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Originally published In Press as doi:10.1074/jbc.M408113200 on September 7, 2004

J. Biol. Chem., Vol. 279, Issue 47, 48495-48504, November 19, 2004
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Phosphorylation of IQGAP1 Modulates Its Binding to Cdc42, Revealing a New Type of Rho-GTPase Regulator*

Katarina Grohmanova{ddagger}, Dominik Schlaepfer{ddagger}, Daniel Hess§, Peter Gutierrez{ddagger}, Matthias Beck{ddagger}, and Ruth Kroschewski{ddagger}

From the {ddagger}ETH-Zürich, Institute of Biochemistry, Schafmattstrasse 18, Zürich 8093, Switzerland and §Friedrich Miescher Institute, Basel 4002, Switzerland

Received for publication, July 19, 2004 , and in revised form, August 31, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Rho-GTPase Cdc42 is important for the establishment and maintenance of epithelial polarity. Signaling from Cdc42 is propagated via its effector molecules that specifically bind to Cdc42 in the GTP-bound form. The cell-cell contact regulator and actin-binding protein IQGAP1 is described as effector of Cdc42 and Rac. Unexpectedly, we show in this study that IQGAP1 bound also directly nucleotide-depleted Cdc42 (Cdc42-ND). This interaction was enhanced in the presence of phosphatase inhibitors and in epithelial cells without cell-cell contacts. Tandem mass spectrometry analysis and immunoprecipitation experiments revealed that IQGAP1 was Ser1443-phosphorylated in vivo, potentially by protein kinase C{epsilon} and upon loss of cell-cell contacts. In addition, we identified two independent domains of the IQGAP1 C terminus that bound exclusively Cdc42-ND. These domains interacted with each other, favoring the binding to Cdc42-GTP. Moreover, phosphorylation on Ser1443 strongly inhibited this intramolecular interaction. Thus, we unraveled a molecular mechanism that reveals a novel type of Rho-GTPase regulator. We propose that, depending on its phosphorylation state, IQGAP1 might serve as an effector or sequester nucleotide-free Cdc42 to prevent signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Epithelial polarity is regulated by the Rho-GTPase Cdc42, which is implicated in signal transduction, gene regulation, cell cycle progression, and cytoskeletal regulation (1). In epithelial cells, Cdc42 regulates establishment and maintenance of basolateral polarity by controlling endocytic and secretory trafficking (2–4). The basolateral membrane of epithelial cells is formed upon establishment of cell-cell contacts. Trans-activation of nectins and formation of E-cadherin mediated cell-cell contacts lead to the activation of Cdc42 (5, 6). However, how cell-cell contact formation favors the activation of Cdc42 is not known.

The most direct activators of GTPases are guanine nucleotide exchange factors (GEFs),1 which are characterized by four properties. GEFs bind to a GDP-bound GTPase, they catalyze the displacement of GDP and stabilize the resulting nucleotide-free GTPase. Due to the high ratio of GTP to GDP in cells, the nucleotide-depleted GTPase-GEF complex dissociates into GEF and GTP-bound (active) GTPase. The active GTPase can then bind selectively to effectors and elicit downstream effects.

Activation of GEFs for Cdc42 at sites of cell-cell contacts might involve several modes. Some GEFs are activated by phosphorylation, which abolishes intramolecular inhibition (7–9). Other GEFs need additional factors to exhibit GEF activity. For example, interaction of intersectin with the Cdc42 effector protein N-WASP causes the activation of intersectin GDP displacement activity (10). Also, interaction of DOCK180 with the protein ELMO might be necessary for its activation as a GEF for Rac (11, 12).

Mammalian IQGAPs are scaffolding proteins with conserved homologues in budding yeast, Dictyostelium, and Caenorhabditis elegans and have been shown to regulate actomyosin ring formation during cytokinesis, cell motility, and cell-cell contacts (13). Despite the presence of a GAP homology domain in the C terminus, no GAP activity could be demonstrated (14). However, IQGAP1 is known as a potential effector of Cdc42 and Rac. In several biochemical studies, the effect of Cdc42-GTP or Rac-GTP on IQGAP1 interaction properties was described, but the in vivo consequences are less well characterized (14–17). Several areas were identified where IQGAP1 plays a role. IQGAP1 can directly bind and cross-link actin filaments in dependence on Cdc42-GTP (15, 18). Cdc42-GTP abolishes the binding of IQGAP1 to E-cadherin and {beta}-catenin, increasing cell-cell adhesion (16, 19). IQGAP1 was also shown to capture microtubule plus ends with activated GTPases Cdc42 and Rac1 via the microtubule-associated protein CLIP-170 (20), and finally, it seems to be connected to the regulation of membrane trafficking in gastric parietal cells (21).

In this study, we investigated the mechanism of IQGAP1 interaction with Cdc42. We demonstrate that IQGAP1 represents a novel type of interactor for Rho-GTPases. Besides its binding to Cdc42-GTP, it could also interact with Cdc42-ND. This later binding was increased by phosphorylation and by structural opening of the IQGAP1 C terminus and in the absence of cell-cell contacts. Therefore, we unraveled a new level of regulation for the interaction of IQGAP1 with Cdc42. Our data also suggest that IQGAP1 might act as negative regulator of Cdc42 in single cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Antibodies—pGEX-6p1-IQGAP1-C1, -C2, and -C3 and pGEX-6p1-IQGAP1-C2-S/E and pGEX-6p1-IQGAP1-C3-S/E (encoding GST fusion proteins) originate from PCRs on human IQGAP1 and PCR-based mutagenesis followed by in vivo recombination, respectively (see Tables I and II). Briefly, PCR fragments of mutated GST-C2 and -C3 were cloned into a BamHI-linearized yeast shuttle vector pCJ362 followed by recloning (BamHI) of the mutated C2 and C3 into pGEX-6p1. All vectors were sequenced. His-tagged C2 and C2-S/E constructs were obtained by recloning (BamHI) from pGEX-6p1 into pIVEX2.4a. pRSET-Cdc42 (encoding His-tagged Cdc42) was generated (BamHI/EcoRI) from pRK5myc-Cdc42hs. Plasmids were transformed into BL21(DE3)pLysS for expression. The following antibodies (Abs) were used: IQGAP1 mouse monoclonal Ab (BD Biosciences); Cdc42Hs, IQGAP1, and nPKC{epsilon} rabbit polyclonal Abs (Santa Cruz Biotechnology); and RGS-His mouse monoclonal Ab (Qiagen).


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  		TABLE I
Primer pairs used for cloning of IQGAP1

 


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  		TABLE II
Oligonucleotide sequences

 

Cell Culture—Breast epithelial cells (MCF10A) were cultured as described by Debnath et al. (22). Starvation of cells was performed for 18 h, and stimulation with complete medium was performed for 5 min. If indicated, cells were treated with 2.5 µM Latrunculin B for 30 min. Deprivation of cell-cell contacts was performed for 15 min in the presence of 1 mM pervanadate or for 8–10 h in the presence of 2.5 mM EGTA.

Purification of Recombinant Proteins—GST fusion proteins were purified using GSH-Sepharose 4B. For the recombinant GDP displacement assay, GST was cleaved by PreScission Protease. His-C2, -C2-S/E, and -Cdc42 were purified on Ni2+-nitrilotriacetic acid-agarose and His-Cdc42 was additionally gel-filtrated. All purification methods were according to standard procedures.

GST Pull-down Assays—Immobilized GTPase was incubated four times for 10 min at 4 °C in buffer ND. Beads with nucleotide-depleted GTPase were divided into three aliquots, resuspended in 0.3 ml of buffer ND, buffer N plus 0.5 mM GDP, or buffer N plus 0.5 mM GTP{gamma}S and incubated for 30 min at 4 °C. Confluent or single MCF10A cells were lysed in buffer N or calf intestine phosphatase (CIP) buffer (CIP lysate), respectively. Buffers were prepared with or without phosphatase inhibitors (PI) (1 mM pervanadate, 20 mM NaF). If indicated, CIP lysate without PI was supplemented with 0.8 units of CIP/µg of total protein, and all CIP lysates were incubated at 37 °C for 1 h. Cell lysates were supplemented with 20 mM EDTA, 0.5 mM GDP, or 0.5 mM GTP{gamma}S and added to ND-, GDP-, and GTP{gamma}S-GTPase, respectively. GTPase beads were washed after 1 h with buffer W (for buffers, see Table III).


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  		TABLE III
Buffers used in experiments

 
Immunoprecipitations—MCF10A cells were lysed in NETN buffer (Table III). For PKC{epsilon} immunoprecipitation, 1% Triton X-100 was added to NETN buffer. Cell lysate was incubated for 2 h with IQGAP1 or overnight with PKC{epsilon} antibodies at 4 °C. Antibodies were precipitated with Protein A-Sepharose and washed with NETN buffer.

MS/MS Identification of Proteins and IQGAP1 Phosphorylation— Coomassie-stained gel bands of Cdc42-bound proteins and immunoprecipitated IQGAP1 were cleaved with trypsin as described by Schrimpf et al. (23). The extracted peptides were analyzed by capillary liquid chromatography-MS/MS. Individual MS/MS spectra, containing sequence information for a single peptide, were compared with the program TurboSequest against the combined protein sequence databases SwissProt 43.5 and TrEMBL 26.5. This resulted in the identification of the peptides and, by association, the proteins in the lanes. The IQGAP1 phosphopeptide was further analyzed by an MS3 experiment. The peptide ion was isolated and fragmented in the ion trap, where dehydroalanine was formed from phosphoserine.

Recombinant Binding to Cdc42-ND—100 pmol of immobilized GST-C3, GST-C3-S/E, and control proteins were incubated for the indicated times at 37 °C with 100 pmol of His-Cdc42 in buffer R with or without 5mM EDTA. Samples were washed at 4 °C with buffer WN (for buffers, see Table III).

Recombinant Binding Assay—100 pmol of immobilized GST-C1, GST-C2, GST-C3, GST-C3-S/E, and control proteins were incubated for 1 h at 30 °C with 100 pmol of His-Cdc42-ND, His-Cdc42-GDP, or His-Cdc42-GTP{gamma}S and washed at 4 °C with buffer W. Before binding, ND and nucleotide-bound Cdc42 were prepared by incubating 0.9 µM Cdc42 in 1 ml of buffer E and buffer M for 20 min at 30 °C, respectively. 20 mM MgCl2 was added to loaded Cdc42, incubated for 10 min at room temperature, and cooled on ice. Unbound nucleotides were removed by desalting columns, and 20 µM nucleotides were added to samples with loaded Cdc42 (for buffers, see Table III).

Intramolecular Interaction Assay—100 pmol of immobilized GST-C1 and control proteins were incubated for 1 h at 4 °C with the indicated amounts of His-C2, His-C2-S/E, and control proteins in buffer WN (Table III) and washed at 4 °C.

GDP Displacement Assay—0.84 µM His-Cdc42 was loaded with [8-3H]GDP in buffer A (Table III) plus 5 mM EDTA and 8.4 µM [8-3H]GDP for 20 min at 30 °C. Then 20 mM MgCl2 was added, incubated for 10 min at room temperature, and cooled on ice. Unbound nucleotides were removed by desalting columns. Cdc42-[8-3H]GDP was eluted with buffer A. 100 nM immunoprecipitated or recombinant IQGAP1 was mixed with 10 µM GTP{gamma}S and 100 nM Cdc42-[8-3H]GDP and transferred to room temperature. The amount of bound radionucleotides at the indicated time points was determined using a nitrocellulose filter-binding method and normalized as the percentage of [8-3H]GDP bound at time 0. For each time point, the samples were assayed in triplicates.

Computer Analysis—The algorithm GenTHREADER (24, 25) from the World Wide Web-based server PSIPRED (bioinf.cs.ucl.ac.uk/psipred) was used for threading analysis of the C-terminal part of IQGAP1 (26). The PSIPRED version 2.4 (27) algorithm was used to perform the secondary structure prediction of aa 1244–1460 of IQGAP1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IQGAP1 Is an Unconventional Interactor with Cdc42 and Rac1 in Human Epithelial Cells—To identify novel regulators of Cdc42 in epithelial cells, we carried out pull-down experiments using GST-Cdc42 bound to Sepharose beads. To characterize the full spectrum of possible interactors, beads carrying the GTP- and GDP-bound forms of Cdc42 as well as the nucleotide-free form of the GTPase were used.

The three different forms of Cdc42 were incubated with lysate of confluent breast epithelial cells (MCF10A) in the absence or presence of PI, since phosphorylation frequently regulates protein-protein interactions. Bound proteins were separated by SDS-PAGE and visualized by silver staining. On these gels, we identified a protein with an apparent molecular mass of 195 kDa whose interaction with the different forms of Cdc42 varied depending on the presence or absence of PI (p195; Fig. 1A). Quantification of five experiments revealed that in the absence of PI p195 bound to all three forms of Cdc42, with a clear preference for Cdc42-GTP{gamma}S. In contrast, in the presence of PI p195 bound preferentially to Cdc42-ND.



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  		FIG. 1.
Differential binding of IQGAP1 toward Cdc42. A, quantification of IQGAP1 band intensities of pull-downs with GST-Cdc42. GST-Cdc42-ND, -GDP, and -GTP{gamma}S beads were incubated with MCF10A lysate in the presence (+) or absence (–) of PI and washed, and proteins were separated by SDS-PAGE and silver-stained. Five independent pull-down experiments were quantified. Within each group, the most prominent band was set to 100%. B, pull-downs with GST-Cdc42, -Rac1, and -RhoA were performed as in A. Presented are Western blot slices stained with anti-IQGAP1 and GTPase-containing Coomassie-stained gel slices as loading controls. C, a phosphatase activity, absent in single cells, regulates the binding of IQGAP1 toward Cdc42. Single MCF10A cells were obtained by pervanadate treatment for 15 min (Pervanadate) or starvation and EGTA treatment for 10 h (EGTA). Some cells were treated with 100 nM PMA for 3 h after starvation and EGTA treatment (EGTA -> PMA). Cell lysates with (+) and without (–) PI were prepared. Some cell lysates were incubated for 1 h at 37 °C with or without CIP before pull-down (EGTA 1h 37 °C CIP, EGTA 1h 37 °C). Pull-downs with GST-Cdc42 were performed as in A. Shown are silver-stained gel slices containing IQGAP1 and GST-Cdc42 as a loading control.

 
Next, we sought to determine the molecular identity of p195. Purified p195 was partially sequenced using MS/MS analysis and found to correspond to human IQGAP1. The identity of p195 was confirmed by immunoblot using a specific IQGAP1 antibody (Fig. 1B). To determine the specificity, we asked whether IQGAP1 also binds to the nucleotide-free forms of the GTPases Rac1 and RhoA. In the absence of PI, IQGAP1 bound preferentially to the GTP{gamma}S forms of Cdc42 and Rac1, as reported before (14). In contrast, in the presence of PI, IQGAP1 bound predominantly to Cdc42-ND. Weaker binding was detected toward Cdc42-GDP and Cdc42-GTP{gamma}S. In addition, IQGAP1 in the presence of PI bound weakly to Rac1-ND and Rac1-GTP{gamma}S. IQGAP1 bound more strongly the different forms of Cdc42 than those of Rac1 (the presented expositions can be directly compared). It did not bind to any form of RhoA (Fig. 1B). Thus, a phosphatase activity causes IQGAP1 to behave predominantly as an effector for Cdc42 and Rac1 in confluent MCF10A cells. When this activity was inhibited, IQGAP1 started to bind preferentially the nucleotide-free form of Cdc42.

Since IQGAP1 acts as a scaffolding protein that integrates signaling between cell-cell adhesion and the cytoskeleton (28), we tested whether cell-cell contacts regulate phosphorylation-dependent binding of IQGAP1 to Cdc42. Pull-down assays were carried out using lysates of cells deprived of cell-cell contacts either after pervanadate (29, 30) or EGTA treatment. In these experiments, IQGAP1 bound predominantly to Cdc42-ND regardless of the presence or absence of PI (Fig. 1C, panels Pervanadate, EGTA, and EGTA 1h 37 °C). Treatment of single cells with phorbol 12-myristate 13-acetate (PMA) leads to the formation of cell-cell contacts (31). Therefore, we analyzed the binding behavior of IQGAP1 from EGTA-treated cells that were subsequently exposed to PMA (Fig. 1C, panel EGTA -> PMA). Under these conditions, IQGAP1 revealed similar binding patterns as in confluent cells (Fig. 1A). These results indicated that the binding of IQGAP1 to Cdc42-ND was reversibly dependent on loss of cell-cell contacts and the absence of a phosphatase in single cells. Indeed, the addition of nonspecific CIP to the cell lysate of single cells prevented the binding of IQGAP1 to Cdc42-ND (Fig. 1C, panel EGTA 1h 37 °C CIP), confirming that this binding was dependent on phosphorylation. Interestingly, phosphatase-treated cell lysate did not prevent the binding of IQGAP1 to Cdc42-GTP, indicating that this interaction was phosphorylation-independent. Taken together, phosphorylation affecting IQGAP1 seemed to be increased in cells lacking cell-cell contacts and was at least in part due to a reduction in phosphatase activity.

Direct Phosphorylation of IQGAP1—Since the binding of IQGAP1 to Cdc42-ND was dependent on phosphorylation, we asked whether IQGAP1 was directly phosphorylated. The proprietary fluorescent Pro-Q Diamond phosphoprotein gel stain provides a method for selectively staining phosphoproteins in acrylamide gels. Using this reagent, we compared the phosphostain (P-stain) intensities of IQGAP1 isolated from confluent and single cells (cells deprived of cell contacts) by binding to Cdc42-ND or Cdc42-GTP{gamma}S (Fig. 2A). The intensity of the staining was strongest when IQGAP1 was isolated from single versus confluent cells. Likewise, the staining was also enhanced in the fraction of IQGAP1 that bound Cdc42-ND versus Cdc42-GTP{gamma}S. MS/MS analysis identified IQGAP1 as the most abundant protein in both P-stained bands (a and c in lane 3). The P-stain signal was absent where IQGAP1 was most abundant (b, lane 3), indicating that only a fraction of IQGAP1 was phosphorylated (Fig. 2, A and B). Since only weaker P-stain was visible when IQGAP1 was isolated from confluent cells and bound to Cdc42-ND and hardly any P-stain was detectable when IQGAP1 was bound to Cdc42-GTP{gamma}S, we conclude that IQGAP1 is predominantly phosphorylated in single cells. It is important to mention that the amount of bound IQGAP1 did not always correlate with P-stain intensity (lanes 1 and 2), indicating specificity of the P-stain. Even stronger evidence that the P-stain truly reflected phosphorylation is provided by the absence of P-staining after treatment of Cdc42-ND-bound IQGAP1 with CIP (lane 5). Thus, IQGAP1 bound to Cdc42-ND was phosphorylated. This experiment also indicated that dephosphorylation by CIP was not sufficient to dissociate IQGAP1 from Cdc42-ND.



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  		FIG. 2.
IQGAP1 is in vivo phosphorylated at S1443, a PKC{epsilon} site. A, GST-Cdc42-ND, -GTP{gamma}S pull-downs were performed with MCF10A lysate of confluent or single cells generated by treatment with EGTA for 8.5 h. As control, GST-Cdc42-ND pull-down with single cell lysate was treated with (+) or without (–) CIP for 1 h at 37 °C. Proteins were separated by SDS-PAGE and stained with Pro-Q Diamond Phosphoprotein Gel Stain (P-stain) analyzed for fluorescent phosphosignals (at heights a and c). Afterward, the gel was stained with Coomassie (only band visible at height b). All three spots (a–c) of lane 3 were analyzed by MS/MS, and peptide ion intensities representing the amount of IQGAP1 are shown in the table. B, identification of the phosphorylated peptide isolated from IQGAP1. Upper panel, MS/MS spectra of 596. The tryptic phosphopeptide was identified by TurboSequest. The major fragment generated by a neutral loss of H3PO4 is marked [M+3H-H3PO4]3+. The loss of H3PO4 of y13 is also shown. The y- and b-fragments detected are indicated in the sequence. The putative phosphoserine identified by b2, y13, and y8 is marked as pS in the sequence. Lower panel, confirmation of the phosphorylation site by an MS3 experiment. The major ion 563 of the MS/MS 596 was isolated and subjected to fragmentation again. Dehydroalanine formed from phosphoserine, was identified by b2, b3, b5, y8, and y10–13 in position 3 of this peptide and is marked as dA in the sequence. C, phosphorylation of Ser1443 is within a PKC{epsilon} consensus site (32). X represents any amino acid apart from Trp, Cys, Ser, or Thr. Phosphorylation sites are shaded gray. D, increasing amounts of IQGAP1 co-precipitatated with PKC{epsilon} during loss of cell-cell contacts. PKC{epsilon} was immunoprecipitated from MCF10A cells, which were treated with EGTA for the indicated times. IgG immunoprecipitation was used as a negative control. Presented are Western blot slices stained with anti-IQGAP1 antibody and heavy chain-containing Coomassie-stained gel slices as loading controls. 1/65 of input, the volume fraction of total cell lysate used in the precipitation.

 
Next we aimed to determine the phosphorylation site in IQGAP1. Starved confluent MCF10A cells were either stimulated with complete medium or not. IQGAP1 was immunoprecipitated, separated by SDS-PAGE, and digested. Analysis of the tryptic peptides by liquid chromatography-MS/MS resulted in sequence coverage of 50–60% for stimulated and nonstimulated samples. Only in the sample of stimulated cells was a phosphopeptide identified. This peptide (SKSVKEDSNLTLQEK) was phosphorylated at position 3 (Ser1443 of IQGAP1) as shown in Fig. 2B. The phosphorylation was confirmed by an MS3 experiment, where dehydroalanine formed from phosphoserine was detected. These experiments revealed Ser1443 as the only detectable phosphorylation site in IQGAP1. Residues 1436–1448 of IQGAP1 are 46% identical and 69% similar to the ideal substrate for PKC{epsilon} (Fig. 2C), suggesting that Ser1443 is a substrate for this kinase (32).

Our data indicated that IQGAP1 was phosphorylated during loss of cell-cell contacts and that PKC{epsilon} is a candidate kinase. Therefore, we investigated whether PKC{epsilon} associated with IQGAP1 in vivo during loss of cell-cell contacts. PKC{epsilon} was immunoprecipitated from lysates of cells treated with EGTA for various amounts of time. Increasing amounts of IQGAP1 co-precipitated with PKC{epsilon} as loss of cell-cell contacts progressed, but not with IgG alone (Fig. 2D). Thus, during the generation of single cells, PKC{epsilon} interacts with IQGAP1 and may phosphorylate it. This phosphorylation could cause the increased binding of IQGAP1 to Cdc42-ND observed in cells deprived of cell-cell contacts.

IQGAP1 Phosphorylation Increases Direct Binding to Cdc42-ND—Our analysis of interactions between IQGAP1 and Cdc42 indicated that IQGAP1 bound to Cdc42-ND and was phosphorylated at a C-terminal serine residue. To determine whether this binding was direct and whether it was regulated by the phosphorylation on Ser1443, we employed an in vitro binding assay using recombinant fragments of IQGAP1 and recombinant Cdc42. Plasmids encoding GST fusion C-terminal fragments of IQGAP1 were generated (Fig. 6A). Two fragments were tested, C3 (aa 956–1657) and an analogous fragment in which Ser1443 was mutated to Glu to mimic phosphorylation (C3-S/E).



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  		FIG. 6.
Conformation-specific binding of C terminus of IQGAP1 to Cdc42. A, scheme of full-length IQGAP1 and recombinant C-terminal IQGAP1 proteins used in binding experiments. CHD, calponin homology domain; WW, polyproline binding domain; IQ, calmodulin binding motifs; GRD, RasGAP-related domain. The asterisk indicates the position of Ser1443. B, tertiary structure of the C terminus of IQGAP1 determines binding specificity toward Cdc42. Immobilized GST fusion C-terminal proteins of IQGAP1, GST, and GST-PAK1-CRIB (GST-CRIB) were incubated with ND-, GDP-, or GTP{gamma}S-His-Cdc42. Left column, anti-Cdc42 Western blot of bound Cdc42. Right column, Coomassie-stained gel slices show the equal input of the GST fusion proteins.

 
Isolated nucleotide-depleted GTPases are structurally unstable and tend to denature. To test whether the interaction IQGAP1-Cdc42 occurs under nucleotide-depleted conditions, we used an assay developed by Beraud-Dufour et al. (33). In this method, nucleotide depletion is directly coupled with stabilization of the depleted GTPase through the binding of a second interactor, an approach that copies a GEF reaction. Immobilized C3 or C3-S/E was incubated with an equal molar ratio of bacterially purified His-tagged Cdc42 in the presence of EDTA, CIP, or both together. EDTA chelates Mg2+ and therefore destabilizes nucleotides bound to GTPases. CIP hydrolyzes phosphate groups, abolishing the presence of nucleotides. In the presence of CIP, EDTA, or both, the C-terminal IQGAP1 fragments bound Cdc42. In contrast, in the absence of CIP and EDTA, neither IQGAP1 fragment bound Cdc42 significantly, supporting the assumption that bacterially expressed Cdc42 is predominantly GDP-bound (Fig. 3A). To test the specificity of this interaction, we incubated Cdc42 with GST-PAK1-CRIB (GST-CRIB), which binds specifically to Cdc42-GTP, and GST alone. As expected, neither GST nor GST-PAK1-CRIB bound Cdc42 in these assays, indicating that the binding of Cdc42 to the GST-C3 and GST-C3S/E fragments was specific. Thus, Cdc42-ND bound directly to both recombinant C-terminal fragments of IQGAP1.



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  		FIG. 3.
Ser1443 phosphorylation of IQGAP1 regulates the binding to nucleotide-free Cdc42. A, immobilized GST-C3, GST-C3-S/E, GST, and GST-PAK1-CRIB (GST-CRIB) were incubated in buffer R containing 1 mM MgCl2 and His-Cdc42 in the presence (+) or absence (–) of EDTA and CIP as indicated. Shown are anti-Cdc42 Western blot slices and GST fusion protein-containing Coomassie-stained gel slices as loading controls. B, immobilized GST-C3, GST-C3-S/E, GST, and GST-PAK1-CRIB were incubated with His-Cdc42 in the presence or absence (–E) of EDTA. Samples were taken at the indicated time points. Shown are anti-His Western blot slices of bound His-Cdc42 and GST fusion protein-containing Coomassie-stained gel slices as loading controls.

 
We next examined whether the phosphorylation on Ser1443 might alter the kinetic of the interaction of C3 with Cdc42-ND. The binding of phospho-mimicking C3-S/E and nonphosphorylated C3 toward Cdc42 was compared over time. This assay was performed as before in an equal molar ratio in the presence of EDTA, and samples were taken after 6, 8, 15, 20, and 30 min (Fig. 3B). The phospho-mimicking fragments bound more Cdc42-ND over time than the wild type fragments. As expected, GST or GST-PAK1-CRIB did not bind Cdc42-ND under these conditions (the presented expositions can be directly compared). This binding assay demonstrated that aa 956–1657 of IQGAP1 bound directly to Cdc42-ND, one property demanded for a GEF, and that the affinity of the interaction was increased by Ser1443 phosphorylation. Thus, IQGAP1 phosphorylated on Ser1443 stabilizes Cdc42-ND better than nonphosphorylated IQGAP1.

IQGAP1 Is Associated with GEF Activity and Stabilizes Nucleotide-free Cdc42—The binding to Cdc42-ND suggested that phosphorylated IQGAP1 (IQGAP1-P) might act as a GEF on Cdc42. Until recently, all known Rho-GEFs were characterized by the presence of a catalytic Dbl homology (DH) domain followed by a Pleckstrin homology domain (PH). A group of unconventional GEFs (Zizimin, Dock180, and SopE) does not contain the canonical DH domain. Instead, these GEFs contain CZH2 domains or a GAGA-loop, respectively, to which in the C-terminal IQGAP1 region no obvious sequence homology could be identified (11, 34, 35). Interestingly, however, threading analysis by GenTHREADER suggested the presence of a DH domain fold between aa 1244 and 1460 of IQGAP1 similarly to the DH domain fold of {beta}-PIX (E-value 0.033, medium confidence level) (Fig. 4). In addition, a DH domain resembling the fold of Intersectin was identified in this fragment, albeit with a low confidence level (data not shown). Secondary structure prediction indicated that aa 1244–1460 of IQGAP1 are likely to be highly {alpha}-helical, similar to known structures of other Rho-GEFs (data not shown) (36). The solved crystal structure of the DH-PH domain of Dbs in complex with nucleotide-free Cdc42 demonstrated that the last {alpha}-helix of the DH domain (K-helix or {alpha}6) is part of the major binding surface for Cdc42 (37). In the threaded IQGAP1, Ser1443 lies at the end of the K-helix. Thus, phosphorylation of Ser1443 would possibly have an influence on the interaction with Cdc42, supporting our previous data (Fig. 3B).



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  		FIG. 4.
Structural similarity between the DH domain of {beta}-PIX and IQGAP1 using GenTHREADER. Identical and similar aa are boxed. Identical aa are shown in boldface type. The secondary structure of {beta}-PIX is shown above the aligned sequences (C, coiled; H, helical). The location of S1443 in IQGAP1 (aa 1244–1460) is indicated with an asterisk.

 
To test the possibility that IQGAP1 might be a GEF for Cdc42, we immunoprecipitated IQGAP1 from confluent cells and subjected the isolated protein to a GDP displacement assay. Immunoprecipitated IQGAP1 exhibited GDP displacement activity under conditions where the nucleotide was not stabilized on Cdc42 (Fig. 5A). As IQGAP1 binds to actin filaments (15), actin co-precipitating proteins could cause the GEF activity. To eliminate this possibility, we isolated IQGAP1 in the presence of Latrunculin B, which sequesters monomeric actin. We found no differences in GEF activity, supporting the notion that IQGAP1 harbors the GEF activity. However, immunoprecipitated IQGAP1 from single or confluent MCF10A cells, which were starved or stimulated, and recombinant phospho-mimicking fragments of IQGAP1 did not displace GDP from Cdc42-GDP if the nucleotide was stabilized via bovine serum albumin on Cdc42 (data not shown).



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  		FIG. 5.
IQGAP1 is an unconventional regulator of Cdc42. A, immunoprecipitated IQGAP1 exhibits GEF activity. IQGAP1 was immunoprecipitated from lysate of starved (18 h) and complete medium-stimulated (5 min) MCF10A cells treated with (+Lat) or without Latrunculin B for 30 min and used in a GDP displacement assay. Immunoprecipitation with an irrelevant antibody was used as a negative control (IP control). SopE was used as a positive control. Depicted is the protein-bound radioactivity as a percentage relative to the counts at time 0, which are set to 100%. Values are means of triplicate samples. The error bars range from 0.06 to 1.89% and are masked by symbols. B, IQGAP1 cannot be eluted with GTP{gamma}S. GST-Cdc42-ND beads were incubated with lysate of confluent (upper panel) or single MCF10A cells obtained by starvation and EGTA treatment for 8 h (lower panel) in the presence of PI. Bound IQGAP1 was eluted at room temperature with elution buffer (Table III) containing a 100-fold molar excess of GTP{gamma}S (+) to Cdc42 for 20 min (upper panel) or with buffer C (Table III) containing a 100-fold molar excess of GTP{gamma}S(+) to Cdc42 for 15 and 60 min (lower panel). Buffer without GTP{gamma}S (–) was used as a control. Shown are silver-stained gel slice (silver) and Western blot slices (anti-IQGAP1) containing IQGAP1. B, beads; E, eluate; BaE, beads after elution. C, IQGAP1 inhibits incorporation of GTP{gamma}S to Cdc42. GST-Cdc42-ND·IQGAP1 (Cdc42-ND/IQGAP1) complex was obtained by pull-down with starved and treated MCF10A cells with EGTA for 8.5 h in the presence of PI. Ice-cold buffer C (Table III) containing a 5-fold molar excess of GTP{gamma}S/[35S]GTP{gamma}S mix to Cdc42 was added, and triplicates were taken immediately (t0) and after 21 min (t21) of incubation at room temperature. Depicted is the protein-bound radioactivity at 1000x cpm (y axis). Values are means of triplicate samples. GST-Cdc42-ND (Cdc42-ND) was used as a positive, and GST was used as a negative control.

 
If IQGAP1 were a typical GEF, one would expect that the complex IQGAP1-Cdc42-ND could be dissociated by the addition of excess GTP{gamma}S. We tested this by two different assays starting with a complex of IQGAP1 bound to immobilized Cdc42-ND. In the first approach, an excess of GTP{gamma}S was added and the presence of IQGAP1 in the supernatant analyzed (Fig. 5B). In the second approach, the incorporation of radiolabeled GTP{gamma}S was monitored over time and compared with the incorporation on Cdc42-ND alone (Fig. 5C). We show that IQGAP1 cannot be solubilized, and the presence of IQGAP1 inhibits the incorporation of radiolabeled GTP{gamma}S into Cdc42-ND.

These results demonstrated that IQGAP1-P did not harbor significant GDP displacement activity and did not behave as a classical GEF but could act to stabilize the nucleotide-free form of Cdc42.

Structural Changes upon Phosphorylation of IQGAP1—To determine regions in IQGAP1 responsible for differential binding toward Cdc42, we compared the binding of Cdc42-ND, -GDP, and -GTP{gamma}S to various recombinant C-terminal fragments of IQGAP1 (Fig. 6). In this assay, Cdc42-GTP{gamma}S bound strongest to C3, followed by Cdc42-ND binding. The weakest interaction was seen between Cdc42-GDP and C3. Thus, C3 bound as an effector- and GEF-like molecule. Surprisingly, Cdc42-GTP{gamma}S did not bind to shorter fragments of IQGAP1. Cdc42 bound exclusively in the nucleotide-depleted form to the first (aa 956–1274; C1) and the last C-terminal part of IQGAP1 (aa 1276–1657; C2). The known effector of Cdc42 and Rac1, PAK1-CRIB, bound as expected exclusively to Cdc42-GTP{gamma}S. Together, these results demonstrate that the C terminus (aa 956–1657) of IQGAP1 harbors two binding domains for Cdc42-ND. Furthermore, the binding of Cdc42-GTP to IQGAP1 depends on the tertiary structure of the C terminus.

To investigate the possibility that the tertiary structure of C3 is determined by direct binding of the region encompassing C1 to the region encompassing C2, we set up a direct interaction test. GST-C1 was immobilized, increasing amounts of His-tagged C2 were added, and bead-bound C2 was analyzed (Fig. 7A, upper part). Increasing input of C2 correlated with increased binding to C1, demonstrating a direct interaction of these two fragments (for controls, see Fig. 7C). Phospho-mimicking C2 fragments (C2-S/E) bound also to C1, albeit the amount did not significantly increase with increasing input (Fig. 7A, lower part; for controls, see Fig. 7D). Quantification of the binding data revealed that the percentage of C2-S/E bound to C1 was significantly lower compared with C2 (Fig. 7B). These results showed that aa 956–1274 interacted with aa 1276–1657 of IQGAP1 and that the phosphorylation in the second half of the C terminus of IQGAP1 strongly reduced this interaction. Taken together, in vivo phosphorylation of IQGAP1 causes an opening of the structure of the C terminus of IQGAP1 to allow binding and sequestration of Cdc42-ND.



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  		FIG. 7.
Phosphorylation regulates intramolecular interaction in the C terminus of IQGAP1. A, the indicated amounts of His-C2 (upper block) or His-C2-S/E (lower block) were incubated with 100 pmol of GST-C1. Shown are anti-His Western blot slices of bound His-C2 (upper block) or His-C2-S/E (lower block) and GST-C1-containing Coomassie-stained gel slices as loading controls. 1/60 of input, one-sixtieth of total His-C2 (upper block) or His-C2-S/E (lower block) used in the reaction. B, quantification of C1/C2 and C1/C2-S-E interactions. The Western blot signals of bound His-C2 and His-C2-S/E were quantified with respect to standard curves calculated from input signals. Shown is the dependence of C1-bound proteins (C2 or C2-S/E) on the amount of input represented as percentage. C, controls for the C1/C2 interaction assay. The indicated amounts of His-tagged proteins were incubated with 100 pmol of GST, GST-C1, or GST-Cdc42-ND as indicated. Shown are anti-His Western blot slices of bound His proteins and GST fusion protein-containing Coomassie-stained gel slices as loading controls. D, controls for C1/C2-S/E interaction assay analogous to C.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study was initiated to identify novel regulators for Cdc42 in human epithelial cells. We identified phosphorylated IQGAP1 as an unconventional interactor with Cdc42-ND, suggesting a role upstream of Cdc42.

To date, IQGAP1 has been described as an effector of Cdc42 and Rac. However, already previous results suggest that IQGAP1 function on Cdc42 might be more complex. For example, IQGAP1 inhibits GTPase activity of Cdc42 in vitro (14), and overexpression of IQGAP1 in cells causes an increased level of Cdc42-GTP (38). Also, down-regulation of IQGAP1 levels in epithelial cells by small interfering RNA reduced the amount of active Rac1 (39). However, the nature of the interaction underlying these results was not defined. Our analysis provides an explanation for these results and supports the idea that IQGAP1 acts both upstream and downstream of Cdc42.

Binding to nucleotide-free GTPases is a property of GEFs. We took great care to avoid the possibility that the interaction of IQGAP1 with Cdc42-ND represents an in vitro artifact by using a coupled nucleotide deloading and binding assay (Fig. 3) and comparison with a bona fide effector fragment PAK1-CRIB (Figs. 3 and 6). We detected GEF activity of immunoprecipitated IQGAP1 toward Cdc42 in vitro. The absence of lipids or other factors, like ELMO, as in the case of DOCK180, required for stimulation of GEF function might explain the weak GEF activity of IQGAP1 in our assays. Another such factor could be a kinase necessary for the phosphorylation of IQGAP1. Indeed, we showed that PKC{epsilon} associated with IQGAP1 during loss of cell contacts and might be the relevant kinase. The net balance between kinase and phosphatase activities is important for cell-cell contact formation and the regulation of IQGAP1-Cdc42 interaction as our analysis shows. Despite the direct interaction of IQGAP1 with Cdc42-ND, weak GEF activity, and medium structural homology to a DH domain, the complex of IQGAP1 with Cdc42-ND could not be dissociated with an excess of GTP, another requirement for GEFs. Therefore, additional factors might be necessary first to cause the generation of IQGAP1-Cdc42-ND complexes and second to dissociate them.

Alternatively, IQGAP1 might represent a novel type of protein. A structural superfamily comprising the MSS4/Dss4 proteins and the translationally controlled tumor protein (TCTP) family exhibits striking biochemical similarities to IQGAP1. MSS4 binds to nucleotide-free Rab1a, like IQGAP1 to Cdc42-ND, and exhibits only minor GEF activity. Due to its unusual characteristics, MSS4 was termed a "guanine nucleotide-free chaperone" (40). The physiological function of the TCTPs as well as the biological relevance of the MSS4 interaction is not clear. In any case, IQGAP1 seems to act up- and downstream of Cdc42.

In our analysis, we reveal a molecular mechanism explaining the binding selectivity of IQGAP1 toward Cdc42-ND or Cdc42-GTP. The short C-terminal fragments of IQGAP1, C1 and C2, bound exclusively to Cdc42-ND. However, the entire C terminus of IQGAP1 (aa 956–1657; C3) bound predominantly to Cdc42-GTP{gamma}S (Fig. 6B). Further, C3 did not exhibit double binding capacity for Cdc42-ND as would be expected for a fragment encompassing the sum of C1 and C2. Thus, the tertiary structure of IQGAP1 seems to predict the differential binding properties of IQGAP1. We examined the possible structure determinants by testing the interaction of C1 with C2 and the influence of Ser1443 phosphorylation. Clearly, C1 and C2 fragments of IQGAP1 interacted specifically, and the interaction was reduced using phospho-mimicking C2 fragments. Therefore, Ser1443 phosphorylation causes an opening of the IQGAP1 structure, exposing the two Cdc42-ND binding determinants, as supported by Figs. 3 and 6. Conformational change based on this intramolecular interaction might be even more pronounced in full-length IQGAP1 and with a real phosphorylation.

What might be the biological significance of IQGAP1 bound to nucleotide-free Cdc42? IQGAP1 localized in cells with and without cell-cell contacts at the plasma membrane (data not shown). This suggests that the complex of IQGAP1-P and Cdc42-ND is already at the plasma membrane, where cell-cell contacts are initiated or broken, and thus at a biologically relevant location. A long lifetime of the binary complex, as observed for DSS4 (41), would cause low levels of Cdc42-GTP in cells deprived of cell-cell contacts. Thus, the chaperone function of IQGAP1 inhibits in cells without cell-cell contacts Cdc42 activation, a novel mechanism of Cdc42 regulation. Consequently, the reduction of Cdc42-GTP levels causes the loss of the biological effects of the effectors Par6 and IQGAP1, which results in the loss of cell-cell contacts. Thus, in addition to its effector function, IQGAP1 has the potential to act as inhibitor of Cdc42. It will be important to understand the regulation of IQGAP1 phosphorylation and how Cdc42-ND is generated.


   FOOTNOTES
 
* This work was supported by the Swiss National Science Foundation (to K. G.), the Schwyzer Foundation (to D. S.), the Novartis Foundation (to M. B.), and the Roche Research Foundation (to P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 41-1-632-6346; Fax: 41-1-632-1591; E-mail: kroschewski{at}bc.biol.ethz.ch.

1 The abbreviations used are: GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; Ab, antibody; GST, glutathione S-transferase; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate; PKC, protein kinase C; MS/MS, tandem mass spectrometry; Cdc42-ND, nucleotide-depleted Cdc42; PI, phosphatase inhibitor(s); PMA, phorbol 12-myristate 13-acetate; CIP, calf intestine phosphatase; P-stain, phosphostain; DTT, dithiothreitol. Back

2 Y. Barral, unpublished plasmid and method. Back


   ACKNOWLEDGMENTS
 
We thank B. Antonny for constructive suggestions, Y. Barral for yeast shuttle vector and help by in vivo recombination, W.-D. Hardt for catalytic SopE, J. Collard for GST-PAK1-CRIB plasmid, I. Mellman for IQGAP1 plasmids, and A. Hall for plasmids encoding Rho-GTPases.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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