COMMUNICATION
A Built-in Arginine Finger Triggers the Self-stimulatory GTPase-activating Activity of Rho Family GTPases*

Baolin Zhang, Yaqin Zhang, Cheryl C. CollinsDagger , Douglas I. JohnsonDagger , and Yi Zheng§

From the Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163 and the Dagger  Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405

    ABSTRACT
Top
Abstract
Introduction
References

Signal transduction through the Rho family GTPases requires regulated cycling of the GTPases between the active GTP-bound state and the inactive GDP-bound state. Rho family members containing an arginine residue at position 186 in the C-terminal polybasic region were found to possess a self-stimulatory GTPase-activating protein (GAP) activity through homophilic interaction, resulting in significantly enhanced intrinsic GTPase activities. This arginine residue functions effectively as an "arginine finger" in the GTPase activating reaction to confer the catalytic GAP activity but is not essential for the homophilic binding interactions of Rho family proteins. The arginine 186-mediated negative regulation seems to be absent from Cdc42, a Rho family member important for cell-division cycle regulation, of lower eukaryotes, yet appears to be a part of the turn-off machinery of Cdc42 from higher eukaryotes. Introduction of the arginine 186 mutation into S. cerevisiae CDC42 led to phenotypes consistent with down-regulated CDC42 function. Thus, specific Rho family GTPases may utilize a built-in arginine finger, in addition to RhoGAPs, for negative regulation.

    INTRODUCTION
Top
Abstract
Introduction
References

The Rho family small GTPases of the Ras superfamily are molecular switches controlling a variety of intracellular signaling events (1, 2).

To understand how these molecular switches are turned off, much effort has been focused on the elucidation of the mechanism of RhoGAP1 actions (3). Recent x-ray crystallography and mutagenesis studies have helped to establish that, like the case of Ras-RasGAP interaction (4), and to certain extent the case of heterotrimeric G-protein-RGS interaction (5, 6), Rho GTPases undergo a transition state mimicked by a combination of AlF4- and RhoGAP in the GTPase-activating reaction catalyzed by RhoGAP (7-9). Furthermore, Rho GTPases appear to utilize a conserved arginine residue, termed "arginine finger", of RhoGAP to stabilize the transition state and to catalyze the cleavage of gamma Pi from the bound GTP (3, 8). This arginine finger mechanism of GTPase activation is apparently shared by both Ras and Galpha i, since critical arginine residues in RasGAP and Galpha i itself (Arg789 in RasGAP and Arg178 in a built-in domain of Galpha i) have been demonstrated to play essential roles in the GTPase-activating process (10, 11). Here we report an unexpected finding that Arg186 in Rho GTPases functions effectively as a built-in "arginine finger" to confer a self-stimulatory GAP activity through homophilic interaction.

    EXPERIMENTAL PROCEDURES

Materials-- MESG and mantGDP were synthesized as described previously (12, 13). Recombinant human RhoA, RhoB, RhoC, Cdc42Hs, and Caenorhabditis elegans and Drosophila melanogaster Cdc42 (Cdc42Ce and Cdc42Dm, kind gifts of Drs. L. Lim and L. Luo) were expressed in Escherichia coli as (His)6-tagged fusions using the pET expression system (12-15). The posttranslationally modified RhoA, Cdc42Hs, S. cerevisiae Cdc42 (Cdc42Sc) and the Cdc42ScK186R mutant were obtained in an insect cell expression system similarly as described (16).

The RhoA, Cdc42Hs, and Cdc42Sc mutants at the amino acid 186 position (RhoAS(R), Cdc42HsR(K), and Cdc42ScK186R) were generated by oligonucleotide-directed mutagenesis of the respective cDNAs (17). The C-terminal truncation mutations of Cdc42Hs (C-7) and RhoA (Rho-8) were generated previously (13).

GTPase Activity Assay-- The GTPase activities of the small GTPases were measured by the MESG/phosphorylase system monitoring the free gamma Pi release from the GTP-bound G-proteins as described for the cases of Cdc42, Rac1, and RhoA (12-15) and by the nitrocellulose filter-binding method (9).

Gel Filtration Chromatography-- A Superdex 200HR 10/30 gel filtration column (Amersham Pharmacia Biotech) coupled to a Bio-Rad Biologic liquid chromatography system was used to analyze the homophilic interactions of the small GTPases as described (13).

Transition-state Complex Formation Assay-- The fluorescence change of small G-protein bound mantGDP in the presence of AlF4- and the activated form of the G-protein was used to detect a GAP-reaction transition-state complex formation (13).

Generation of CDC42K186R Mutant S. cerevisiae Strain-- The cdc42K186R mutant was cloned into the pRS306 plasmid and integrated into the genome of the diploid strain DJD6-11 (cdc42Delta ::TRP1/CDC42 ura3-52/ura3-52) at the ura3-52 locus. Stable Ura+ transformants were subjected to tetrad analysis to follow the ura3-52:: cdc42K186R::URA3 and cdc42Delta ::TRP1 marked loci. A Ura+, Trp+ segregant (CCY1-8A) that contained the cdc42K186R allele as the sole copy of CDC42 within the cell was backcrossed against wild-type strain Y763 two times to generate the strain CCY3-3B.

    RESULTS AND DISCUSSION

The Ras superfamily small GTP-binding proteins are monomeric GTP-hydrolyzing enzymes that contain slow intrinsic GTPase activities (20). Certain members of the Rho family GTPases of the Ras superfamily, e.g. RhoA, Rac2, and Cdc42Hs, form reversible homodimers under physiological buffer conditions in vitro (13). Others such as RhoB and RhoC were found in either monomeric or oligomeric form (data not shown). Although sharing a very high degree of sequence homology, RhoA and RhoB behaved like Ras proteins showing a slow intrinsic GTPase reaction rate at ~0.02 min-1 that was GTPase dose-independent, whereas the rate of GTP hydrolysis by RhoC was found to increase significantly with increasing concentrations of the G-protein bound to GTP (Fig. 1A). Addition of GTPgamma S-bound RhoC to RhoC-GTP resulted in a further enhanced rate of gamma Pi release similar to that seen with the addition of RhoGAP, whereas GTPgamma S-bound RhoA or RhoB had no detectable effect on the respective G-proteins bound to GTP (Fig. 1B), indicating that the activated form of RhoC, but not RhoA or RhoB, can provide a specific GAP activity toward itself. This self-stimulatory GAP activity may account for the faster intrinsic GTP-hydrolysis rate of RhoC compared with RhoA and RhoB (Fig. 1, A and B). Previous studies of the Rho family members Cdc42Hs and Rac2 also showed that these two GTPases contain a self-stimulatory GAP activity when in the activated GTP-bound form (13). Sequence alignment analysis revealed that the presence of a C-terminal polybasic motif, located immediately N-terminal to the CAAX isoprenylation sequences (Fig. 1C), correlated with the homophilic interaction of Rho family members. All Rho family proteins containing the polybasic sequences, including the additional mammalian members Rac1, Rac2, Cdc42Hs, and RhoG, have been found in reversible homodimer or higher oligomer and monomer states, whereas others lacking the polybasic residues, e.g. TC10 and RhoB, are exclusively monomeric like Ras2 (13). Together with the finding that removal or substitution of the C-terminal polybasic residues led to the monomeric form only (see below) and a loss of self-stimulatory GAP activity2 (13), these results suggest that the polybasic nature of the C-terminal domain is critical in mediating the homophilic interactions of specific Rho family GTPases. However, the homophilic interaction itself apparently is not sufficient for the enhanced GTP hydrolysis rate because RhoA does not display detectable GAP activity toward itself albeit containing C-terminal polybasic residues (Figs. 1B and 1C); rather, additional unique structural determinant(s) of the Rho GTPases are required for the observed self-stimulatory GAP activity.


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Fig. 1.   Comparison of the intrinsic and GTPase-activating activities of Rho GTPases. A, concentration dependence of the apparent intrinsic rates of GTP hydrolysis of RhoA, RhoB, and RhoC at 23 °C. Data were fitted into a single exponential to derive the apparent rate constant Kapp. B, the self-stimulatory GAP effects of the active forms of Rho GTPases. gamma Pi release by 5 µM Rho proteins bound to GTP were determined at the 5 min time point in the absence or presence of 5 µM respective stimulants (RhoGAP was at 10 nM) under single turnover conditions. C, amino acid sequence alignment of the polybasic domain of mammalian Rho family proteins. The C-terminal region lysine and arginine residues are underlined. Arrow indicates the position corresponding to Arg186 in Cdc42Hs.

Specific arginine residues of RasGAP, RhoGAP, and a built-in GTPase-activating domain of Galpha i1, known as "arginine fingers," have been shown to facilitate the effective cleavage of phosphodiester bond linking gamma Pi of the G-protein-bound GTP in their respective GTPase-activating reactions (3). In addition, sequence motifs that contain invariant arginine residues suspected to play a role in catalyzing GTP-hydrolysis have also been identified in the GAPs for the Rap, Ypt, Ran, and Arf proteins (4), and an invariant arginine residue in the built-in ArfGAP domain of ARD1, an Arf family GTPase, has been shown to be critical for the ARD1 GAP activity (28). An examination of the polybasic region of Cdc42Hs, RhoC, RhoG, Rac1, and Rac2 (Fig. 1C), which all displayed a faster intrinsic GTPase activity, suggested that a highly conserved arginine residue, arginine 186 (numbered by the sequences of Cdc42Hs), might be involved in the observed self-stimulatory GAP activity and the enhanced intrinsic GTPase activity of the Rho proteins. Mutation of the corresponding residue in RhoA, serine 188, to arginine (RhoAS(R)), resulted in a significant increase of the intrinsic rate of GTP hydrolysis (Fig. 2A) and a gain of self-stimulatory GAP activity (Fig. 2B). Substitution of the arginine 186 residue of Cdc42Hs by lysine (Cdc42HsR(K)) effectively decreased the intrinsic rate of GTPase activity of Cdc42Hs to that of the wild-type RhoA (Fig. 2A) and led to a loss of the self-stimulatory GAP activity (Fig. 2B). Both RhoAS(R) and Cdc42HsR(K) behaved indistinguishably in their GTP-binding and GDP/GTP-exchange properties from the wild-type proteins (data not shown). Moreover, posttranslational modification of the Rho proteins by C-terminal geranylgeranylation did not change the GTPase-activating effect of the arginine residue (data not shown). These results indicate that arginine 186 of the Rho family GTPases constitutes a critical determinant for the self-stimulatory GAP activity that controls their intrinsic GTPase activities.


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Fig. 2.   Effect of Arg186 on the intrinsic GTPase and the self-stimulatory GAP activities of Rho GTPases. A, the intrinsic GTPase activity of wild-type RhoA protein (open circle ) or Cdc42Hs (down-triangle) was compared with RhoAS(R) (bullet ) or Cdc42HsR(K) (black-down-triangle ) mutant. B, the GAP effects of GTPgamma S-bound mutants compared with wild-type RhoA and Cdc42Hs. Reaction conditions were similar to those in Fig. 1B. Data are representative of at least three independent measurements.

To determine whether arginine 186 plays a role primarily in the catalytic or homophilic binding interaction of the G-proteins, the RhoAS(R) and Cdc42HsR(K) mutants were further examined by a fluorescence assay originally designed to detect the formation of a transition-state complex for the Ras-RasGAP and Rho-RhoGAP interactions (19, 21) and by gel-filtration analysis. In the case of the small G-protein-GAP interaction, addition of GAP together with AlF4- causes a change of the emission spectrum of the G-protein bound to the fluorescent GDP-analog, mantGDP, both in maximum absorption wavelength and in intensity (19, 21). Similarly, this was observed when GTPgamma S-bound RhoAS(R) mutant (as GAP) together with AlF4- was added to RhoAS(R)-mantGDP (Fig. 3). This result is indicative of the formation of an analog of the GTP-bound transition state of the GTPase-activating reaction involving RhoAS(R)-mantGDP, AlF4-, and RhoAS(R)-GTPgamma S. Wild-type RhoA, in contrast, failed to form a transition-state complex with AlF4- when RhoA-mantGDP and RhoA-GTPgamma S were incubated together (Fig. 3, insert). This result is consistent with its inability to act as a self-stimulatory GAP. When Cdc42Hs and Cdc42HsR(K) were examined in similar experiments, it was found that the Cdc42Hs mutant had lost the ability to form the transition-state complex with Cdc42HsR(K)-mantGDP and AlF4- when bound to GTPgamma S, whereas wild-type Cdc42Hs behaved like the RhoAS(R) mutant (Fig. 3, insert). However, no change in the gel-filtration profiles was detected for Cdc42HsR(K) and RhoAS(R), with each mutant protein showing a similar monomer/dimer distribution pattern as the respective wild-type proteins (data not shown). Truncation of the last seven and eight amino acids from Cdc42Hs (C-7) and RhoA (Rho-8), respectively, resulted in only the monomeric form of the proteins (13). These results provide evidence that the self-stimulatory GAP reaction of Rho family GTPases employs an arginine finger-like mechanism similar to the RhoGAP-catalyzed reaction, i.e. the arginine 186 residue is essential for the formation of a GAP-reaction intermediate, but is not required for homophilic binding interaction.


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Fig. 3.   The role of Arg186 in self-stimulatory GAP reaction. The ability of RhoAS(R)-GDP to form a GAP-reaction transition-state complex with AlF4- and the GTPgamma S-bound GTPase was measured by monitoring the fluorescence emission spectra of mantGDP-bound G-protein (0.1 µM) upon addition of AlF4- (150 µM AlCl3 and 15 mM NaF) and 1 µM GTPgamma S-bound GTPase. Insert, titration of AlF4--induced fluorescence responses of respective mantGDP-bound RhoA, Cdc42Hs, and mutant GTPases by various concentrations of respective GTPases bound to GTPgamma S.

To determine the potential physiological relevance of the arginine finger-mediated self-regulation of Rho family GTPases, we examined the role of arginine 186 in the Cdc42 subfamily. The eleven members of the Cdc42 subfamily that have been identified from S. cerevisiae to Homo sapiens share over 80% amino acid sequence identify, and they can functionally complement each other under defined conditions (2, 22, 23). Interestingly, arginine 186 is found in all the Cdc42 sequences from D. melanogaster and higher organisms, whereas a lysine or serine residue is present at the 186 position of Cdc42 from C. elegans and lower eukaryotes (Fig. 4A). The arginine finger theory would predict that Cdc42 from higher eukaryotes than D. melanogaster should contain a faster intrinsic GTP hydrolysis rate than that of lower eukaryotes because of the catalytic effect of arginine 186. Indeed, as shown in Fig. 4B, the Cdc42Sc and Cdc42Ce proteins displayed a slow intrinsic GTPase activity similar to that of RhoA and Ras with a GTP hydrolysis rate of 0.02 min-1, whereas Cdc42Dm and Cdc42Hs demonstrated a marked higher intrinsic GTPase activity that can be further stimulated by the respective Cdc42 bound to GTPgamma S. These results raised the possibility that possession of Arg186 contributes to the mechanism of regulation of the Cdc42 GTPases of higher eukaryotes. To determine the in vivo effect of the built-in arginine finger, a lysine to arginine mutation was introduced into Cdc42Sc at position 186. Cdc42Sc is an essential gene product in S. cerevisiae and plays a critical role in polarized cell growth and cell division cycle regulation (24, 25). Purified wild-type Cdc42Sc showed an intrinsic GTP hydrolysis ability similar to that of RhoA, whereas the Cdc42ScK186R mutant protein displayed a significantly enhanced GTPase activity similar to that of Cdc42Hs (Figs. 5A and 2B). Genomic integration of cdc42K186R mutation into a S. cerevisiae Delta cdc42 strain resulted in a temperature-sensitive lethal phenotype at 37 °C (Fig. 5B). At the permissive temperature of 23 °C, the cdc42K186R mutant led to a pleiotropic phenotype of elongated, multibudded, multinucleated cells (68%) and large, round unbudded cells (13%) (Fig. 5C, and data not shown), which is similar to phenotypes observed with S. cerevisiae CDC42 effector cla4 and ste20 mutants (26, 27), and indicative of a G2 cell cycle delay and/or a cytokinesis defect. Given that the cdc42K186R mutant seems to be able to interact with regulators of Cdc42 such as Cdc24 and Bem3 like the wild-type protein,2 it is likely that the introduced arginine finger is involved in an abnormal negative regulation of Cdc42Sc function, contributing to the temperature-sensitive phenotype and morphological abnormalities.


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Fig. 4.   The intrinsic GTPase activity of Cdc42 is governed by the presence of Arg186. A, sequence alignment of the C-terminal polybasic domain of Cdc42 from representative eukaryotes. Arrow indicates the 186 position. B, comparison of the intrinsic GTPase and self-stimulatory GAP activities of Cdc42Sc, Cdc42Ce, Cdc42Dm, and Cdc42Hs. Conditions were similar to those in Fig. 1B.


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Fig. 5.   A regulatory role of Arg186 of Cdc42Sc in S. cerevisiae. A, time courses of [gamma -32P]GTP hydrolysis were compared between Cdc42Sc and Cdc42ScK186R mutant protein at ~2 µM concentration at 23 °C. B, cdc42K186R mutation in S. cerevisiae causes temperature-sensitive lethality. The cdc42K186R mutant strain CCY3-3B and wild-type strain C276-4A were streaked onto yeast extract-peptone-dextrose plates and incubated at 23 or 37 °C for 3 days. C, the phenotype of cdc42K186R mutant at the permissive temperature of 23 °C.

Rho family GTPases utilize a conserved GAP-stimulated GTP-hydrolyzing machinery similar to that of Ras for negative regulation (2-4). We demonstrate here that specific Rho family proteins may employ an additional built-in arginine finger-like mechanism through homophilic interaction to effectively accelerate the basal intrinsic GTPase activity. This additional negative regulation mechanism seems to be absent from many Rho GTPases from lower eukaryotes, and introduction of this arginine finger into such GTPases results in abnormal negative regulatory effects leading to morphological defects and lethality. Because the currently available three-dimensional structures of Rho family GTPases were all derived from various C-terminal truncated forms of the proteins, it remains to be seen how the homodimers are configured so that the C-terminal polybasic domain containing the critical arginine residue of one molecule may interact with the GTP binding core of another molecule. To this end, we have started to map the residues on Rho GTPases that may serve as sites for Arg186 action and have identified Tyr32 of Cdc42Hs at the GTP hydrolytic center as one of such sites.2 It will be important in the near future to obtain a complete picture of the structural configurations of the Rho family homodimers and to compare it with that of the Rho GTPase-GAP complexes. We propose that the built-in arginine finger mechanism may provide an alternative to RhoGAP-mediated negative regulation for Rho GTPases from higher eukaryotes, thereby increasing the flexibility for the regulatory circuit of these small GTPases. The discovery of this novel type of negative regulation may provide valuable information concerning the many diverse roles of Rho GTPases and may generate an interesting paradigm for possible negative therapeutics involving these GTPases.

    FOOTNOTES

* This work was supported by American Cancer Society Grant RPG-97-146 and National Institutes of Health Grant GM53943 (to Y. Z.) and by National Science Foundation Grants MCB-9405972, MCB-9723071, and MCB-9728218 (to D. I. J.).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.

§ To whom correspondence should be addressed. Tel.: 901-448-5138; Fax: 901-448-7360; E-mail: yzheng{at}utmem.edu.

The abbreviations used are: GAP, GTPase-activating protein; GST, glutathione S-transferase; mantGDP, 2'(3')-O-(N-methylanthraniloyl) GDP; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

2 B. Zhang and Y. Zheng, unpublished observations.

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