The intrinsic GTPase activity of the Rho family
GTP-binding protein Rac1 is drastically stimulated upon interaction
with its GTPase-activating proteins (GAPs) and is significantly
inhibited when coupled to certain effector targets such as the
p21-activated kinases (PAKs) and IQGAPs. Here we have characterized the
interaction of Rac1 with a panel of mammalian GAPs and putative
effectors by measuring the kinetic and binding parameters involved and
made comparisons with similar interactions for Cdc42 and RhoA. In
contrast with Cdc42 (for which the GAP domain of p50RhoGAP is 50-fold
more efficient than those of p190, Bcr, and 3BP-1) and with RhoA
(toward which only p50RhoGAP and p190 displayed high efficiencies), the catalytic efficiencies
(Kcat/Km) of the GAP
domains of p50RhoGAP, p190, Bcr, and 3BP-1 on Rac1 are found to be
comparable in a range between 0.9 and 2.6 min
1
µM
1. However, similar to the cases of Cdc42
and RhoA, the Km values of the GAP domains on Rac1
compare well to the binding affinity to the guanylyl
imidodiphosphate-bound Rac1, which ranges from 10.5 to 40.5 µM, suggesting a rapid equilibrium reaction mechanism.
The dissociation constants of the p21-binding domains of PAK1, PAK2,
and the RasGAP-related domain of IQGAP1, which all cause significant
reduction of the intrinsic rate of GTP hydrolysis upon binding to
Rac1-GTP, are found to be 0.71, 0.26, and 2.13 µM for
Rac1-GTP, compared with that determined for Cdc42-GTP at 2.9, 20.5, and
0.39 µM, respectively, under similar conditions. These
results suggest that p50RhoGAP, p190, Bcr, and 3BP-1 are all capable of
acting as a negative regulator for Rac1-mediated signaling, and that,
although PAK1 and IQGAP1 can couple tightly with both Rac1 and Cdc42,
PAK2 is likely to be a specific effector for Rac1 instead of Cdc42.
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INTRODUCTION |
Rac1 belongs to the mammalian Rho family of Ras superfamily small
GTP-binding proteins, which includes the additional members of RhoA,
RhoB, RhoC, RhoE, RhoG, Cdc42, TC10, and Rac2 (1, 2). The Rho family
proteins appear to be key components in the transduction of
intracellular signals that induce actin-cytoskeleton reorganization and
gene activation (1-3). The initial studies of the role of Rac, Rho,
and Cdc42 in fibroblast cells have provided evidence that Rac1
regulates lamellipodium formation and membrane ruffling (4), RhoA
regulates the formation of actin stress fibers and focal adhesion
complexes (5), and Cdc42 regulates filopodium formation (6, 7). All
three GTPases were subsequently shown to be required for G1
to S phase cell cycle progression (8) and to be capable of mediating
gene transcription through the Jun N-terminal kinase module of the
mitogen-activated protein kinase pathways (9-11). Rac1, in particular,
may also be involved in the regulation of exocytic and endocytic
pathways, and has a role in activation of the NADPH oxidase enzyme
complex (12, 13).
The biochemical mechanisms underlying the biological functions of Rho
GTPases have been under intensive scrutiny. Like other types of
G-proteins, the GDP-bound forms of Rho proteins are in an inactive
state while the GTP-bound forms serve to transduce signals to the
immediate downstream effector targets (14). A family of
GTPase-activating proteins
(GAPs)1 that stimulates the
intrinsic GTPase activity and thereby facilitates the deactivation of
the small G-proteins has emerged over the past few years by molecular
cloning and biochemical studies (15). Primary sequence analysis
revealed an ~170-amino acid homology region, designated the RhoGAP
domain, in GAP family molecules that was found to be necessary and
sufficient for GAP activity (16). Recently available x-ray crystal
structures of the RhoGAP domains of the p85 regulatory subunit of
phosphatidylinositol 3-kinase and the p50RhoGAP (also known as
Cdc42GAP) suggest that these critical domains involved in interaction
with Rho GTPases adopt a highly conserved structural folding in three
dimensions (17, 18). Moreover, the structure of the complex of Cdc42 with p50RhoGAP further revealed that the mechanism of Rho protein interaction with RhoGAPs are different from the Ras-RasGAP or the
heterotrimeric G-protein-RGS interaction in a manner that the small
G-protein contacts mostly through its switch I and II regions with a
unique shallow pocket of RhoGAP domain formed between the B and F
helices, the A-A1 loop, and the F-G loop (19). However, detailed
mechanistic proposal regarding how RhoGAPs enhance the GTPase activity
of Rho proteins remains lacking, especially when it is realized that
certain unique features of each Rho protein-GAP pair apparently
contribute to the specificity of individual reactions (20,
43).
The recent identification of putative effectors of Rho family GTPases
has helped to provide molecular links to the diverse biological
activities of Rho proteins. Among a growing panel of the small
G-protein targets, p21-activated kinase (PAK) was initially found to be
activated upon binding to the GTP-bound forms of Rac1 and Cdc42 (22,
23), and later was implicated in mediating signaling from Rac1 and
Cdc42 to the Jun N-terminal kinase (24, 25) and in certain cases, to
actin cytoskeleton (26). IQGAP1, a RasGAP domain containing protein
with selective affinity to the GTP-bound Rac1 and Cdc42 (27), binds to
F-actin directly and cross-links the actin filaments into
interconnected bundles (28), therefore may serve as a link between
these GTPases and the actin cytoskeleton. Furthermore, in analogy to
that suggested by the studies of p120RasGAP, which was implicated as an
effector for Ras signaling in defined cases (29), there is also
evidence that certain RhoGAPs may act as potential effectors of Rho
GTPases (30, 31).
To examine the mechanism of Rho family GTPase interaction with GAPs and
effectors in detail and to make direct comparison of these interactions
among Rac, Cdc42, and Rho, we have set out to determine the kinetic
parameters and binding affinities of a panel of mammalian RhoGAPs,
i.e. p190, p50RhoGAP, Bcr, and 3BP-1, and a few putative
effectors for Rac1 and Cdc42, i.e. PAK1, PAK2, and IQGAP1,
to the small GTPases. Previous studies of Cdc42 and RhoA suggest that
there exists a unique mechanism for each specific interaction between a
Rho protein and a RhoGAP, and that relative tight binding to the small
G-proteins may be necessary to constitute an effective effector target
(32, 43). In the present work, we have determined the intrinsic and
GAP-stimulated rates of GTP hydrolysis by Rac1, obtained the apparent
binding affinities of the RhoGAP domains to both GTP- and GDP-bound
forms of Rac1, and made direct comparisons with similar interactions of
Cdc42 and RhoA. In addition, we have also determined the affinities of
Rac1-GTP and Cdc42-GTP to the p21-binding domains (PBDs) of PAK1, PAK2, and IQGAP1 based upon the property of the effectors to inhibit the
intrinsic rate of GTP hydrolysis of Rac1. Our results suggest that
p50RhoGAP, p190, Bcr, and 3BP-1 are all capable of acting as a negative
regulator for Rac1-mediated signaling and that, although PAK1 and
IQGAP1 can couple tightly with both Rac1 and Cdc42, PAK2 is likely to
be a specific effector for Rac1 instead of Cdc42.
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EXPERIMENTAL PROCEDURES |
Materials--
GDP, GTP, and bacterial purine nucleoside
phosphorylase were purchased from Sigma. GMP-PNP was obtained from
Boehringer Manheim. The phosphorylase substrate,
2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), was
synthesized as described (33), and the linearity of its absorbance
response at 360 nm for measuring a range of Pi
concentrations from 1 µM to 70 µM was
ensured by using the coupling reaction of purine nucleoside
phosphorylase in a buffer containing standard phosphate solution
(33).
Recombinant human Rac1, Cdc42, and RhoA proteins were expressed in
Escherichia coli as (His)6-tagged fusions by
using the pET28 expression system (Novagen). Briefly, the cDNAs of
the small G-proteins were cloned into pET28 vector at the in-frame
BamHI-EcoRI sites, and the resulting constructs
were transformed into competent BL21 cells. The induction and
purification of (His)6-tagged proteins by
isopropyl-1-thio-
-D-galactopyranoside and
Ni2+-charged agarose beads were carried out following the
instructions provided by Novagen. The glutathione
S-transferase fusions of GAP domain and the PBD of
p50RhoGAP, Bcr, 3BP-1, p190, PAK1, PAK2, and IQGAP1 were expressed in
E. coli using the pGEX-KG system (32). The GAP domains of
p50RhoGAP, Bcr, 3BP-1, and p190 contain the amino acid residues
205-439, 1010-1271, 185-410, and 1249-1513 of the native proteins,
respectively, and the PBD of human PAK1, PAK2, and IQGAP1 contain amino
acid residues 51-135, 35-134, and 864-1657, respectively.
Purification of the glutathione S-transferase fusions from
E. coli were carried out as described by glutathione affinity chromatography (16). All proteins prepared for measurements were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and Coomassie Blue staining analysis, and the contents
of each were at least 90% pure. Concentrations of the recombinant GAP and effector p21-binding domains were determined using the BCA protein
assay reagents from Pierce with bovine serum albumin (BSA) as a
standard, and the effective concentrations of the small
G-proteins were determined using the MESG/phosphorylase system under
single turnover conditions as described below.
Spectroscopic Measurements of GTPase Activity--
The rates of
GTP hydrolysis of the small GTPases were measured by the
MESG/phosphorylase system monitoring the absorbance increase of the
reaction mixture at 360 nm as described for the cases of Ras and Cdc42
(32, 35). Briefly, a 0.8-ml solution containing 50 mM
HEPES, pH 7.6, 150 mM NaCl, 0.1 mM EDTA, 0.2 mM MESG, 10 units of purine nucleoside phosphorylase, 200 µM GTP, and the indicated amount of recombinant
G-proteins was mixed in a 4-mm width, 10-mm pathlength cuvette, and the
time courses of absorbance change at 360 nm, which reflect the rates of
multiple turnover of the G-proteins, were recorded. Single turnover
GTPase reactions were initiated by the addition of MgCl2 to
a final concentration of 5 mM. For measurements of
GAP-catalyzed reactions, 5-50 µl of stock solution containing the
indicated amount of GAP domains were added together with
MgCl2 to the reaction mixtures. A control experiment in
which the GTPases were omitted was carried out in each independent
measurement to provide a background of absorbance to be subtracted from
the sample signals. The concentration of Pi in the reaction
solution was calculated by a factor of extinction coefficient
360 nm = 11,000 M
1
cm
1 at pH 7.6 from the absorbance change (33), and was
used to determine the effective concentrations of the small G-proteins after one round of single turnover reaction. Because the phosphorylase coupling reaction is extremely fast with a rate constant of 40 s
1 (33), the slope of the absorbance in the time course
is treated as proportional to the rate of the GTPase activity.
Competition Binding Assay--
Recombinant Rac1 was preloaded
with [
-32P]GTP (10 µCi, 6000 Ci/mmol, NEN Life
Science Products) in a 100-µl buffer containing 50 mM
HEPES, pH 7.6, 0.2 mg/ml BSA, and 0.5 mM EDTA for 10 min at
ambient temperature before the addition of MgCl2 to a final concentration of 5 mM. An aliquot of the
[
-32P]GTP-loaded Rac1 (~20 nM) was mixed
with a reaction buffer containing 50 mM HEPES, pH 7.6, 0.2 mg/ml BSA, and 5 mM MgCl2 in the presence or
absence of GAP domains and Rac1 preloaded with GMP-PNP or GDP at
indicated concentrations. At the 5-min time point, the reactions were
terminated by filtering the reaction mixture through nitrocellulose filters followed by washing with 10 ml of ice-cold buffer containing 50 mM HEPES, pH 7.6, 150 mM NaCl, and 10 mM MgCl2. The radioactivity retained on the
filters was then subjected to quantitation by scintillation counting
(16).
Data Analysis--
Kinetic data were analyzed by nonlinear
regression using equations derived before for Cdc42-GAP interactions
(32) with the program Enzfitter (Elsevier Biosoft). The apparent rate
constants (Kc) of intrinsic GTP hydrolysis by the
small GTPases were determined by fitting data to a single exponential
function for single turnover reactions or to a linear equation for
EDTA-induced multiple turnover reactions. A modified Michaelis-Menten
equation was used to derive kinetic parameters for GAP-catalyzed GTP
hydrolysis assuming GAP acting as the enzyme catalyst, Rac1-GTP as the
substrate, and the Rac1-GDP and Pi as the products.
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(Eq. 1)
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V0 is the initial rate of hydrolysis,
[Rac1]0 is the total Rac1 concentration,
Kc is the apparent rate constant of intrinsic GTPase
activity, and the term Kc[Rac1]0 is a
correction for the rate of intrinsic GTP hydrolysis by Rac1. Kcat is derived by
Vmax/[GAP]0, with
[GAP]0 representing total GAP concentration.
The dissociation constants (Kd) of GAP binding to
Rac1 were derived by fitting data of the competition assay to the following derived equation under conditions that the total
concentration of competitor [C]0 (C representing
GMP-PNP- or GDP-bound Rac1) and reaction Km values
are orders of magnitude larger than the concentration of
Rac1-[
-32P]GTP.
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(Eq. 2)
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The binding constants of the PBD of effectors to Rac1-GTP and
Cdc42-GTP were extracted by fitting data of the initial rate of GTP
hydrolysis of the GTPases at various concentrations of the effector
domains to the following derived equation, assuming that the rate of
hydrolysis of the small GTPase in complex with effector is much slower
than Rac1-GTP or Cdc42-GTP alone.
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(Eq. 3)
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V0 and [E]0 are the initial
rate of GTP hydrolysis and the total concentration of PBD added
to the assay, respectively, and Ki represents the
binding affinity (dissociation constant) of the PBD to GTP-bound
G-proteins.
 |
RESULTS |
The rate of GTP hydrolysis by Rac1 directly affects the time span
in which Rac1 remains active, and therefore may have a major influence
on the duration of signaling output from the small G-protein. To
determine the rate of intrinsic GTP hydrolysis, the time courses of
GTPase reaction of Rac1 under both multiple and single turnover conditions were monitored by the MESG/phosphorylase assay (Fig. 1). The continuous absorbance trace at
360 nm wavelength reflects
Pi release from Rac1-GTP
detected by the phosphorylase coupling reaction with MESG as a
substrate, and the initial presence of 0.1 mM EDTA and 200 µM GTP and the absence of Mg2+ provide a
condition at which the G-protein undergoes rapid GDP/GTP exchange.
Interestingly, a very slow phase of GTP hydrolysis by Rac1 at 5 µM concentration was observed at this condition, with an
apparent rate of 0.054 × 10
2 min
1, in
contrast with the over 60- and 8-fold faster rates displayed by Cdc42
and RhoA, respectively (Fig. 1; Table I).
The differences in the rate of EDTA-induced multiple turnover of the
small GTPases may be attributed to their distinction in
Mg2+ binding affinity and the subsequent GTP binding
ability, since Rac1 seems to bind most weakly to Mg2+ among
the three GTPases, whereas their rates of GDP dissociation under these
conditions are similar.2 When
5 mM MgCl2 was added to the mixture to initiate
single turnover reaction (Fig. 1), an apparent rate constant of 0.030 min
1 by Rac1 was obtained by fitting the data to a single
exponential function (Table I). This falls between that of Cdc42 (0.064 min
1) and RhoA (0.015 min
1) under similar
conditions (Table I). Thus, Rac1 behaves similarly to other members of
Rho family GTPases in its intrinsic ability to hydrolyze GTP which is
~10-fold faster than that of Ras, and seems to possess a unique
property in Mg2+ and/or GTP binding, which affects its rate
of EDTA-induced multiple turnover.

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Fig. 1.
Comparison of kinetics of intrinsic GTP
hydrolysis of Rac1 with that of Cdc42 and RhoA. Pi
released from GTP-bound small G-proteins were measured by the
continuous spectroscopic assay of MESG/phosphorylase system as
described under "Experimental Procedures." The absorptions at 360 nm of 5.0 µM solution of small GTPases were monitored
during the time course in a buffer containing 50 mM HEPES,
pH 7.6, 150 mM NaCl, 0.1 mM EDTA, 200 µM GTP, and 0.2 mM MESG with 10 units
coupling phosphorylase at 20 °C which provides a condition for the
G-proteins to undergo multiple turnovers. Arrow indicates
the time point at which a final concentration of 5 mM
MgCl2 was added to initiate single turnover reactions. The
dotted lines represent the periods of addition and
mixing.
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Table I
Apparent rate constants of intrinsic GTP hydrolysis of Rac1
compared with that of Cdc42 and RhoA
The GTPase activities of the small G-proteins were measured by the
MESG/phosphorylase method under conditions described in Fig. 1. The
earlier traces of GTP hydrolysis depicted in Fig. 1 (in the presence of
EDTA) were fitted into a linear equation to derive the rate constants
for EDTA-induced multiple turnover reactions and the later traces (upon
addition of excess MgCl2) were fitted into a single exponential
function to derive the rate constants for single turnover hydrolysis.
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When p50RhoGAP was added together with Mg2+ to the single
turnover reactions of the small GTPases, increases in the rate of GTP
hydrolysis were observed (Fig.
2A). The scale of response to
p50RhoGAP by Rac1 was apparently different from that of Cdc42 and RhoA,
reflecting differences in kinetics for the respective GAP-reactions. To
determine the kinetic parameters of GAP-stimulated GTP hydrolysis by
Rac1, the initial rate of
Pi release by Rac1-GTP was
measured as a function of Rac1 concentration at a fixed concentration of GAP, as shown for the case of p50RhoGAP catalysis in Fig.
2B. Since the amount of the small G-protein (treated as a
substrate) was in large excess of GAPs (treated as enzymes), the
reactions can be treated by Michaelis-Menten kinetics with the
adjustment of the intrinsic rate of GTP hydrolysis by Rac1 (see
Equation 1 under "Experimental Procedures"). Fitting of the data
obtained for p50RhoGAP, p190, Bcr, and 3BP-1 (Fig. 2C) gave
Km values at 18.7-49.3 µM range
(Table II). The
Kcat values were further derived from
Vmax (Table II) assuming that all GAPs present were active, and ranged from 23.4 to 72.3 min
1 under
these conditions. When the catalytic efficiencies
(Kcat/Km) of these GAPs for
Rac1 were compared, the differences were found to be less than 3-fold
(ranging from 0.9 to 2.6 min
1µM
1). These catalytic
properties of GAPs on Rac1 apparently differ from those on Cdc42 for
which p50RhoGAP demonstrated greater than 50-fold preference over the
other GAPs (32), and from RhoA for which both p190 and p50RhoGAP were
20-fold more active while Bcr and 3BP-1 showed only marginal activities
(43).

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Fig. 2.
Determination of the kinetic parameters of
GAP-stimulated Rac1-GTP hydrolysis. A, time courses of
Pi release from Rac1, Cdc42, and RhoA under stimulation
by p50RhoGAP. Reaction conditions were similar to Fig. 1, and GAP
reactions were initiated by adding p50RhoGAP at 5 nM final
concentration together with MgCl2 (arrow) to 5.0 µM small GTPases. B, time courses of GTP
hydrolysis by Rac1 in the presence of 18.8 nM p50RhoGAP at
four different Rac1 doses as indicated. C, initial rates of
GTP hydrolysis by Rac1 under GAP catalysis as a function of Rac1 concentration. The initial rates
of GTP hydrolysis monitored by Pi release were measured
in the presence of a constant amount of the GAP domain of p50RhoGAP
(18.8 nM, filled circles), p190 (66.0 nM, filled triangles), Bcr (37.2 nM,
open circles), or 3BP-1 (137 nM, open
triangles) and increasing concentrations of Rac1-GTP. The
GAP-catalyzed GTP hydrolysis rates were fitted into the modified
Michaelis-Menten equation (Equation 1 under "Experimental
Procedures") to yield Km and
Vmax values of the reactions.
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Table II
Kinetic parameters of GAPs that regulate Rac1 under single turnover
conditions
GAP activities of p50RhoGAP, p190, Bcr, and 3BP-1 analyzed in Fig.
2C by nonlinear regression yielded the
Vmax and Km values listed, and
the Kcat and
Kcat/Km were derived thereon. GAP
reactions were carried out at 20 °C under single turnover conditions
under which MgCl2 (5 mM) was in great excess of
EDTA (0.1 mM). Concentrations of the GAP domains used in
the measurements were 18.8 nM, 66.0 nM, 37.2 nM, and 137 nM for p50RhoGAP, p190, Bcr, and
3BP-1, respectively. These concentrations of GAPs fell within the
linear range of the absorbance increase in response to increasing doses
of GAPs.
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To determine the affinity of Rac1 binding to GAPs, the ability of
GMP-PNP (a nonhydrolyzable GTP analog)-bound Rac1 to inhibit competitively the GAP-stimulated hydrolysis of
Rac1-[
-32P]GTP was measured (Fig.
3). The concentration range in which Rac1-GMP-PNP inhibits GAP-mediated GTP hydrolysis is an indication of
its affinity (Kd) for GAP. By fitting data to
Equation 2 as described under "Experimental Procedures," we derived
that p50RhoGAP bound to Rac1-GMP-PNP with an Kd of
26.4 µM, p190 bound with an Kd of 40.5 µM, Bcr bound with 10.5 µM, and 3BP-1 bound
with 27.7 µM (Table III).
Similar to the cases for Cdc42 and RhoA (32, 43), the
Kd values of these GAPs on Rac1 show a general
correlation with the Km values (Tables II and III),
suggesting rapid equilibrium binding of the GAPs to Rac1-GTP, which is
not a rate-limiting step in the GAP-catalyzed reactions. However,
distinct from Cdc42 or RhoA, Rac1 does not seem to recognize any of the
GAPs with micromolar affinity (the tightest binding occurs for Bcr with
a Kd value of 10.5 µM), whereas the
Cdc42-p50RhoGAP, RhoA-p50RhoGAP, and RhoA-p190 interactions were found
to be at over 5-fold higher affinity (Table III). To see if there is an
effect of product inhibition in the time courses of GAP-stimulated GTP
hydrolysis as has been observed for the RhoA-p50RhoGAP reaction (43),
we measured the affinity of Rac1-GDP to various GAPs by a similar
assay, and found that both p50RhoGAP and 3BP-1 bound to Rac1-GDP with
negligible affinity (160 µM and 97.6 µM,
respectively), while Bcr and p190 recognize Rac1-GDP with affinities at
21.7 µM and 59.3 µM, respectively. We
conclude that there were minimal, if any, Rac1-GDP inhibitory effect on the GAP reactions of Rac1 at up to 20 µM
concentration.

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Fig. 3.
Inhibition of GAP-stimulated
Rac1-[ -32P]GTP hydrolysis by GMP-PNP-bound Rac1.
~20 nM Rac1-[ -32P]GTP together with 200 nM p50RhoGAP (filled triangles), 90 nM Bcr (filled circles), 500 nM
3BP-1 (open circles), or 350 nM p190 (open
triangles) were present in a GAP assay buffer containing 50 mM HEPES, pH 7.6, 0.2 mg/ml BSA, and 5 mM
MgCl2 when Rac1 preloaded with GMP-PNP at the indicated
concentrations were added. The reactions were terminated after 5 min at
20 °C.
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Table III
Affinity of GAPs for Rac1 compared with that for Cdc42 and RhoA
The competition reactions were carried out under conditions described
in Fig. 3. The dissociation constants (Kd) were
derived by fitting data shown in Fig. 3 to Equation 2 under
"Experimental Procedures."
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A few Rho GAPs have been speculated to act as effectors for the
G-protein functions (30, 31), and Rac1 and Cdc42 seem to share
additional downstream effectors based upon the binding profiles of
certain PBD of putative effectors (2). We sought to quantify the
interactions of Rac1 with some of the putative effectors and to make
comparisons with that of the GAPs, and with Cdc42. To determine the
direct binding affinity of the effector domains to Rac1-GTP, we
conducted GTPase assays of Rac1 in the presence of various doses of
purified PBDs of PAK1, PAK2, and IQGAP1. As shown in Fig.
4A, the intrinsic GTPase
activity of Rac1 is inhibited by the addition of PAK1 PBD, and this
inhibitory effect is dependent on the concentrations of the PBD. The
initial rates of GTP hydrolysis as a function of the concentrations of the inhibitory PBD were fitted by a non-linear regression (Equation 3)
to extract the binding constants (Ki) to Rac1-GTP and Cdc42-GTP (Fig. 4B), and Ki values of
0.71, 0.26, and 2.13 µM for PAK1, PAK2, and IQGAP1
binding to Rac1-GTP, and 2.9, 20.5 and 0.39 µM for
binding to Cdc42-GTP, respectively, were obtained (Table
IV). Both of the PBDs of PAK1 and IQGAP1 were able to bind to Rac1-GTP and Cdc42-GTP with affinities at micromolar range, whereas the PBD of PAK2 demonstrated an apparent preference to Rac1-GTP over Cdc42-GTP. When the interactions of Rac1
with the effector domains were compared with that of the GAP domains,
it appears that Rac1-GTP binds to GAP domains with significantly lower
affinity than to the effector PBDs, suggesting that the GAPs may
function as negative regulators rather than effectors for Rac1
signaling.

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Fig. 4.
Determination of the affinities of PBD of
effectors to Rac1 and Cdc42 by inhibition of intrinsic GTP hydrolysis
of the small GTPases. A, time courses of Rac1-GTP hydrolysis
in the presence of various amounts of PBD of PAK1 under single turnover
conditions. Reaction condition was similar to that described in Fig. 1,
and 15.0 µM Rac1 and the indicated amount of PAK1 PBD
were present when reactions were initiated by the addition of 5 mM MgCl2. B, comparison of binding affinities
of Rac1-GTP to effector domains with that of Cdc42-GTP. The initial
rates of GTP hydrolysis by Rac1 and Cdc42 as a function of the
concentrations of PBD of PAK1 and PAK2 are shown. 15.0 µM Rac1 (triangles) or Cdc42
(circles) and increasing concentrations of PBD of PAK1
(filled symbols) or PAK2 (open symbols) were
present in the reactions. Data were fitted to Equation 3 described
under "Experimental Procedures" to derive the binding constants
Ki in Table IV.
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Table IV
Affinity of the PBD of effectors for Rac1 and Cdc42
The GTPase-inhibitory reactions of the PBDs were carried out as shown
in Fig. 4A. The initial rate of GTP-hydrolysis as a function
of the PBD was treated by nonlinear regression fitting to Equation 3
under "Experimental Procedures" as shown in Fig. 4B to
derive the dissociation constants (Ki) for the
interaction between Rac1-GTP and Cdc42-GTP and respective effector
domains.
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 |
DISCUSSION |
In the present study, we have investigated the kinetic properties
of the GTPase reaction of Rac1 under catalysis of a panel of mammalian
GAPs, i.e. p50RhoGAP, p190, Bcr, and 3BP-1, and made comparisons with similar reactions of two closely related members of
Rho family GTPases, Cdc42 and RhoA, by using a quantitative assay
measuring
Pi release from Rac1-GTP in real time. The
data obtained show that there is no significant difference in kinetic mechanism between the GAP reactions of Rac1 involving these GAP molecules and that the four GAPs examined are approximately equally capable of acting as a negative regulator of Rac1. There are, however,
some significant differences between these GAP-catalyzed reactions of
Rac1 and those of Cdc42 and RhoA. For example, the fact that all four
GAPs can function efficiently to stimulate the GTPase activity of Rac1
is in contrast with the case for Cdc42, in which p50RhoGAP contains
over 50-fold supreme GAP activity over the other GAPs (32) and with the
case for RhoA, in which both p50RhoGAP and p190 are orders of magnitude
more potent than Bcr and 3BP-1 (43), reflecting distinct specificities
of these GAPs toward individual small G-proteins. In addition, by
comparing the affinities of the PBD of putative effectors,
i.e. PAK1, PAK2, and IQGAP1, to the active forms of Rac1 and
Cdc42, we found that while all three effectors can couple to Rac1-GTP
with dissociation constants at close to micromolar, PAK2 binds to
Cdc42-GTP with ~100-fold lower affinity than binding to Rac1-GTP.
These results suggest that while PAK1 and IQGAP1 may function as
potential targets to transduce signals from both activated Rac1 and
Cdc42, PAK2 is likely to be a specific effector for Rac1 instead of
Cdc42.
The similarities in kinetic parameters of the interactions of the four
GAPs with Rac1-GTP suggest that the basic reaction mechanisms involved
in activating Rac1 GTPase are analogous in these cases. The
Km values of the GAP reactions show good agreement
with the Kd values of GAP binding to Rac1-GMP-PNP, which is reminiscent of the Cdc42-GAP interactions (32) and the
RhoA-p190GAP and RhoA-p50RhoGAP interactions (43) and is consistent
with a mechanism of GAP-catalyzed GTP hydrolysis, which involves fast
equilibrium binding to Rac1 followed by a rate-limiting step of
Pi cleavage of bound GTP. This is also similar to the well characterized Ras-RasGAP interaction, for which it has been shown
that a fast equilibrium between Ras and RasGAP proceeds the
GAP-catalyzed bond cleavage reaction (36). Recently available x-ray
crystal structure of RhoA in a transition state complex with p50RhoGAP,
GDP, and AlF4
has highlighted the role
of a conserved arginine residue (Arg-85) in the GAP domains
contributing to the catalytic reactions of Rho GTPases (37). Indeed, in
three distinct GTPase-catalyzing machines, i.e. RhoGAP,
RasGAP, and a built-in domain of G
i, the arginine
fingers are found to converge at the same point of the GTPase
stabilizing
-phosphate oxygens and the leaving group, albeit from
different directions (38). It seems highly likely that Rac1 and the
GAPs adopt a similar mechanism of intermolecular interaction involving
an arginine finger in the catalytic core of the small GTPase.
The difference between the seemingly non-selective GAP activity of the
GAPs toward Rac1 and the extraordinary specificity demonstrated by
p50RhoGAP toward Cdc42 (32) and the preference of p190 for RhoA (43) is
likely to reside in the structural differences between Rho proteins. We
have shown in a recent study of the p190-RhoA interaction that the
specificity of this coupling involves unique structural determinants
outside of the switch I domain of the GTPase which is required for the
GAP reaction (20). The charged residue Asp-90 of RhoA, in particular,
makes over 10-fold contribution to the binding affinity and thereby specificity of p190 recognition. Comparison of the three dimensional structures of Rac1 and RhoA reveals that although they adopt highly conserved tertiary and secondary folding patterns, Rac1 displays a
relatively neutral surface, whereas RhoA shows a predominantly electronegative surface mostly due to aspartates at residues 13, 87, 90, and 124 (39, 40). The lack of unique salt bridges between Rac1 and
the GAPs, therefore, may partly account for the mediocre and similar
catalytic efficiencies and binding affinities of these GAPs toward
Rac1.
The in vivo functions of Rho GAPs remain to be determined,
but the kinetic parameters of Bcr and 3BP-1 are in agreement with previously observed phenotype in fibroblast cells (41, 42) and are
consistent with Bcr being a negative regulator of Rac function in
in vivo situations (21). Microinjections performed using
RhoGAP domains suggest that, whereas p50RhoGAP and p190 seem to be able
to inhibit the RhoA regulated actin-stress fiber formation in
fibroblast cells, both Bcr and 3BP-1 can down-regulate Rac-mediated
membrane ruffling (41, 42). It is not clear why p50RhoGAP or p190 was
not observed to inhibit Rac1 function under the microinjection
conditions given their similar in vitro behaviors toward
Rac1 as Bcr and 3BP-1. One possible explanation is that the 2-3
µM binding affinity and high catalytic efficiency of
p190GAP and p50RhoGAP for RhoA (43) compared with the over 10 µM affinity for Rac1 have caused a preferential
recognition and catalysis of RhoA GTPase by these GAPs, whereas the
down-regulation of Rac1 would require a higher threshold of the GAP
concentrations to be detected in cellular context.
The effector molecules responsible for the cellular functions of Rac1,
which include inductions of membrane ruffling and lamellipodia, focal
complex formation, and gene activation, remain unknown. However, the
two human p21-activated kinases, PAK1 and PAK2, and the RasGAP domain
containing actin-associated molecule IQGAP1 are likely candidates for
some of these effects of Rac1, given the recent findings that PAK can
induce both actin reorganization and the activation of a
mitogen-activated protein kinase cascade involving the Jun N-terminal
kinase (24-26) and that IQGAP1 can serve to induce bundling of
filamentous actins in cells (28). To quantify these potentially
important interactions involving Rac1 and to compare with that of
Cdc42, with which these Rac1 effector candidates share similar binding
profiles, we have measured the binding affinities of the PBDs of these
putative targets to Rac1-GTP and Cdc42-GTP under similar conditions.
Based on their ability to inhibit the intrinsic rates of GTP hydrolysis
upon binding to the small GTPase, we derived that PAK2 binds to Rac1 with a dissociation constant of 0.26 µM, PAK1 binds with
1-fold weaker affinity, and IQGAP1 binds with an ~8-fold weaker
affinity. These binding profiles fall within an affinity of the
micromolar range and are likely to bear physiological significance
comparing to other well characterized protein-protein interactions of
the Ras signaling pathway (29). By comparison, a marked difference of
binding profiles of these effector domains to Cdc42-GTP was observed.
IQGAP1 demonstrates the tightest binding with an affinity at 0.39 µM, PAK1 binds with an 2.9 µM affinity,
while PAK2 shows a much reduced affinity at 20.5 µM.
Therefore, in vitro, IQGAP1 and PAK1 may constitute as
potential effectors for both Rac1 and Cdc42, whereas PAK2 is likely to
be a specific effector for Rac1 instead of Cdc42. One important
question that must be addressed in the future is how small GTPases like
Rac1 differentially recognize these potential effectors, which differ
by up to 8-fold binding affinity under physiological conditions. It is
possible that distinct patterns of tissue distribution and/or cellular
localization of these effectors contribute to the occurrence of some of
the interactions leading to defined physiological effects and/or that
the interactions involving the effector domains are highly regulated in
the context of the full-length molecules in response to specific
stimulatory signals.
One of the criteria for being a candidate effector of small GTPases is
the relative tight binding capability to the activated form of GTPases.
The four Rho GAPs characterized in this study all contain a binding
affinity at above the 10 µM Kd value
to Rac1, comparing to the typical micromolar range of affinities of
PAKs and IQGAP1, suggesting that they may not be recognized by Rac1-GTP
as effectors. However, given the observed effect by one of the
Rac1-specific GAPs, n-chimaerin, which is able to act synergistically with Rac1 to induce lamellipodia formation in fibroblast cells (31), and the conditional effector functions demonstrated by RasGAP in certain biological systems, which also possesses an over 10 µM binding affinity toward Ras-GTP
(29), it remains to be seen if any of the Rho GAPs, especially in its full-length environment, can play an effector role in Rac1 signaling. How the interaction of Rac1 with multiple regulators and effectors, which may bear opposite effects on its guanine-nucleotide binding state, influences the outcome of Rac1 signaling represents another important area of future work.
We thank Dr. Andre Bernards for providing the
IQGAP1 cDNA.