on a Mono-Q column, following the heparin column (Fig. 5B). The activities eluted at identical positions by gel filtration, at an apparent mass of 500 kDa, following a heparin column (data not shown). These data suggest that the kinases are present as a complex.


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  		FIG. 5.   The PtdInsP 5-kinase and DGK interact with Rac as a complex. GST-Rac bound to GSH-agarose beads was incubated with rat brain homogenate. The beads were washed and eluted with a NaCl gradient. (A) The fractions containing PtdInsP 5-kinase and DGK activities were pooled, diluted, and loaded onto a heparin-Sepharose column. The column was eluted with a 0.25 to 0.8 M NaCl gradient, and the fractions were assayed for PtdInsP 5-kinase (circles) and DGK (squares) activities. (B) The active fractions were pooled, desalted on a Sephadex G-25 column, and applied to a Mono-Q column. The Mono-Q column was eluted with a 0 to 1 M linear NaCl gradient, and the fractions were assayed for PtdInsP 5-kinase (squares) and DGK (circles) activities. The results are representative of four experiments. The salt concentrations were monitored with a conductivity meter, and the protein concentration was determined by monitoring absorbance at 280 nm.

PtdInsP 5-kinase and DGK associate with RhoGDI. RhoGDI binds Rho family G proteins and is thought to function by inhibiting nucleotide dissociation and hydrolysis and by sequestering Rho family proteins in the cytosol (6). Since DGK and PtdInsP 5-kinase interact with Rac bound to both GTP and GDP, we investigated whether these kinases are found in a complex with RhoGDI. GST-RhoGDI bound to GSH-agarose beads was incubated with rat brain homogenate, washed, and assayed for lipid kinase activities. We found both PtdInsP 5-kinase and DGK activities associated with GST-RhoGDI (Fig. 6A). In contrast, no lipid kinase activity bound to GST alone. This result indicates that RhoGDI can form a complex with PtdInsP 5-kinase and DGK. To determine if these interactions also occur in vivo, we immunoprecipitated RhoGDI from rat brain homogenate and assayed it for associated lipid kinase activities. We detected both PtdInsP 5-kinase and DGK activities in the RhoGDI immunoprecipitate, whereas no lipid kinase activity was found in the control immunoprecipitate (Fig. 6B). This result confirms that RhoGDI associates with PtdInsP 5-kinase and DGK in vivo.


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  		FIG. 6.   RhoGDI associates with both PtdInsP 5-kinase and DGK. (A) GST and GST-RhoGDI bound to GSH-agarose beads were incubated with rat brain homogenate. The beads were washed and assayed simultaneously for DGK and PtdInsP 5-kinase activities. (B) RhoGDI was immunoprecipitated from rat brain homogenate, washed, and assayed for DGK and PtdInsP 5-kinase activities. An immunoprecipitation using nonimmune (NI) serum was done as a negative control. The migration positions of the lipid standards in both experiments are indicated. The data are representative of five experiments.

To further investigate the role of RhoGDI in the formation of the complex, we determined whether RhoGDI associated with bacterially expressed Rac and if RhoGDI was present in fractions containing the lipid kinase activities separated on a heparin-agarose column. We found by Western blotting that a small amount of RhoGDI associated with bacterially expressed GST-Rac. A fraction of the Rac-associated RhoGDI was detected in the flowthrough of the heparin column, but no RhoGDI was found in the fractions containing lipid kinase activities (data not shown). This result indicates that RhoGDI is not necessary for formation of a complex between PtdInsP 5-kinase and DGK. The possibilities remain that RhoGDI can either bind separately or mediate Rac binding to the lipid kinases or that Rac and RhoGDI are both necessary to form a complex with the lipid kinases.

To determine if RhoGDI is necessary for Rac binding to the lipid kinases, we compared lipid kinase binding of bacterially expressed GST-Rac to that of GST-Rac produced in 293 cells. Rac produced in mammalian cells is geranylgeranylated and therefore binds RhoGDI with high affinity. The G proteins were matched for GTP binding and incubated with rat brain homogenate. After washing, the samples were assayed and blotted for RhoGDI. We found equal amounts of the kinase activities in both conditions. However, Rac produced in 293 cells was associated with a vast excess of RhoGDI compared to the bacterial Rac (data not shown). Since the presence of RhoGDI does not correlate with the ability of Rac to bind to the lipid kinases, we conclude that RhoGDI is not necessary for Rac to bind to the lipid kinases.

To confirm that the interaction between RhoGDI and the lipid kinases is dependent on Rac, we preincubated the Rac C-terminal peptide (Rac 166-188) or a control peptide (Rac 130-148) at various concentrations with rat brain homogenate. GST-RhoGDI, bound to GSH-agarose, was added to the homogenate and incubated for an additional hour. The beads were then washed and assayed for lipid kinase activities. We found that the Rac C-terminal peptide (Rac 166-188) competed with GST-RhoGDI for DGK (Fig. 7A) and PtdInsP 5-kinase (Fig. 7B) activity with apparent Kis of 30 and 80 µM, respectively, whereas the control Rac peptide did not compete. In contrast to the GST-Rac peptide competition experiment, we found that 30% of the lipid kinase activities remained bound to GST-RhoGDI, even in the presence of 1 mM Rac C-terminal peptide (Fig. 7A and B). The remaining lipid kinase activities associated with RhoGDI may be due to RhoA and Cdc42, which also bind both lipid kinases to a lesser extent. To determine whether the loss of lipid kinase activities that we detected in the peptide competition experiment with RhoGDI was due to peptide-mediated release of Rac from RhoGDI, we determined the effect of the Rac peptides on the association of Rac with RhoGDI. We found that the association between Rac and RhoGDI was not disrupted by either peptide (Fig. 7C). These results argue that Rac is necessary for the complex formation and that RhoGDI alone is not likely to bind the complex.


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  		FIG. 7.   Rac mediates the association between RhoGDI and the lipid kinases. Increasing concentrations of the Rac C-terminal peptide (Rac 166-188) were preincubated with rat brain homogenate for 30 min. GST-RhoGDI bound to GSH-agarose beads was added to the homogenate and incubated for an additional hour. The beads were then washed and assayed for DGK (A) and PtdInsP 5-kinase (B) activities. A control peptide corresponding to residues 130 to 148 of Rac (Rac 130-148) was also used in the experiment at 1 mM. The activity of the lipid kinases associated with GST-RhoGDI in the absence of peptide was taken as 100%. Data shown are the mean ± standard error of the mean of three experiments. (C) GST-RhoGDI bound to GSH-agarose was incubated with SF9 cell lysate expressing Rac1, washed, and then treated with or without 1 mM Rac peptides. After incubation with peptides, the supernatant was collected from each sample (S). The RhoGDI beads were washed, and then the beads (B) and supernatant from each sample were analyzed by Western blotting using Rac1 antiserum. The results shown are representative of three experiments.

Phospholipids promote the association of the lipid kinases with Rac. The C termini of Rho family GTPases are positively charged and resemble phosphoinositide binding sequences (22, 34). Zheng et al. have recently shown that phospholipids bind to the C terminus of Rho GTPases and stimulate nucleotide release (68). Since the binding of lipid kinases to Rac is also mediated by the C terminus, we investigated whether phospholipids could influence the ability of Rac to associate with the lipid kinases. We used Rac expressed in Cos cells for this set of experiments, since geranylgeranylation could affect phospholipid binding. GST-RacV12 expressed in Cos cells was purified with GSH-agarose beads, washed, treated with phospholipids, and then incubated with rat brain homogenate. Following the incubation, the beads were washed and assayed for DGK and PtdInsP 5-kinase activities. Preincubation of Rac with PS, PA, PtdIns4P, and PtdIns-3,4-P2 significantly enhanced the association of both the PtdInsP 5-kinase and the DGK (Fig. 8). Phosphatidylcholine, PI, and PI-3,4,5-P3, in contrast, had no effect. The lipids increased the PtdInsP 5-kinase activity associated with Rac to a greater extent than the DGK activity. The same phospholipids promoted the association of the lipid kinases with Cos cell-expressed RhoA and Cdc42, but about half as well as for Rac (data not shown). Since addition of phospholipid directly to the kinase assay had no effect on the activity of either PtdInsP 5-kinase or DGK and the lipid added during the incubation should be washed away, the increased activity was due to enhanced binding of the lipid kinases (not shown). The interactions between bacterially expressed Rac and the PtdInsP 5-kinase and DGK were also enhanced by lipids, but the effect was less marked (data not shown). This difference suggests that geranylgeranylation of Rac stabilizes the G protein-phospholipid interaction and/or the lipid kinase-G protein interaction in the presence of lipids. The finding that phospholipids promote the association of PtdInsP 5-kinase and DGK with Rac suggests that phospholipids might regulate these associations in vivo.


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  		FIG. 8.   Effects of lipids on the interaction between Rac and the PtdInsP 5-kinase and DGK. GST-RacV12 expressed in COS 7 cells was purified with GSH-agarose beads, washed, preincubated with 25 µM lipid, and then incubated with rat brain homogenate. Following the incubation, the beads were washed and assayed for DGK and PtdInsP 5-kinase (PIPK) activities. The data shown are the mean ± standard error of the mean of three experiments. PC, phosphatidylcholine.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have shown that Rac binds to both a PtdInsP 5-kinase and a DGK. The association is independent of GTP and requires the C terminus of Rac. While some lipid kinase binding was also seen with RhoA and Cdc42, sixfold more activity associated with Rac. The PtdInsP 5-kinase and DGK appear to bind as a complex to Rac, since they copurify once eluted from Rac. The association of these lipid kinases with Rac is enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. RhoGDI also interacts with the PtdInsP 5-kinase and DGK, primarily as a result of their association with Rac.

DGKs appear to play a number of roles in signal transduction. Since they phosphorylate the second messenger DG, DGKs are thought to attenuate the activation of DG-dependent protein kinases C (4). In addition, the product of DGKs, PA, may function as a second messenger itself. PA is mitogenic (45) and has been shown to activate a number of enzymes, including type I PtdInsP 5-kinases, n-chimaerin, and an unidentified protein kinase which phosphorylates the NADPH oxidase protein p47phox (3, 29, 66).

To date, eight DGK genes have been cloned. The protein products of these genes all contain a C-terminal catalytic domain and two cysteine-rich zinc finger domains. Three highly homologous DGK isozymes, DGKalpha , DGKbeta , and DGKgamma , are further characterized by a conserved N-terminal domain of unknown function and EF hands that bind Ca2+ (16, 19, 20, 32, 57, 58). DGKvarepsilon resembles this group of DGKs but lacks an EF hand motif and is highly selective for arachidonate-containing substrates (63). Three other DGKs isoenzymes, DGKdelta , DGKeta , and DGKtheta , contain a pleckstrin homology domain, implying that they bind to phospholipids (27, 35, 56). In addition, DGKtheta possesses a third cysteine-rich domain, a proline- and glycine-rich domain, and a putative Ras-binding domain (27). Finally, DGKzeta is characterized by four tandem ankyrin repeats, a nuclear targeting motif, and a sequence homologous to the phosphorylation site domain of the myristoylated alanine-rich C kinase substrate protein (9, 17).

So far we have not been able to identify the DGK that associates with Rac. Its biochemical characteristics indicate that it is not activated by calcium like the DGKalpha , -beta , and -gamma (18), nor is it specific for 1-stearoyl-2-arachidonyl-sn-diacylglycerol, like DGKvarepsilon (63). DGKdelta is not present in the brain, ruling it out as the Rac-associated DGK (56). While DGKzeta is not inhibited by R59949, its other characteristics are consistent with the Rac-associated DGK, suggesting that it is a potential candidate (9). The Rac-associated DGK could also be DGKtheta or a new isoform (27).

The role of Rac in the regulation of the actin cytoskeleton is well established, and recent evidence suggests that this effect is mediated, at least in part, by an increase in the synthesis of PI-4,5-P2 (23). The simplest model to explain our data is that PtdInsP 5-kinase, DGK, and Rac exist as a preformed complex bound to RhoGDI (Fig. 9). Upon stimulation, the complex is shuttled to the membrane and released from RhoGDI as has been previously proposed (5). Once in proximity to its substrate, the DGK synthesizes PA, which stimulates PtdInsP 5-kinase to produce PI-4,5-P2. These phospholipids may mediate dissociation of the Rac-RhoGDI complex (13) and/or stimulate nucleotide exchange either directly (68) or through activation of a guanine nucleotide exchange factor (43). Newly synthesized PI-4,5-P2 could also bind to actin regulatory proteins and induce actin uncapping and new actin polymerization.


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  		FIG. 9.   Model for the function of the Rac-associated lipid kinase complex. PtdInsP (PIP) 5-kinase, DGK, and Rac may exist as a preformed complex bound to RhoGDI. Upon stimulation, the complex may be shuttled to the membrane, where the DGK could synthesize PA, which would stimulate PI-4,5-P2 production. These phospholipid products may mediate dissociation of the Rac-RhoGDI complex and/or stimulate nucleotide exchange. Newly synthesized lipids could also bind to actin regulatory proteins and induce actin uncapping and new actin polymerization as well as target other Rac signaling pathways such as the NADPH oxidase. GEFs, guanine nucleotide exchange factors.

The PtdInsP 5-kinase and DGK may also have a function distinct from, or in addition to, actin regulation. The C terminus of Rac is required for activation of the NADPH oxidase in neutrophils (15, 30, 31, 38). It has been proposed that this region of Rac may be necessary to bind acidic phospholipids and stabilize its membrane association (22). Given our finding that the Rac C terminus mediates binding to PtdInsP 5-kinase and DGK, it is possible that these lipid kinases are effectors of Rac, necessary for the activation of the NADPH oxidase. PI-4,5-P2 could play a role in the activation of the oxidase, and a PA-stimulated protein kinase involved in NADPH oxidase activation has recently been described (66).

Rac, RhoGDI, and a type I PtdInsP 5-kinase have also been implicated in secretion (24, 49, 50). The complex that we described may regulate secretion by increasing the levels of PI-4,5-P2 which can stimulate the activities of both ADP-ribosylation factor and phospholipase D (reviewed in reference 41). Alternatively, the negatively charged phospholipid products of the complex may drive vesicle fusion by altering the properties of the membrane itself.

Diekmann et al. found that the Rac178Rho chimera caused membrane ruffling when injected into cells (15). We would predict that if the PtdInsP 5-kinase and DGK are important for actin polymerization, this chimera would not cause ruffling. It is possible that when present at a high concentration, as is the case in an injection experiment, sufficient amounts of the PtdInsP 5-kinase and DGK are bound to the chimera to mediate ruffling (we find that this chimera binds lipid kinases fivefold more than GST but sixfold less than full-length Rac). Other signals that substitute for the PtdInsP 5-kinase and DGK, such as PI 3-kinase, could also be activated. The finding that Rac stimulates PI-4,5-P2 synthesis and uncapping of the barbed ends of actin filaments in permeabilized platelets supports a role for this complex in actin regulation (23).

RhoGDI is thought to form a complex with Rho family G proteins and keep them sequestered in the cytosol. Our data suggest a more complicated role for RhoGDI. When the RhoGDI-Rac-lipid kinase complex contacts the membrane, synthesis of PA and/or PI-4,5-P2 could stimulate release of Rac from RhoGDI. These phospholipids have been shown to specifically enhance release of G proteins from RhoGDI (5, 13). It is possible that once released from RhoGDI, the Rac-lipid kinase complex is stabilized by phospholipids, such as PS, at the membrane. This would explain the increase in activities associated with Rac in the presence of particular phospholipids. The ability of specific phospholipids to enhance lipid kinase binding to Rac could reflect an ability of these phospholipids to localize the complex at different sites in the cell. PS might bring the complex to the plasma membrane, and PI-3,4-P2 might localize the complex to areas of PI 3-kinase activity. Since the interaction of the lipid kinases with RhoGDI is dependent primarily on Rac, we have been unable to determine if there is a distinct effect of the phospholipids on the lipid kinase activities associated with RhoGDI. RhoGDI has recently been found in another protein complex. Hirao et al. detected RhoGDI in the immunoprecipitated CD44-ERM protein complex from BHK cells (26). This complex was found to be regulated by the product of the PtdInsP 5-kinase, PI-4,5-P2. It is possible that the formation of this complex is dependent on the presence of PtdInsP 5-kinase associated with RhoGDI.

The identification of a PtdInsP 5-kinase-DGK complex bound to Rac and RhoGDI and the mapping of its interaction to the C terminus of Rac should allow for further elucidation of the role of lipid kinases in Rho family signaling.

    ACKNOWLEDGMENTS

We are grateful to Alan Hall and Dagmar Diekmann for providing the cDNAs of Rho, RacV12, RacA38, and the Rac-Rho chimeras. We also thank Larry Feig for the Rac1 and Cdc42 cDNAs, Margaret Chou for the Rac, Rho, and Cdc42 mammalian expression constructs, and Bing Lim for RhoGDI. We express our appreciation to James A. Fuchs and members of the Carpenter and Cantley laboratories for helpful discussions.

This work was supported by NIH grant GM54389 to C.L.C. and GM36624 to L.C.C.

    FOOTNOTES

* Corresponding author. Mailing address: Division of Signal Transduction, Harvard Institute of Medicine, 1007, 330 Brookline Ave., Boston, MA 02215. Phone: (617) 667-0941. Fax: (617) 667-0957. E-mail: ktolias{at}bidmc.harvard.edu.

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Top
Abstract
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Materials & Methods
Results
Discussion
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Mol Cell Biol, February 1998, p. 762-770, Vol. 18, No. 2
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