COMMUNICATION
A Built-in Arginine Finger Triggers the Self-stimulatory
GTPase-activating Activity of Rho Family GTPases*
Baolin
Zhang,
Yaqin
Zhang,
Cheryl C.
Collins
,
Douglas
I.
Johnson
, and
Yi
Zheng§
From the Department of Biochemistry, University of
Tennessee, Memphis, Tennessee 38163 and the
Department of
Microbiology and Molecular Genetics, University of Vermont,
Burlington, Vermont 05405
 |
ABSTRACT |
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 |
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
Pi from the bound GTP (3,
8). This arginine finger mechanism of GTPase activation is apparently
shared by both Ras and G
i, since critical arginine residues in
RasGAP and G
i itself (Arg789 in RasGAP and
Arg178 in a built-in domain of G
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
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 (cdc42
::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
cdc42
::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 GTP
S-bound RhoC to RhoC-GTP resulted in a further enhanced rate of
Pi release similar to that seen with the addition of
RhoGAP, whereas GTP
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. 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 G
i1, known as "arginine fingers," have been shown to facilitate the effective cleavage of phosphodiester bond linking
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 ( ) or Cdc42Hs ( ) was compared with RhoAS(R) ( ) or
Cdc42HsR(K) ( ) mutant. B, the GAP effects of
GTP 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.
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|
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 GTP
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)-GTP
S. Wild-type
RhoA, in contrast, failed to form a transition-state complex with
AlF4
when RhoA-mantGDP and
RhoA-GTP
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 GTP
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 GTP 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
GTP 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 GTP S.
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|
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 GTP
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
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 [ -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.
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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; GTP
S, guanosine 5'-3-O-(thio)triphosphate.
2
B. Zhang and Y. Zheng, unpublished observations.
 |
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