Characterization of the Interactions between the Small GTPase Cdc42 and Its GTPase-activating Proteins and Putative Effectors
COMPARISON OF KINETIC PROPERTIES OF Cdc42 BINDING TO THE Cdc42-INTERACTIVE DOMAINS*

(Received for publication, April 16, 1997)

Baolin Zhang , Zhi-Xin Wang Dagger and Yi Zheng §

From the Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163 and Dagger  Institute of Biophysics, Academia Sinica, Beijing, China

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The small GTPase Cdc42 interacts with multiple factors to transduce diverse intracellular signals. The factors that preferentially recognize the GTP-bound, active state of Cdc42 include a panel of GTPase-activating proteins (GAPs), the Cdc42/Rac interactive binding (CRIB) motif-containing molecules, and the RasGAP domain containing IQGAP1 and IQGAP2. In the present study, we have determined the kinetic parameters underlying the functional interactions between the Cdc42-binding domains of some of these factors and Cdc42 by monitoring the continuous release of gamma Pi and have compared the ability of the domains to bind to Cdc42. The catalytic efficiencies (Kcat/Km) of the GAP domains of Bcr, 3BP-1, and p190 on Cdc42 are found to be 60-, 160-, and over 500-fold less than that of Cdc42GAP, respectively, and the differences are due, to a large part, to differences in Km. The Km values of the GAP domains compare well to the binding affinity to the guanylyl imidodiphosphate-bound Cdc42, suggesting a rapid equilibrium reaction mechanism. The affinity of the Cdc42-binding domains of the CRIB motif of Wiskott-Aldrich Syndrome protein and p21cdc42/rac-activated kinase 1, and the RasGAP-related domain of IQGAP1, which all inhibit the intrinsic rate of GTP hydrolysis of Cdc42, are found to be 4, 0.7, and 0.08 µM, respectively. These quantitative analysis provide insight that Cdc42GAP functions as an effective negative regulator of Cdc42 by fast, relatively tight binding to the GTP-bound Cdc42, whereas IQGAP1 interacts with Cdc42 as a putative effector with over 10-fold higher affinity than the CRIB domains and GAPs, and suggest that various GAPs and effectors employ distinct mechanism to play roles in Cdc42-mediated signaling pathways.


INTRODUCTION

The Rho family small GTP-binding protein Cdc42 appears to be an essential component in the transduction of intracellular signals that induce actin-cytoskeleton reorganization and gene activation (1-3). Genetic analysis of bud-site mutants revealed that Cdc42 is a key component in an actin-dependent polarization process during budding (4), as well as in mating (5) in Saccharomyces cerevisiae. The mammalian homolog of yeast Cdc42 has been shown to regulate the formation of the actin structure filopodia in Swiss 3T3 cells (6, 7) and cell polarity in T cells (8). Mutations of cdc42 in yeast (4) and introduction of Cdc42 mutants to mammalian cells (9) and to Drosophila (10) render large multinucleated cells, suggesting a possible role for Cdc42 in cytokinesis. Furthermore, constitutively activated form of Cdc42, like some of the other members of Rho family proteins (1-3), has been found to activate the Jun NH2-terminal kinase (11-14), the p70 S6 kinase (15), serum response factors (16), and DNA synthesis pathway (17).

In cells receiving the appropriate stimuli, Cdc42 GTPase is converted to the active GTP-bound state by guanine nucleotide exchange factors (18), whereas the GTP-bound form is in turn rendered inactive due to its intrinsic GTPase activity that is further stimulated by the GTPase-activating proteins (GAPs)1 (19). The RhoGAP family of proteins represents one of the major groups of regulators for the small GTPase, of which over 17 members have been identified to date (19). Primary sequence analysis revealed an ~170-amino acid homology region, designated as RhoGAP domain, in these proteins (20). Biochemical analysis of the RhoGAP activity indicated that this domain contains the minimum structural unit that is necessary and sufficient for the GAP activity (21). The RhoGAP domains share about 20-40% amino acid identity, and the proteins in which they are contained are generally large and multifunctional. The RhoGAP family members include Cdc42GAP (also known as p50RhoGAP), which may form a complex with SH3 domain-containing signaling molecules such as PI-3 kinase and Src through an additional proline-rich motif (22, 23); the breakpoint cluster region gene product (Bcr), which also possesses serine/threonine kinase (24) and guanine nucleotide exchange factor activities in its amino-terminal sequences (25); the RasGAP-binding phosphoprotein p190, which binds to GTP at its amino terminus (26, 27); and the SH3 domain-binding protein 3BP-1, which was identified by a screen for molecules interacting with the Abl oncoprotein with high affinity (28, 29). Recent x-ray crystallography studies of the RhoGAP domains of Cdc42GAP and p85, the regulatory subunit of PI-3 kinase, have provided further evidence that these critical domains involved in interaction with Rho GTPases adopt a highly conserved structural folding in three dimensions (30, 31).

The incoming signals of Cdc42 may bifurcate at the activated small G-protein itself through multiple effector targets to lead to the diverse biological effects. A panel of candidate effectors, identified by an overlay assay using the [gamma -32P]GTP-bound Cdc42 (32), turned out to be a class of novel protein serine/threonine kinases termed p21-activated kinases (PAKs) (33-35). Molecular cloning and data base searches led to the discovery of a family of proteins sharing a conserved Cdc42/Rac interactive binding (CRIB) motif of PAK (36), many of which have since been shown to be candidate effectors for Cdc42. The CRIB motif-containing putative effectors include PAK1, the kinase activity of which is activated upon binding to the active form of the GTPase (12, 34), and the Wiskott-Aldrich Syndrome protein (WASP), which binds to Cdc42-GTP and has been implicated in Cdc42-mediated actin polymerization process (37-39). Additional putative Cdc42 effectors have recently been identified by Cdc42-GTP affinity binding approach, and these include the IQGAP1 and IQGAP2, two RasGAP domain-containing proteins with selective affinity to the GTP-bound Cdc42 (40, 41). Moreover, in analogy to that suggested by the studies of p120RasGAP (42), the RhoGAPs may also serve as effectors of the small GTPases. Direct evidence of this comes from the findings that the RhoGAP domain of p85 can bind to the GTP-bound states of Cdc42 and Rac1 resulting in PI-3 kinase activation (43), and that the brain specific RhoGAP member n-chimaerin acts synergistically with Cdc42 and Rac1 to induce the formation of filopodia and lamellipodia (44).

The Cdc42-GAP and Cdc42-effector interactions have not been investigated in detail. In particular, little quantitative information of these seemingly important interactions has been made available and been compared. In the present work, we have developed a real-time spectroscopic method to study the kinetics of Cdc42-GTP hydrolysis in the context of its coupling to GAP domains of Cdc42GAP, Bcr, 3BP-1, and p190, to the CRIB motifs of PAK1 and WASP, and to the Ras-GAP related domain of IQGAP1. By an enzyme-coupled phosphorolysis reaction that allows direct measurement of gamma Pi release from Cdc42, we show that the differences of catalytic efficiency of GAPs reside largely in their different Km values, which compare well with their dissociation constants (Kd) for Cdc42-GTP. In addition, we have also obtained the affinity of Cdc42-GTP for the Cdc42-interactive domains of WASP, PAK1, and IQGAP1 based upon the property of the effectors to inhibit the intrinsic rate of GTP hydrolysis of Cdc42. The results indicate that Cdc42GAP functions as an effective negative regulator of Cdc42 by fast, relatively high affinity binding to the GTP-bound Cdc42, whereas IQGAP1 interacts with Cdc42 as a putative effector with over 10-fold higher affinity than the CRIB domains and GAPs, and suggest that various GAPs and effectors employ distinct mechanism to play roles in Cdc42-mediated signaling pathways.


EXPERIMENTAL PROCEDURES

Materials

GMP-PNP was from Boehringer Mannheim. GDP, GTP, bacterial purine nucleoside phosphorylase, and the ingredients to generate the phosphorylase substrate, 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), were purchased from Sigma. MESG was synthesized following the published protocol (45) and was dried to a yellow solid in a lyophilizer for storage at -20 °C. An estimated ~60% product of the final synthesis procedure was MESG, 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 (pH 7.6) (45).

Expression and Purification of Recombinant Proteins

Human Cdc42 protein was expressed in Escherichia coli as His6-tagged fusion by the pET expression system 28 (Novagen). Briefly, the cDNA of Cdc42 was cloned into pET28 vector at the in-frame BamHI-EcoRI sites, and the resulting construct was transformed into competent BL21 cells. The induction and purification of His6-tagged Cdc42 by isopropyl-1-thio-beta -D-galactopyranoside and Ni2+-charged agarose beads were carried out following the instructions provided by Novagen. The glutathione S-transferase (GST) fusion of GAP domains and the Cdc42-interacting effector domains of Cdc42GAP, Bcr, 3BP-1, p190, PAK1, WASP, and IQGAP1 were expressed in E. coli using the pGEX system (21). The GAP domains of Cdc42GAP, 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 Cdc42-interacting domains of human PAK1, WASP, and IQGAP1 contain amino acid residues 51-135, 215-295, and 864-1657, respectively. The GST-Cdc42GAP and GST-3BP-1 were generated as described previously (22, 29). The GST-Bcr, GST-p190, GST-PAK1, and GST-WASP constructs were made by polymerase chain reaction amplification of cDNAs corresponding to the indicated amino acid regions with primers that contain the BamHI and EcoRI sites, followed by digestion and ligation into the pGEX-KG vector. The GST-IQGAP1 was generated by cloning the BamHI-EcoRI fragment of cDNA (nucleotides 2703-5670) derived from pJOmega -IQGAP1 (a kind gift of A. Bernards) into pGEX-KG. Production and purification of the GST-fusions from E. coli were carried out as described by glutathione affinity chromotography (21). If necessary, the GST moiety of the fusions was cleaved by thrombin digestion, followed by incubation with p-aminobenzamidine immobilized on agarose beads (Sigma) to remove the thrombin (46).

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 judged at least 90% pure. Concentrations of the recombinant GAP and effector Cdc42-binding domains were determined using the BCA protein assay reagents from Pierce with bovine serum albumin (BSA) as standard, and the effective concentration of Cdc42 was measured using the MESG system under single-turnover conditions as described below.

Spectroscopic Measurements of GTPase Activity

The hydrolysis of GTP by Cdc42 was measured by the MESG system monitoring the absorbance increase of the reaction mixture at 360 nm, based on the method described by Webb and Hunter (47). Briefly, a 0.8-ml solution containing 50 mM HEPES, pH 7.6, 0.1 mM EDTA, 0.2 mM MESG, 10 units of purine nucleoside phosphorylase, 200 µM GTP, and the indicated amount of recombinant Cdc42 (>1 µM) was mixed in a 4-mm width, 10-mm pathlength cuvette, and the time courses of absorbance change at 360 nm were recorded using a Pharmacia Ultraspec III spectrometer equipped with compatible computer software for data manipulations. Single-turnover GTPase reactions were initiated by addition of MgCl2 to a final concentration of 5 mM. For measurements of GAP-catalyzed reactions, 5-50 µl of stock solution containing indicated amounts of GAP were added together with MgCl2 to the reaction mixtures. A control experiment in which Cdc42 was left out was carried out to provide a background of absorbance in each independent measurement to be subtracted from the sample signals. Moreover, experiments at a broad range of concentrations of GAPs were performed to minimize the possibility that the release of the reaction product, Pi, from Cdc42-GDP, rather than the GTPase reaction, is the rate-limiting step.

The concentrations of Pi in the reaction solution are proportional to the net absorbance change by a factor of extinction coefficient epsilon 360 nm = 11,000 M-1 cm-1 at pH 7.6 (47), and were used to determine the concentration of Cdc42 after one round of single-turnover reaction. Because the phosphorylase coupling reaction is extremely fast with a rate constant of 40 s-1 (45), the slope of the absorbance in the time course is treated as proportional to the rate of the GTPase activity of Cdc42: rate of Cdc42-GTP hydrolysis = 1/epsilon  × Delta A cm-1min-1.

Fluorescence Measurements of GTPase Activity

The fluorescence measurements were made using an SLM-Aminco Series 2 Luminescence Spectrometer. To monitor the tryptophan fluorescence emission of Cdc42, samples containing the indicated amount of Cdc42-GTP in the absence or presence (0.94 nM) of Cdc42GAP in a buffer with 50 mM HEPES, pH 7.6, and 10 mM MgCl2 were mixed and thermostated at 20 °C. The excitation was at 295 nm, and emission was at 330 nm. The increase of fluorescence intensity of a unique tryptophan residue (Trp-97) of Cdc42 due to the shift to a higher quantum yield of the GDP-bound state (48) was followed during the time course of GTP hydrolysis. The difference in fluorescence intensities (in defined arbitrary units) between the GTP- and GDP-bound forms of Cdc42 was calibrated by a factor of 48,000 M-1 cm-1. The background fluorescence as determined by omitting Cdc42 in the reaction mixture (which includes GAP and free GTP) was negligible comparing to the sample signal of Cdc42.

Filter-binding Assay of GTPase Activity

Recombinant Cdc42 was preloaded with [gamma -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 [gamma -32P]GTP-loaded Cdc42 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. At different time points the reaction was terminated as described (21) by filtering the reaction mixture through nitrocellulose filters, followed by washing with 10 ml of ice-cold buffer with 50 mM HEPES, pH 7.6, and 10 mM MgCl2. The radioactivity retained on the filters were then subjected to quantitation by scintillation counting. In the competition assays to determine the binding affinity of GAP domains to Cdc42, GST-GAP domains at the indicated concentrations together with the indicated amount of Cdc42 preloaded with GMP-PNP or GDP were present in the reaction mixture in addition to the components described including 5 nM Cdc42-[gamma -32P]GTP, and the reactions were terminated after a 5-min incubation at 20 °C.

Data Analysis

Kinetic data were analyzed by nonlinear regression using equations derived below with the program Enzfitter (Elsevier Biosoft). The rate constant (Kc) of intrinsic GTP hydrolysis by Cdc42 was determined by fitting data obtained by the MESG system, by tryptophan fluorescence, or by filter-binding assay to a single exponential function. Since the amount of Cdc42 present in the GAP reaction is in great excess of GAPs, a modified Michaelis-Menten equation was used to derive kinetic parameters assuming GAP acting as the enzyme catalyst, Cdc42-GTP as the substrate, and the Cdc42-GDP and Pi as the products.
V<SUB>0</SUB>=&egr; V<SUB><UP>max</UP></SUB>[<UP>Cdc</UP>42]<SUB>0</SUB>/(K<SUB>m</SUB>+[<UP>Cdc</UP>42]<SUB>0</SUB>)+&egr; K<SUB>c</SUB>[<UP>Cdc</UP>42]<SUB>0</SUB> (Eq. 1)
V0 is the initial rate of hydrolysis, epsilon  is the extinction coefficient at 360 nm for the phosphorylase reaction product, [Cdc42]0 is the total Cdc42 concentration, Kc is the rate constant of intrinsic GTPase activity, and the term epsilon Kc[Cdc42]0 is a correction for the rate of intrinsic GTP hydrolysis by Cdc42. Kcat is derived by Vmax/[GAP]0, with [GAP]0 representing total GAP concentration.

To determine the affinity of GAPs to Cdc42, the competition assay involving Cdc42-[gamma -32P]GTP, GST-GAP, and competitor C (GMP-PNP- or GDP-bound Cdc42) was described by two simultaneous reactions.
<UP>GAP</UP>+[&ggr;-<SUP>32</SUP><UP>P</UP>]<UP>GTP-Cdc</UP>42 ⇌ [&ggr;-<SUP>32</SUP><UP>P</UP>]<UP>GTP-Cdc</UP>42 · <UP>GAP ⇌ GAP</UP>+<UP>Cdc42-GDP</UP>+[&ggr;-<SUP>32</SUP><UP>P</UP>]<UP>P<SUB>i</SUB></UP>
<UP><SC>Reaction</SC> 1</UP>
<UP>GAP</UP>+<UP>C</UP> ⇌ <UP>GAP-C</UP>
<UP><SC>Reaction</SC> 2</UP>
Kd = [GAP][C]/[GAP-C]. The dissociation constants (Kd) were derived by fitting data to the following derived equation under the conditions that the total concentration of competitor [C]0 and reaction Km value are orders of magnitude larger than the concentration of Cdc42-[gamma -32P]GTP.
<UP>Inhibition of GAP-catalyzed GTP-hydrolysis %</UP>= (Eq. 2)
1−K<SUB>d</SUB>/(K<SUB>d</SUB>+[<UP>C</UP>]<SUB>0</SUB>)

To determine the affinity of effector domains to Cdc42-GTP, the GTPase-inhibitory reaction involving Cdc42-GTP and an effector domain (E) was treated as two simultaneous steps.
<UP>Cdc42-GTP ⇌ Cdc42-GDP</UP>+<UP>P<SUB>i</SUB></UP>
<UP><SC>Reaction</SC> 3</UP>
and
<UP>E</UP>+<UP>Cdc42-GTP ⇌ E-Cdc42-GTP</UP>
<UP><SC>Reaction</SC> 4</UP>
The binding constants of the Cdc42-binding domain of effectors to Cdc42-GTP were extracted by fitting data of the initial rate of GTP hydrolysis by Cdc42 at various concentrations of the effector domain to the derived equation, assuming that the rate of hydrolysis of Cdc42-GTP in complex with E is much slower than Cdc42-GTP alone.
V<SUB>0</SUB>=1/2&egr;K<SUB>c</SUB>(([<UP>Cdc</UP>42]<SUB>0</SUB>−K<SUB>i</SUB>−[<UP>E</UP>]<SUB>0</SUB>)+{(K<SUB>i</SUB>+[<UP>E</UP>]<SUB>0</SUB>+ (Eq. 3)
[<UP>Cdc</UP>42]<SUB>0</SUB>)<SUP>2</SUP>−4[<UP>E</UP>]<SUB>0</SUB>[<UP>Cdc</UP>42]<SUB>0</SUB>}<SUP>1/2</SUP>)
V0 and [E]0 are the initial rate of GTP hydrolysis and the total concentration of effector domain added to the assay, respectively, and Ki represents the binding affinity (dissociation constant) of the effector domain to Cdc42-GTP.


RESULTS

A Spectroscopic Assay for gamma Pi Release from Cdc42-GTP

The MESG/phosphorylase assay was developed to measure the kinetics of Pi release from GTPases and ATPases (45), and has been applied to the studies of Ras and RasGAP interaction (47). To determine the suitability of this assay system for measuring the Cdc42-GTP hydrolysis, the time course of intrinsic Cdc42 GTPase reaction as reflected by the absorbance trace of gamma Pi release was monitored in the presence of phosphorylase and MESG substrate (Fig. 1A). In the presence of 0.1 mM EDTA when nucleotide exchange occurs fast, Cdc42 undergoes multiple turnover resulting in a linear increase of Pi release. Upon the addition of 5 mM final concentration of MgCl2, Cdc42 is stabilized at the GTP-bound state due to the presence of excess GTP (200 µM) and starts single-turnover GTP hydrolysis. The response of the absorbance increase is linear up to 70 µM Cdc42 (data not shown), and the conditions used here for the loading of GTP and the initiation of single-turnover GTPase reaction were adopted for all subsequent measurements.


Fig. 1.

A, continuous spectroscopic assay of gamma Pi release from the Cdc42-GTP complex measured by absorbance at 360 nm with the MESG assay system. The absorption of 8.0 µM solution of Cdc42 in a buffer containing 50 mM HEPES, pH 7.6, 0.1 mM EDTA, 200 µM GTP, and MESG with 10 units of coupling phosphorylase was monitored during the time course. Reactions were initiated by the addition of 5 mM MgCl2. Arrow indicates the time point at which a final concentration of 5 mM MgCl2 was added to the reaction mixture. The dashed line represents the period of addition and mixing. B, comparison of the time courses for single-turnover GTP hydrolysis by Cdc42 measured by the MESG system (squares), the tryptophan fluorescence change (diamonds), and the [gamma -32P]GTP filter-binding assay (triangles). Experimental conditions for each assay were as described under "Experimental Procedures." Data of GTP hydrolysis were best fitted into a single exponential to derive the intrinsic rate constant of Cdc42 (Kc). C, temperature dependence of the intrinsic GTPase activity of Cdc42. gamma Pi release from 8.0 µM Cdc42 was monitored by the MESG system during the time couse at 20 °C, 30 °C, and 37 °C, respectively, upon the addition of 5 mM MgCl2 as indicated by the arrow.


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To ensure that the observed rate of Cdc42-GTP hydrolysis measured by the MESG/phosphorylase method is valid, the time course for gamma Pi-elicited absorbance change was compared with that obtained by two other established GTPase assays for Cdc42, the [gamma -32P]GTP filter binding and the Cdc42 tryptophan fluorescence methods (Fig. 1B). The time courses of Cdc42-GTP hydrolysis at 20 °C measured by the three different assays show excellent agreement, and fittings of the data to a single exponential function yield intrinsic rate constants (Kc) of 0.073, 0.075, and 0.065 min-1 by the MESG/phosphorylase, the fluorescence, and the filter binding methods, respectively (Table I). These data validify the MESG/phosphorylase assay as a reliable means for measurement of the GTPase activity of Cdc42.

Table I. Rate constants of intrinsic GTPase activity of Cdc42 determined by different GTPase assays

The GTPase activity of Cdc42 was measured by the three different methods under otherwise identical conditions as described under "Experimental Procedures," and the traces of GTP-hydrolysis depicted in Fig. 1B were fitted into a single exponential function to derive the intrinsic rate constant Kc. GTP-hydrolysis of Cdc42 measured at different temperatures by the Pi/MESG method as depicted in Fig. 1C were also analyzed similarly. ND, not determined. Results are representative of three independent experiments.

Assay Kc
20 °C 30 °C 37 °C

min-1
Pi release/MESG 0.073  ± 0.001 0.120 ± 0.004 0.193 ± 0.005 
Tryptophan fluorescence 0.075  ± 0.002 ND ND
Filter binding 0.065  ± 0.001 ND ND

To see how Cdc42 would behave at more physiological temperatures, the rate of GTPase activity of Cdc42 was determined at 20, 30, and 37 °C (Fig. 1C). A ~1.6-fold increase in was observed when temperature was raised from 20 °C to 30 °C, or from 30° to 37 °C, and the GTP hydrolysis occurs ~2.6-fold faster at 37 °C than at 20 °C (Table I). To allow accurate assessment of the initial rates of GAP-stimulated GTP hydrolysis, all subsequent GAP-catalyzed GTPase assays were performed at 20 °C, which provides an optimal window of absorbance increase.

Kinetics of Interaction of Cdc42-GTP with Cdc42GAP

To determine the kinetics of Cdc42GAP-stimulated hydrolysis by Cdc42, we first made sure that the amount of Cdc42GAP employed is within the linear range with regard of the GAP-catalyzed rate increase. Fig. 2A shows the absorbance traces for three different reaction with the same amount of Cdc42 but by addition of different amounts of Cdc42GAP to initiate the reactions. The initial rates of the reactions were linear with Cdc42GAP concentrations up to 4 nM (Fig. 2A, insert). In the following experiments to investigate the interaction of Cdc42GAP with Cdc42-GTP, the initial rate of Pi release was measured as a function of Cdc42 concentration at a fixed concentration of 3.6 nM Cdc42GAP (Fig. 2B). Since the amount of Cdc42 is in far excess of Cdc42GAP, the reaction can be treated by Michaelis-Menten kinetics with the adjustment for the intinsic rate of GTP hydrolysis by Cdc42 (see Equation 2 in the Experimental Procedures). The data were fitted to give a Km of 3.08 µM and a Vmax 2.02 µM/min (Fig. 2C), and the Kcat value of 2103.9 min-1 was derived thereon (Table II). The assumption that all GAPs purified were active was applied when calculating Kcat values in the current study, although this may lead to an underestimation if a fraction of the GAP was rendered inactive during the preparation procedures; there is currently no established method to determine this. Additional experiments were carried out with GST-Cdc42GAP fusion protein to see if the GST moiety in GST-GAP fusions may affect the kinetics of the reaction. Essentially identical results were obtained (Km value at 2.96 ± 0.98 µM, and at 2151 ± 198 min-1), indicating that the GST portion of the fusion does not interfere with the GAP-Cdc42 interaction. Thereafter we used GST-fused GAPs for further investigation.


Fig. 2.

Determination of kinetic parameters of Cdc42GAP by the MESG system. A, time courses of gamma Pi release from Cdc42-GTP stimulated by different doses of Cdc42GAP. Reaction conditions were similar to in Fig. 1A at 20 °C, and the reactions were initiated by adding Cdc42GAP at nM concentrations indicated together with MgCl2 (arrow). Inset, initial rate of Pi release from the Cdc42-GTP complex as a function of the Cdc42GAP concentration. B, time courses of GTP hydrolysis by Cdc42 in the presence of 3.6 nM Cdc42GAP at three different Cdc42 doses (µM) as indicated. Arrow indicates the time when Cdc42GAP and MgCl2 were added. C, rate of GTP hydrolysis by Cdc42 as a function of Cdc42 concentration. The intrinsic rate of GTP hydrolysis (in the absence of GAP, squares) was best-fitted into a linear function with a slope of Kc, and the Cdc42GAP-catalyzed GTPase rates (triangles) were fitted to the modified Michaelis-Menten equation (Equation 1 under "Experimental Procedures") to give the Km and Vmax values of the reaction.


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Table II. Kinetic parameters of GAPs that regulate Cdc42 under single turnover conditions

GAP activities of Cdc42GAP, Bcr, 3BP-1, and p190 analyzed in Figs. 2, 3, 4 by nonlinear regression yielded the Vmax and Km values listed, and the Kcat and Kcat/Km were derived thereon. Data represent results obtained by the MESG/phosphorylase method unless otherwise indicated. 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 0.96 nM, 11.4 nM, 34.7 nM, and 36.3 nM for Cdc42GAP, Bcr, 3BP-1, and p190, respectively, by the MESG/phosphorylase method, and 0.94 nM Cdc42GAP by the fluorescence method. These concentrations of GAPs fell within the linear range of the absorbance or fluorescence increase in response to increasing doses of GAPs. Results are representative of at least three independent measurements.

GAP Vmax Km Kcat Kcat/Km

µM/min µM min-1 min-1 µM-1
Cdc42GAP 2.02  ± 0.09 3.08  ± 0.48 2103.9  ± 93.7 683.1  ± 30.4
Cdc42GAPa 2.38  ± 0.10 2.61  ± 0.29 2578.3  ± 108.3 987.8  ± 41.5
Bcr 3.02  ± 0.16 23.72  ± 3.2 265.5  ± 14.1 11.2  ± 0.6
3BP-1 7.55  ± 0.77 50.47  ± 9.42 217.3  ± 22.2 4.3  ± 0.4
p190 2.52  ± 0.32 59.19  ± 12.41 69.4  ± 8.8 1.2  ± 0.2

a Determined by the tryptophan fluorescence method.

To compare the kinetic parameters obtained by the MESG/phosphorylase assay with that by the fluorescence method, we also determined the initial rate of tryptophan fluorescence increase, which correlates with the rate of GTP hydrolysis at increasing concentrations of Cdc42-GTP (Fig. 3). The best fit of the data yielded a Km of 2.61 µM and a Vmax of 2.38 µM/min, and Kcat was calculated to be 2578 min-1. These are in good agreement with the kinetic constants determined by the MESG/phosphorylase system (Table II), thus further legitimize the use of the MESG/phosphorylase assay to examine other Cdc42-GAP or Cdc42-effector interactions described below.


Fig. 3. Single-turnover kinetic analysis of the interaction between Cdc42-GTP and Cdc42GAP by tryptophan fluorescence method. The fluorescence measurements were carried out as described under "Experimental Procedures," the initial rates of tryptophan fluorescence change of the Cdc42-GTP resulting from the hydrolysis of GTP when mixed with 0.94 nM Cdc42GAP were determined at increasing concentrations of Cdc42-GTP, and data were best-fitted into the modified Michaelis-Menten equation (Equation 1).
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Kinetics of Interaction of Cdc42-GTP with the GAP Domains of Bcr, 3BP-1, and p190

The interactions of the GAP domains of Bcr, 3BP-1, and p190 with Cdc42-GTP were examined by the MESG/phosphorylase system and compared with that of the Cdc42GAP. Similar to that shown in Fig. 2, Fig. 4 shows the initial rates of Pi release measured as a function of concentrations of Cdc42-GTP at fixed concentrations of GST-GAP domains. Data analysis revealed the Km values of Bcr, 3BP-1, and p190 to be 23.72, 50.47, and 59.19 µM, and the Vmax values to be 3.02, 7.55, and 2.52 µM/min, respectively (Table II). The catalytic efficiencies of the GAP domains of Bcr, 3BP-1, and p190 are at least 60, 160, and over 500-fold less than that of the GAP domain of Cdc42GAP, respectively, and the difference in Km seems to be a major factor for the varied ability of the GAPs to stimulate GTP hydrolysis of Cdc42.


Fig. 4. Determination of the single-turnover kinetic parameters of the GAP domains of GST-Bcr, GST-3BP-1, and GST-p190. The initial rates of GTP hydrolysis as monitored by the gamma Pi release with the MESG system were measured with a constant amount of the GAP domain of Bcr (11.4 nM, diamonds), 3BP-1 (34.7 nM, solid squares), or p190 (36.3 nM, triangles) and increasing concentrations of Cdc42-GTP, similar to that described in Fig. 2. Km and Vmax values were extracted by nonlinear fitting as in Fig. 2C. The intrinsic rates of GTP hydrolysis (open square) were fitted into a linear function to yield Kc.
[View Larger Version of this Image (23K GIF file)]

Cdc42 Binding to GAPs

The interaction of GAPs with Cdc42 at steady state was investigated by measuring the ability of GMP-PNP-bound (a nonhydrolyzable GTP anolog) or GDP-bound Cdc42 to inhibit competitively the GAP-stimulated hydrolysis of [gamma -32P]GTP-bound Cdc42. The concentration range in which Cdc42-GMP-PNP or Cdc42-GDP inhibits GAP-mediated GTP hydrolysis is an indication of its affinity (Kd) for GAP. By fitting data to Equation 3 described under "Experimental Procedures" (Fig. 5), we determined that Cdc42GAP bound to Cdc42-GMP-PNP with an Kd of 2.78 µM, Bcr bound with an Kd of 24 µM, 3BP-1 bound with an Kd of 46.6, whereas p190 bound with the lowest affinity of 55 µM (Table III). These Kd values compare well with the Km values obtained for the GAPs (Table II), suggesting a rapid equilibrium binding of GAPs to Cdc42-GTP, which is not a rate-limiting step in the GAP-catalyzed reactions. The correlation between catalytic efficiency and binding affinity of the GAPs examined suggests that tight binding of a GAP domain may contribute to the stabilization of a transition state of Cdc42 in facilitating GTP hydrolysis.


Fig. 5. Interaction of GAP domains with Cdc42 bound to GMP-PNP measured by inhibition of GAP-stimulated Cdc42-[gamma -32P]GTP hydrolysis. ~5 nM Cdc42-[gamma -32P]GTP together with 2 nM Cdc42GAP (solid triangles), 14 nM Bcr (squares), 30 nM 3BP-1 (diamonds), or 34 nM p190 (open triangles) were present in the assay buffer containing 50 mM HEPES, pH 7.6, 0.2 mg/ml BSA, and 5 mM MgCl2 when Cdc42 preloaded with GMP-PNP at the indicated concentrations was added. The reactions were terminated after 5 min at 20 °C.
[View Larger Version of this Image (21K GIF file)]

Table III. Affinity of GAPs for Cdc42

The competition reactions were carried out under conditions described in Fig. 5. The dissociation constants (Kd) were derived by fitting data such as shown in Fig. 5 to Equation 2 under "Experimental Procedures." Data are representative of two independent measurements.

GAP Kd
GMP-PNP GDP

µM
Cdc42GAP 2.78  ± 0.06 >500 
Bcr 24.05  ± 0.04 266  ± 13
3BP-1 46.6  ± 1.5 >1000
p190 55.2  ± 1.1 127  ± 6

To see if there is an effect of product inhibition in the time courses of GAP-stimulated GTP hydrolysis, we measured the affinity of Cdc42-GDP to various GAPs in a similar assay, and found that both Cdc42GAP and 3BP-1 bound to Cdc42-GDP with negligible affinity, while Bcr and p190 recognize Cdc42-GDP with ~100-fold and ~2-fold lower affinity, respectively, than the GMP-PNP-bound Cdc42 (Table III). We therefore conclude that there were minimal, if any, Cdc42-GDP-inhibitory effect on the GAP reactions of Cdc42. This is in contrast with the GAP-catalyzed reaction of RhoA in which the reaction product RhoA-GDP demonstrates a comparable affinity to GAP as the reactant Rho-GTP,2 suggesting a diversity of the mechanism of GAP-stimulated GTP hydrolysis among Rho proteins.

Interaction of Cdc42-GTP with Cdc42-binding Domains of Putative Effectors

We are interested in examining the interactions of putative effectors with Cdc42 and making comparisons with that of the GAPs, given that some RhoGAPs have been speculated to act as effectors for the G-protein function (43, 44). The CRIB domain of rat alpha -PAK and the RasGAP-related domain of IQGAP1 were previously observed to inhibit both the intrinsic and GAP-stimulated GTP hydrolysis of Cdc42 (33, 40), and recently, it was reported that the CRIB domain of mouse PAK3 has a similar effect on Cdc42 (49). To determine the direct binding affinity of the effector domains to Cdc42-GTP, we conducted GTPase assays of Cdc42 in the presence of various doses of purified Cdc42-binding domains of IQGAP1, PAK1, and WASP (Fig. 6). As shown in Fig. 6A, the intrinsic GTPase activity is inhibited by the addition of IQGAP1, and this inhibitory effect is dependent on the concentrations of the effector. The initial rates of GTP hydrolysis as a function of the concentrations of the inhibitory effector domains were fitted by a nonlinear regression (Equation 3) to extract the binding constants (Ki) to Cdc42-GTP (Fig. 6B), and Ki values of 0.082, 0.78, and 4.15 µM for IQGAP1, PAK1, and WASP, respectively, were obtained (Table IV). Thus, IQGAP1 seems to bind to Cdc42-GTP with ~10-fold higher affinity than PAK1, and ~50-fold higher affinity than WASP. When the interaction of GAP domains with Cdc42 are compared with these effector domains, Cdc42GAP is the only GAP tested to fall in the range of effector interaction affinity (approximately micromolar range), while Bcr, 3BP-1, and p190 interact with Cdc42 at least 5-fold less tightly than any of the effectors examined.


Fig. 6. Determination of the affinities of putative effector-domain binding to Cdc42 by inhibition of GTP hydrolysis of Cdc42. A, time courses of Cdc42-GTP hydrolysis in the presence of various amount of Cdc42-binding domain of IQGAP1 under single-turnover conditions measured by the MESG system. Reaction conditions were similar to that in Fig. 1. 6.5 µM Cdc42 and the indicated amount of IQGAP1 domain were present when reaction was initiated by the addition of MgCl2. B, initial rates of GTP hydrolysis of Cdc42 as a function of effector domain concentrations. 6.5 µM Cdc42 and the effector domain of IQGAP1 (squares), PAK1 (diamonds), or WASP (triangles) were present. Data were fitted to Equation 3 described under "Experimental Procedures" to derive the binding constants.
[View Larger Version of this Image (16K GIF file)]

Table IV. Affinity of the Cdc42-binding domain of effectors for Cdc42

The GTPase-inhibitory reactions of the effector domains were carried out as in Fig. 6A. The initial rate of GTP hydrolysis as a function of the effector domain was treated by nonlinear regression fitting to Equation 3 under "Experimental Procedures," as shown in Fig. 6B to derive the dissociation constants (Ki) for the interaction between CDC42-GTP and the respective efector domains.

Effector domain Ki

µM
IQGAP1 0.082  ± 0.033
PAK1 0.78  ± 0.10
WASP 4.15  ± 0.06


DISCUSSION

Increasing evidence has emerged implicating Cdc42 as an essential component in a variety of cellular transduction pathways; this prominent member of Rho family small GTPases mediates the reorganization of actin polymerization process in mammalian and yeast cells and regulates critical cellular processes such as mitogen-activated kinase kinase (Fus3/Kss1 in yeast and JNK/p38 in mammals) activation, p70 S6 kinase activation, and cell cycle progression (1-3), and may also have a role in mammalian development (50). Studies aimed at understanding the biochemical mechanisms underlying the biological effects of Cdc42 have led to the discovery of numerous regulators and potential effector targets, which specifically interact with the activated GTP-bound form of Cdc42 (19, 36) and may serve to down-regulate or initiate one or more pathways bifurcating at the GTPase itself. In this paper we describe the quantitations of some of the interactions involving Cdc42 that have been implicated in the regulation or transduction of relevant signals, and provide for the first time a direct comparison of the kinetic and steady state parameters of the interactions with four mammalian RhoGAPs (Cdc42GAP, Bcr, 3BP-1, and p190) and three putative effectors (IQGAP1, PAK1, and WASP). Our data show that both the GAPs and the effectors interact with Cdc42-GTP by a fast equilibrium mechanism, and the affinities of the interaction correlate with the catalytic GAP activities of the GAPs or the GTPase-inhibitory activities of the effectors. The broad spectrum of binding affinities of these cellular factors to Cdc42 (Kd varying from 80 nM to 50 µM) suggest that they employ distinct mechanism to play roles, if any, in Cdc42-mediated signaling pathways.

Due to the relatively fast intrinsic GTP hydrolysis rate of Cdc42 (10-fold faster than Ras), the interactions of Cdc42 with GAPs and with putative effectors, which display GTPase-inhibitory protein function on Cdc42 similar to that by the GDP dissociation inhibitors in this respect (51), can be readily measured by the change in gamma Pi release. The MESG/phosphorylase assay allows us to monitor in real time the GTP hydrolysis process by converting the Pi signal to an absorbance change at 360 nm, and offers a few advantages over two other available methods to measure GTPase activity of Cdc42; it provides faster, continuous, and more quantitative trace of GTP hydrolysis compared with the [gamma -32P]GTP filter binding method, and it does not suffer from the possible interference by inner filter effect at high concentrations of fluorophore (i.e. tryptophan residue of Cdc42) or by nonphysiological artifact of fluorescent GTP-analogs such as the N-methylanthraniloyl-GMP-PNP of the fluorescence method (48, 49). As demonstrated in Figs. 1 and 3, this assay yielded similar kinetic parameters of the intrinsic GTPase activity as well as the GAP-stimulated GTPase activity of Cdc42 as that measured by the radioactive filter-binding and/or the tryptophan fluorescence methods (when the Cdc42 concentration is less than 10 µM), thus establishing it as a reliable, easy-to-perform, and quantitive method to study the molecular interactions involving Cdc42 and other Rho family small GTPases. More attractively, the MESG/phosphorylase assay can be utilized to study the molecular interplays of multiple regulatory proteins of Rho family GTPases under multiple turnover conditions, e.g. when the activation of the small G-proteins is provided by guanine nucleotide exchange factors (Dbl-like proteins) and the deactivation or transduction of signals is mediated by GAPs or effectors.3

Consistent with previous qualitative estimations (21-23), our quantitative analysis of Cdc42-GAP interactions indicates that Cdc42GAP represents the most active GAP specie toward Cdc42, with a catalytic efficiency (Kcat/Km) at least over 60-fold higher than the other GAPs been examined. Given its wide spectrum of tissue distributions, it is likely that Cdc42GAP may serve as one of the major negative regulators of Cdc42 signaling pathway, whereas the significantly lower catalytic efficiencies and binding affinities of 3BP-1 and p190 (~160- and ~600-fold lower, and ~16- and ~19-fold less tight, respectively) suggest that they are probably not involved in direct regulation of Cdc42. So far, in vivo evidence of cellular functions of the GAPs has been lacking. Microinjections performed using the GAP domains suggest that while both Bcr and 3BP-1 can down-regulate Rac-mediated membrane ruffling, Cdc42GAP and p190 seem to be able to inhibit the Rho-regulated actin-stress fiber formation in fibroblast cells (29, 52). Our results that the GAP domains of Bcr and 3BP-1 contain supreme catalytic efficiency toward Rac with Km values at 3-5 µM, and p190 and Cdc42GAP have Km values of 2-3 µM for Rho2 are consistent with these observations in cells. But it remains unclear what effect these GAPs may bear on the Cdc42-regulated processes such as filopodia formation and p70 S6 kinase and JNK/p38 kinase activations.

The mechanism of GAP activation of Cdc42 GTPase activity appears to be similar to that of Ras-RasGAP interaction, i.e. a fast equilibrium binding of GAP to the GTPase followed by GTP hydrolysis (53). The Km values of GAPs examined are in excellent agreement with the Kd values observed under steady state conditions, indicating that GAP binding is not the rate-limiting step. However, the current data do not indicate further which of the consequent steps of gamma Pi cleavage and product Pi release is rate-limiting. Although experimental conditions have been carefully chosen to ensure that the reaction kinetics favor the GTP hydrolysis step, it remains possible that Pi release may become rate-limiting at very high GAP concentrations. The role of GAP in the activation of GTPase reaction may be to stabilize a particular transition state conformation to await for the gamma -phosphate cleavage by intrinsic reactive groups of Cdc42, or to supply active site residues important for efficient catalysis, both of which have supporting evidence in the case of RasGAP action (53-55). Our data do not distinguish between these possibilities.

The binding of putative effectors to Cdc42, including PAK1, WASP, and IQGAP1, invariably led to the inhibition of the relatively fast intrinsic rate of GTP hydrolysis. It is speculated that this GTPase-inhibitory protein activity accompanying the binding and activation of downstream effectors may bear physiological relevance by prolonging the time at which Cdc42 remains active (33, 40). We have taken advantage of this property of effector-Cdc42 interaction to derive a direct binding constant for the Cdc42-binding domains of PAK1, WASP, and IQGAP1, and found that the RasGAP-like domain of IQGAP1 contains over 10-fold higher affinity toward Cdc42-GTP than the CRIB domains of PAK1 and WASP (Table IV). The significant difference (over 7-fold) in binding affinity of the two CRIB domains also suggest that structural variability in the CRIB motif and the residues surrounding it may have an effect on its Cdc42-binding function. Given the demonstrated role of WASP in Cdc42-mediated cytoskeleton regulation (39), it may also be inferred that either functional coupling can occur between Cdc42-GTP and its effector at µM affinity, or WASP may be further regulated under physiological conditions to achieve higher affinity for Cdc42. In light of the recent finding that certain RhoGAPs may contribute to the effector functions of small GTPases (43, 44), a direct comparison of the binding affinities of these effector domains with that of GAP domains measured in this study seems to indicate that, while it is possible for Cdc42GAP to act as a putative effector for Cdc42 with a Kd similar to that of WASP at µM range, it is less likely that 3BP-1, or p190 functions to transduce signals from Cdc42 with a Kd value at over 50 µM. However, the relatively low binding affinity of some GAPs such as Bcr does not rule out their role in down-regulation or effector function of Cdc42 if the case for RasGAP, which has a Kd of ~20 µM for Ras, can be brought up as a comparison (56, 57).

It is important to note that current studies utilizing the limited Cdc42-interactive domains of GAPs and effectors provide only a simplified model which may differ from the situations in which full-length molecules are likely to behave. Recently, the amino-terminal sequences of p120RasGAP containing the SH2 and SH3 domains were found to be required for the full catalytic GAP activity toward Ras (58). Along these lines, many of the RhoGAPs and effectors are large and multifunctional, therefore a much more complicated intra- or intermolecular interactions involving additional structural motifs of the molecules may contribute to the regulation of binding and/or catalytic interaction with Cdc42, in analogy to RasGAP. For example, we have previously observed that the carboxyl-terminal fragment of Bcr containing the GAP domain is a much more active GAP than the full-length Bcr immunoprecipitated from cell lysates (21), and the GAP activity of the RhoGAP member n-chimaerin has been reported to be subjected to regulation by lipid-binding (59). Thus, to measure and compare the binding and catalytic activities of intact RhoGAPs and effectors to Rho family GTPases will be of considerable interests in the future.

In summary, the data presented here provide direct comparison of the binding and catalytic activities of a panel of mammalian RhoGAP domains and putative effector domains to Cdc42. The superb catalytic efficiency of Cdc42GAP and high binding affinity of IQGAP1 constitute them as potentially important players in the regulation or transduction of Cdc42-mediated signals.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant GM53943 and American Cancer Society Grant RPG-97-146-01 (to Y. Z.).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}utmem1.utmem.edu.
1   The abbreviations used are: GAP, GTPase-activating protein; Bcr, the breakpoint cluster region gene product; BSA, bovine serum albumin; GMP-PNP, guanylyl imidodiphosphate; GST, glutathione S-transferase; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside; PAK, p21cdc42/rac-activated kinase; WASP, the Wiskott-Aldrich Syndrome protein; CRIB, Cdc42/Rac interactive binding.
2   B. Zhang and Y. Zheng, unpublished results.
3   Baolin Zhang and Yi Zheng, unpublished observation.

ACKNOWLEDGEMENTS

We thank Drs. Piera Cicchetti, Rong Li, Jonathan Chernoff, and Andre Bernards for providing the cDNAs of 3BP-1, WASP, PAK1, and IQGAP1. We also thank Dr. Bruce Martin for helpful discussion.


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