Research Article 391 A dual role for IQGAP1 in regulating exocytosis

Eric N. Rittmeyer1, Samira Daniel2,3, Shu-Chan Hsu4 and Mahasin A. Osman1,* Departments of 1Microbiology and 2Molecular Medicine, Cornell University, Ithaca, NY 14853-2703, USA 3Division of Endocrinology, Diabetes and Bone Disease, Mount Sinai School of Medicine, New York, NY 10029-6574, USA 4Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA *Author for correspondence (e-mail: [email protected])

Accepted 1 November 2007 Journal of Cell Science 121, 391-403 Published by The Company of Biologists 2008 doi:10.1242/jcs.016881

Summary Polarized secretion is a tightly regulated event generated by terminus of IQGAP1 binds the exocyst-septin complex, conserved, asymmetrically localized multiprotein complexes, enhances secretion and abrogates the inhibition caused by and the mechanism(s) underlying its temporal and spatial CDC42 or the depletion of IQGAP1, the C-terminus, which regulation are only beginning to emerge. Although yeast Iqg1p binds CDC42, inhibits secretion. Pulse-chase experiments has been identified as a positional marker linking polarity and indicate that IQGAP1 influences -synthesis rates, thus exocytosis cues, studies on its mammalian counterpart, regulating exocytosis. We propose and discuss a model in which IQGAP1, have focused on its role in organizing cytoskeletal IQGAP1 serves as a conformational switch to regulate architecture, for which the underlying mechanism is unclear. exocytosis. Here, we report that IQGAP1 associates and co-localizes with the exocyst-septin complex, and influences the localization of Supplementary material available online at the exocyst and the organization of septin. We further show http://jcs.biologists.org/cgi/content/full/121/3/391/DC1 that activation of CDC42 GTPase abolishes this association and inhibits secretion in pancreatic ␤-cells. Whereas the N- Key words: Cell polarity, IQGAP1, Insulin secretion

Introduction Sakai et al., 2002; Vega and Hsu; 2003; Trimble, 1999). As is the Polarized is essential for specialized cellular functions case with all septins, its role in these processes remains unclear. such as migration as well as for cell division and differentiation, Whereas the yeast exocyst communicates with several members and is generated by a core mechanism involving evolutionarily of the Rho subfamily of small GTPases, including Cdc42p conserved multi-protein complexes (Johnson and Wodarz, 2003; (Lipschutz and Mostov, 2002; Novick and Guo, 2002), in

Journal of Cell Science Nelson, 2003). The generation of polarized cell growth is driven mammals, only RAL (RALA) and TC10 (RHOQ) GTPases have by directed exocytosis and requires the action of asymmetrically been shown to bind the exocyst (Inoue et al., 2003), and no link localized positional cues and localized activation of G- has been reported with CDC42. Recently, genetic and biochemical followed by the recruitment of downstream effectors to a specific evidence have shown that both yeast and mammalian exocysts site, leading to the reorganization of the and microtubule communicate with the ER-translocon via an interaction with the and of the secretory pathway (reviewed in Osman Sec61␤ subunit (Lipschutz et al., 2003; Toikkanen et al., 2003), and Cerione, 2005). However, the molecular determinants of its suggesting a role in protein synthesis/translocation events. spatial and temporal regulation are not well understood. Mammalian IQGAP1 was also identified as a CDC42 target, Yeast IQGAP1, Iqg1p, was identified as a target for Cdc42p acting through its C-terminal domain (Brill et al., 1996; Kinoshita GTPase (Osman and Cerione, 1998) and was found to bind Bud4p, et al., 1997; McCallum et al., 1996; Kuroda et al., 1996), and the the septin Cdc12p and the exocyst Sec3p, which serve as positional domain structure of yeast and mammalian IQGAPs is conserved cues for cell polarity events such as axial bud-site selection and (Epp and Chant, 1997; Lippincott and Li, 1998; Osman and (Osman et al., 2002). Although there is no known Cerione, 1998), implying a conserved function. IQGAP1 is a mammalian homolog for Bud4p, a counterpart exists for each member of a three-isoform family of proteins that also includes septin and exocyst subunit. The mammalian exocyst [Sec6-Sec8 IQGAP2 (Yamashiro et al., 2003) and IQGAP3 (Wang et al., 2007). (EXOC3-EXOC4) complex] has been co-purified and IQGAP1, the most studied, binds , cross-links actin immunoprecipitated with septins (Hsu et al., 1996; Hsu et al., 1998; filaments, integrates signaling networks (reviewed in Mateer et al., Hsu et al., 1999); like its yeast counterpart, it was found to 2003; Brown and Sacks, 2006), and regulates cell-cell contacts influence polarized vesicle delivery (Hsu et al., 1999). Mammalian (Fukata et al., 1999; Fukata et al., 2001) and the capture of septins were individually identified and implicated in cytokinesis, microtubule plus-ends via association with CLIP-170 (Fukata et exocytosis, vesicle-targeting and membrane dynamics (Spiliotis al., 2002). The current paradigm is that IQGAP1 regulates actin and Nelson, 2006; Beites et al., 1999; Trimble, 1999). Together, assembly in cooperation with the ARP2/3 complex and the Rho these findings argue that septins can cooperate with the exocyst in GTPases in different cell types to regulate cell outgrowth and the regulation of polarized secretion, perhaps acting as positional migration (Watanabe et al., 2004; Bensenor et al., 2007; Le markers, tethering or tracking filaments. One of the best- Clainche et al., 2007; Wang et al., 2007). This role, however, does characterized mammalian septins is NEDD5, from here on referred not contradict with an IQGAP1 essential function in secretion, to as SEPT2 (Spiliotis and Nelson, 2006; Kinoshita et al., 1997; because the mammalian exocyst subunit EXO70 (EXOC7) has also 392 Journal of Cell Science 121 (3)

been reported to interact with the ARP2/3 complex and modulate terminus in protein synthesis, as indicated by pulse-chase actin-based membrane dynamics (Zuo et al., 2006), similar to experiments. These data raise a possibility that IQGAP1 serves as IQGAP1. Thus, in this work we present a novel role for IQGAP1 a regulator of secretion by acting as a conformational switch. in the regulation of exocytosis. Here, we investigated whether mammalian IQGAP1 associates Results with the exocyst-septin complex and influences secretion in a Association of IQGAP1 with the exocyst-septin complex is CDC42-regulated fashion. We used pancreatic ␤-cell lines for two regulated by CDC42 reasons. First, we found that IQGAP1, the exocyst and septins are Because the domain structure of yeast Iqg1p and mammalian abundant proteins in these cells, offering insulin secretion as a IQGAP1 is conserved (Fig. 1A), the ability of IQGAP1 to interact physiologically relevant functional assay for exocytosis. Second, with the exocyst-septin complex was examined. Because mastoparan, a tetradecapeptide from wasp venom, was reported to mammalian cells contain two ~60% identical IQGAP isoforms in enhance insulin secretion in ␤HC-9 cell lines that overexpress addition to IQGAP3, the interaction with both IQGAP1 and wild-type CDC42 by stimulating its exchange activity (Daniel et IQGAP2 was examined in pancreatic ␤TC-6 cells using antibodies al., 2002). Mastoparan is known to stimulate exocytosis specific for each protein. IQGAP1 but not IQGAP2 (or IQGAP3, independent of Ca2+ by activating G-proteins in a number of not shown) co-precipitated with the exocyst-septin complex (Fig. mammalian cell types, including ␤-cells (reviewed in Kowluru, 1B). 2003). Thus, it seemed plausible that CDC42 also would enhance To examine the effects of CDC42 and its activation by an IQGAP1–exocyst-mediated secretion. mastoparan on the association of IQGAP1 with the exocyst-septin Surprisingly, we found that overexpression or activation of complex, ␤TC-6 cells stably expressing the vector control or wild- CDC42 by mastoparan, dominant-active mutations or IQGAP1 type CDC42 (CDC42WT) that were treated either with mastoparan expression disrupted the endogenous association of IQGAP1 with (Mp) or vehicle alone were used for immunoprecipitation (IP). the exocyst-septin complex and blocked secretion. This effect Unexpectedly, IQGAP1 formed complexes only in control cells appeared to be mediated by the C-terminus of IQGAP1, which lacking CDC42WT that were not treated with mastoparan (Fig. 1C, binds CDC42 and inhibits secretion. By contrast, the N-terminus lane 3). CDC42 did not disrupt the exocyst-septin complex itself of IQGAP1 binds to the exocyst-septin complex, enhances and only IQGAP1 was displaced (Fig. 1C, lanes 1, 2 and 4). In secretion and abrogates the inhibition caused by CDC42 or the addition, antibodies against IQGAP1 or the V5 tag of its depletion of IQGAP1, perhaps via the involvement of the N- recombinant constructs were also able to co-immunoprecipitate Journal of Cell Science

Fig. 1. Association of IQGAP1 with the exocyst-septin complex is regulated by CDC42. (A) IQGAPs are conserved proteins. Domain structure of mammalian IQGAP1 (top) and yeast Iqg1p (bottom) with percent identity indicated on top of each domain. The sequences downstream of the GRD are 24% identical and are well-conserved across species. (B) IQGAP1, but not IQGAP2 (or IQGAP3, not shown), co-immunoprecipitates with the EXO70-SEPT2 complex. Antibodies against EXO70 and SEPT2 were used as indicated on the top of each lane to immunoprecipitate equal amounts of protein from pancreatic ␤TC-6 cells, and western blots were stained with monoclonal antibodies specific for IQGAP2 (middle panel) or IQGAP1 (right panel). Left panels: 5% of the input probed with the indicated antibodies is shown. ‘Mock’ is a negative control using anti-Myc. (C) CDC42 disrupts the interaction between IQGAP1 and the exocyst-septin complex. ␤TC-6 stably expressing wild-type CDC42 (Cdc42WT) or the vector control were either treated with mastoparan (Mp +) to activate CDC42 or with vehicle alone as a control (Mp –), and their lysates were used for co-immunoprecipitation with anti-EXO70 monoclonal antibodies. Right panel: 5% of the proteins used for the IP is shown. The blots represent four experiments with identical results. CHD, homology domain; IR, specific IQGAP repeats; WW, domain resembling the SH3 protein-interacting domains, not well-conserved in yeast; IQ, eight IQ motifs that bind calmodulin; GRD, rasGAP-related domain containing sequences that bind CDC42 and RAC1. IQGAP1 regulates exocytosis 393

Fig. 2. Active CDC42 alleles diminish the association of IQGAP1 with the exocyst. (A) Effects of different alleles of CDC42 on the co-immunoprecipitation of IQGAP1 with EXO70. Lysates from pancreatic ␤-cells transiently expressing equal amounts of different HA-tagged CDC42 alleles (bottom) were used to co- immunoprecipitate IQGAP1 (middle panel) with EXO70 (top panel) monoclonal antibodies. IQGAP1 was blotted from 5% of the input protein (lower panel) as a loading control. Cells expressing the vector alone were used as a positive control and mock is a negative control with anti-Myc antibodies. (B) Quantitation of IQGAP1 association with EXO70 in the presence of CDC42 mutants. The intensity of the IQGAP1 bands co-precipitated with EXO70 in the presence of the CDC42 alleles were quantified by densitometry using Quantity One software and a ChemiDoc XRS system (Bio-Rad), and are expressed as the mean ± s.d. for n=3. (C) Binding of CDC42 to IQGAP1 is necessary for the negative effects of CDC42 on the co-immunoprecipitation of IQGAP1 and EXO70. Co- Journal of Cell Science immunoprecipitation of IQGAP1 and EXO70 was performed from cells expressing either a HA-CDC42-F28LC37A double mutant that diminishes CDC42 binding to IQGAP1 (see D, top), or expressing the mutant V5-IQGAP1-F⌬MK, which lacks the 24 amino acids of the CDC42-binding region (see D, bottom), and from their parental F28L or V5-F1 proteins. The histogram represents the mean ± s.d. for n=3 blots. (D) Confirmation of the binding abilities of the mutants used in C. The double-mutant HA-CDC42-F28LC37A, the deletion-mutant V5-IQGAP1-F⌬MK and their parental constructs transiently expressed in ␤TC-6 cells were used for IP with antibodies for the respective tags as shown on the figure and probed for co-immunoprecipitation of IQGAP1 (top) or CDC42 (bottom). The input is presented in the lower part of each panel. WB, western blot; WCL, whole cell lysate.

endogenous EXO70, SEC8 and SEPT2 in untreated control ␤-cells (F28L; Fig. 2, lane 4) or the empty vector for control (vector; Fig. and in six other mammalian cell lines (Fig. 3C; and supplementary 2, lane 5) were transiently expressed to similar levels (HA-CDC42, material Fig. S1A,C). These data were identical in ␤HC-9 cells and Fig. 2A, lower panel). When endogenous EXO70 was indicate that IQGAP1 interacts with the exocyst-septin complex immunoprecipitated, more endogenous IQGAP1 co-precipitated in and that overexpression or activation of CDC42 by mastoparan cells expressing the vector control or the dominant-negative disrupts this interaction. CDC42 allele (T17N) but little in cells expressing WT or the dominant-active alleles (Q61L and F28L). This result is not due to Active alleles of CDC42 inhibit IQGAP1 association with the differences in IQGAP1 or EXO70 expression, because equal exocyst-septin complex amounts of IQGAP1 (Fig. 2A, bottom of the upper panel) and To ascertain that the disruption of the interaction between IQGAP1 EXO70 (Fig. 2A, top panel) were found in the lysate used for IP and the exocyst-septin complex is specific to the activation of in cells expressing each of the CDC42 alleles. CDC42 and not due to other effects caused by mastoparan, we Three such blots were quantified for the intensities of the undertook a genetic approach using dominant CDC42 mutants in IQGAP1 bands co-precipitated with EXO70 in the presence of a transient-expression assay. We compared the effects of dominant these alleles, and their averages are presented as relative amounts alleles of CDC42 on IQGAP1-EXO70 co-immunoprecipitation in (Fig. 2B). These data were identical in both ␤HC-9 and ␤TC-6 pancreatic ␤-cells that have not been treated with mastoparan (Fig. cell lines, and indicate that dominant-active CDC42 alleles more 2A,B). An HA-tagged pcDNA3 vector encoding the GTPase- effectively diminish the endogenous association between defective, dominant-active mutant of CDC42 (Q61L; Fig. 2, lane IQGAP1 and EXO70. Moreover, expression of the WT allele 1), the dominant-negative (inactive) mutant (T17N; Fig. 2, lane 2), (CDC42-GDP or nucleotide-depleted, CDC42-ND) also blocked the wild-type (WT; Fig. 2, lane 3), the constitutively active mutant the interaction (see Discussion). Therefore, by two independent 394 Journal of Cell Science 121 (3)

Fig. 3. The N-terminus of IQGAP1 interacts with EXO70 and SEPT2. (A) Schematic representation of the V5-His-tagged IQGAP1 constructs cloned into the pcDNA3.1 Topo vector and used for transient and stable expressions in this study: IQGAP1 full-length (F1), N-terminal (N1), C-terminal (C2) and the IR-WW domains. For abbreviations of the domains, see legend to Fig. 1. (B) Endogenous or exogenous IQGAP1 can co-immunoprecipitate two exocyst subunits and SEPT2 from different cell lines. Left: anti-IQGAP1 antibodies were used to co-immunoprecipitate SEC8, EXO70 and SEPT2 subunits from total lysate of the indicated cell lines and 5% of representative input lysate was blotted as a loading control; Mock is a negative control immunoprecipitate. Right: V5 antibodies for the recombinant IQGAP1 constructs expressed in COS7 cells shown in C were used for IP, and western blots (WB) were probed with antibodies for SEC8, EXO70

Journal of Cell Science and SEPT2. Cells expressing the vector alone were included as negative controls (last lane). (C) Pull-down of the recombinant IQGAP1 proteins with GST-SEPT2 or GST-EXO70. Left: expression of the IQGAP1 constructs resolved on a 10-20% gradient SDS-PAGE and blotted with V5 antibodies. Right: the lysates shown on the left were incubated with immobilized GST alone (lysate from N1 cells is represented in the first lane), GST-SEPT2 (upper panel) or GST-EXO70 (lower panel) and processed as described in the Materials and Methods. The blots were probed with V5 antibodies to detect the recombinant IQGAP1 proteins. The bottom section of each panel demonstrates equal input of GST-SEPT2 (upper panel) or GST-EXO70 (lower panel). (D) Pull-down of GST-SEPT2 and GST-EXO70 with 6ϫHis-IQGAP1-N. Proteins from cells expressing V5-His-IQGAP1-N or V5-His vector alone were bound to the TALON cobalt resin, incubated with the bacterial lysates of GST-SEPT2 (upper panel) or GST-EXO70 (middle panel) and blotted with SEPT2 or EXO70 antibodies, respectively. 5% of the purified IQGAP1-N or bacterial lysate of GST-EXO70 or GST-SEPT2 were blotted in the lower panel. Data represent three independent experiments with identical results.

lines of evidence – pharmacologically using mastoparan and CDC42-F28L (Fig. 2D, upper panels) without altering the binding genetically using dominant mutant alleles – our data indicate that of other effectors (Lin et al., 1999) and the F⌬MK deletion mutant active CDC42 disrupts the interaction between IQGAP1 and the significantly reduced binding of CDC42 to IQGAP1 (Fig. 2D, exocyst-septin complex; protein subunits implicated in polarized lower panel). Co-precipitation of IQGAP1 with EXO70 was exocytosis. enhanced significantly by these mutations compared with their cognate parental proteins (Fig. 2C), indicating that binding of A physical interaction between CDC42 and IQGAP1 is CDC42 to IQGAP1 is necessary for its negative effects on IQGAP1 necessary for disrupting the IQGAP1-exocyst complex interaction with the exocyst-septin complex. To examine whether binding of CDC42 and IQGAP1 is necessary for the dissociation of IQGAP1 from the exocyst-septin complex, The region of IQGAP1 mediating the association with the we constructed CDC42 and IQGAP1 mutants that are unable to exocyst and septin bind to each other. The double mutant HA-CDC42-F28LC37A and Next, we investigated whether CDC42 displaces the exocyst-septin the deletion mutant V5-IQGAP1-F⌬MK, in which the 24 amino complex by competing for binding. Thus, to identify the domain(s) acids M1054-K1077 of IQGAP1, required for CDC42 binding, are of IQGAP1 that mediate the interaction with EXO70 and SEPT2, deleted (Mataraza et al., 2003), were transiently expressed in ␤TC- antibodies against the V5 tag of the IQGAP1 constructs shown in 6 cells, verified for binding (Fig. 2D) and co-precipitation of Fig. 3A (supplementary material Fig. S1Ci) were used to co- IQGAP1 with EXO70 was performed (Fig. 2C) as described above. precipitate two different subunits of the exocyst, SEC8 and The C37A mutation specifically reduced IQGAP1 binding to EXO70, and SEPT2 (Fig. 3B, right panels). Exogenous full-length IQGAP1 regulates exocytosis 395

Fig. 4. Confocal images of IQGAP1 co-localization with EXO70. Upper panels: fixed HeLa cells were double-stained with antibodies for IQGAP1 (Texas red) and EXO70 (Alexa- Fluor-488, green) and examined with a confocal microscope. Lower panels: Left, confocal slice (~0.2 ␮m) of the perinuclear area. Middle, thin confocal slice (~0.18 ␮m). Right, high- resolution image of the merged section in the upper panel for the cytoplasmic tubular structures (arrow) of IQGAP1-EXO70. No post-image-acquisition processing was carried out.

by confocal microscopy (Fig. 4). As expected for interacting proteins, in >90% (n=100) of the examined cells, IQGAP1 and EXO70 concentrated together at the leading edge of the cells, consistent with previously reported localization of the individual proteins (Watanabe et al., 2004; Bensonor et al., 2007; Zuo et al., 2006). In addition, they co-localized in a perinuclear meshwork and cytoplasmic tubular structures, presumably the ER (Fig. 4, arrow). This overlapping localization is consistent with their biochemical association. IQGAP1-F (F1; Fig. 3B, first lane), IQGAP1-N (N1; Fig. 3, second We further examined the effect of IQGAP1 on EXO70 lane) and the IR-WW (Fig. 3B, third lane), but not IQGAP1-C (C2; localization by RNA interference (RNAi). Control RNAi did not Fig. 3B, fourth lane) or the empty vector (Fig. 3B, fifth lane), could affect IQGAP1 expression in HeLa cells (Fig. 5A, first lane), associate with the exocyst in vivo. Conversely, antibodies for whereas IQGAP1-RNAi (Fig. 5A, second lane) diminished it by IQGAP1 co-precipitated the two-exocyst subunits and SEPT2 from ~90% without affecting the expression of IQGAP2 (nor IQGAP3, different mammalian cell lines (Fig. 3B, left panels, supplementary not shown), EXO70 or SEPT2, confirming the specificity of material Fig. S1Ai), confirming the functionality of the IQGAP1-RNAi. In >90% (for n=50) of IQGAP1-RNAi cells, recombinant IQGAP1 proteins. EXO70 signal was faint, and not readily detectable at the To verify that IQGAP1-N mediates the interaction with membrane ruffles, requiring a four-times longer exposure to be EXO70-SEPT2, bacterially expressed GST-vector for control, visible (Fig. 5B). These data suggest that EXO70 might depend on GST-SEPT2 (Fig. 3C, upper panel) and GST-EXO70 (Fig. 3C, IQGAP1 or factors organized by it such as actin or microtubules right, lower panel) were used as affinity reagents to pull down for proper localization. Alternatively, EXO70 polarized transport the recombinant V5-IQGAP1 domains expressed in COS7 cells might be impaired, explaining the faint signal at the membrane

Journal of Cell Science (Fig. 3C, left). GST alone (Fig. 3C, represented in lane 1 of both ruffles. panels with N1 cell lysate) did not pull down any domain, whereas GST-SEPT2 and GST-EXO70 pulled down V5-N1 (Fig. Co-localization and effects of IQGAP1 expression on SEPT2 3C, lane 2) but not V5-C2 (Fig. 3C, lane 4). Contrary to the in organization vivo data, an association between the V5-IR-WW domain and Endogenous SEPT2 and IQGAP1 also co-localized to the plasma GST-EXO70 was not detected, and GST-SEPT2 pulled down a membrane both in HeLa and pancreatic ␤-cells (Fig. 6; weak band (Fig. 3C, upper panel, lane 3), suggesting that the supplementary material Fig. S2), in agreement with the reported entire N-terminus is required for efficient in vitro interaction. The SEPT2 localization to the plasma membrane in interphase and role of IQGAP1-N was confirmed further by conversely pulling mitotic MDCK (Spiliotis and Nelson, 2006) and in PC12 cells down GST-SEPT2 or GST-EXO70 with 6ϫHis-IQGAP1-N1 (Beites et al., 1999). immobilized on cobalt resins (Fig. 3D). These results confirm that Further, we measured the effects of IQGAP1 overexpression on IQGAP1-N plays a role in mediating the association with the EXO70 and SEPT2 localization. HeLa cells stably expressing exocyst-septin subunits. Consistent with this finding, exogenous equal but low levels (<10% of WT) of the V5-IQGAP1 domains V5-N1 and V5-IR-WW, but not other IQGAP1 domains, co- did not show any measurable effect on EXO70 or SEPT2 immunoprecipitated with EXO70 in ␤TC-6-CDC42WT stable localizations (not shown), and expression of IQGAP1-F or cell lines (supplementary material Fig. S1B), abrogating the IQGAP1-C had no detectable effects on SEPT2 organization (not inhibitory effects of CDC42 on the endogenous binding. shown). However, cells expressing the IR-WW domain, which Furthermore, these data suggest that the dissociation of the binds to the exocyst-septin complex, exhibited disorganized IQGAP1-exocyst-septin complex is unlikely because of a SEPT2 filaments, as characterized in 200 cells each from four binding-site competition with CDC42, which binds to the C- independent experiments represented in Fig. 6A. In control cells, terminus of IQGAP1. Rather, a different mechanism is involved SEPT2 localized in a perinuclear position and prominently (see Fig. 9). decorated the cell peripheries with a few visible SEPT2 filamentous structures in some cells (Fig. 6A, upper panels), Co-localization of endogenous IQGAP1 with EXO70 and the whereas, in the IR-WW cells, SEPT2 filaments were elaborate, effect of IQGAP1 depletion thick and randomly distributed across the cells or often clustered As an additional measure for IQGAP1 association with EXO70, in the nuclear vicinity (Fig. 6A, lower panel), suggesting the their localization was compared in fixed double-stained HeLa cells involvement of IQGAP1 in septin-filament organization. 396 Journal of Cell Science 121 (3)

Differential effects of IQGAP1 domains on secretion As a readout assay for functional association of IQGAP1-exocyst-septin, we measured insulin secretion in pancreatic ␤TC-6 cells, comparing basal and glucose-induced secretion in cells expressing equal levels of the V5-IQGAP1 domains (Fig. 7). The basal secretion levels were slightly enhanced by the expression of IQGAP1 constructs (Fig. 7A). However, expression of IQGAP1-C blocked the induced insulin exocytosis, whereas that of IQGAP1-F had no significant effect. By contrast, expression of IQGAP1-N or IR- WW enhanced exocytosis significantly (>50%). Similar results were obtained from ␤HC-9 cells using a different kit or using radiolabeled methods (not shown). Furthermore, IQGAP1-C appears to reduce the endogenous association between IQGAP1 and the EXO70-SEPT2 complex Fig. 5. Effects of IQGAP1 depletion on EXO70. (A) Depletion of IQGAP1 with targeted RNAi. (supplementary material Fig. S1Cii,iii), mimicking A representative western blot with the indicated antibodies from equal amounts of total protein the effect of activated CDC42. Thus, the domain of from HeLa cells transfected at 100 nM with either control RNAi (first lane) or IQGAP1-RNAi (second lane). (B) Effects of IQGAP1 depletion on EXO70 localization. Upper panels: co- IQGAP1 that interacts with the exocyst-septin localization of IQGAP1 with EXO70 in control HeLa cells. Fixed cells were double-stained with complex enhances exocytosis, whereas the domain antibodies for IQGAP1 (Texas red) and EXO70 (Alexa-Fluor-488, green). Lower panels: that interacts with CDC42 acts as dominant- localization of EXO70 (right panel) in HeLa cells that were depleted of IQGAP1 (left panel). negative, inhibiting exocytosis. To examine the role of endogenous IQGAP1, we measured the stimulated level of secreted insulin in Next, we investigated the effect of IQGAP1 depletion on SEPT2 ␤TC-6 cells treated with IQGAP1-RNAi (supplementary material localization in 70 cells depleted of IQGAP1 by RNAi (Fig. 6B). Fig. S3). Depletion of IQGAP1 did not affect the expression levels Both at 100 nM (Fig. 6B, left) and 40 nM (Fig. 6B, right) of EXO70 or SEPT2 (Fig. 5A and supplementary material Fig. concentrations of IQGAP1-RNAi, SEPT2 localization was not S3Ai), but decreased induced insulin secretion by ~45%, which readily detectable at the plasma membrane, as was the case with was rescued by co-transfection of an RNAi-refractory IR-WWR EXO70. Therefore, IQGAP1 overexpression or depletion affects domain (supplementary material Fig. S3Aii,B). These data affirm septin localization and filament organization. the dominant-negative effects of IQGAP1-C and indicate further

Journal of Cell Science In pancreatic ␤-cells, we observed a similar localization of that IQGAP1-N serves as a dominant-positive (Fig. 7A). Although IQGAP1 using ER and membrane markers (supplementary in the immune system delivery of secretory lysosomes requires the material Fig. S2). In single cells, IQGAP1 concentrated at a clearing of both actin and IQGAP1 from the target site of the paranuclear position, but when cell-cell contacts were formed plasma membrane (Stinchcombe et al., 2006), depletion of (islet-like cells), it concentrated in the cell peripheries. Although IQGAP1 in mast cells only mildly enhances agonist-stimulated the reason behind this switch in localization is unclear, histamine secretion (Psatha et al., 2007). Together, these data localization of the ER marker inositol 1,4,5-triphosphate receptor, support the concept that the essential role of IQGAP1 in secretion type 3 (IP3R3, ITPR3) revealed an identical pattern to that of is regulatory. IQGAP1 in these cells (supplementary material Fig. S2A). IP3R3 is an ER resident protein shown to switch localization from the IQGAP1 influences protein synthesis ER to the plasma membrane when MDCK cells polarize Our secretion results, however, do not differentiate between (Colosetti et al., 2003), presenting a possibility that pancreatic ␤- whether the enhancement of secretion is a result of increased cells undergo polarization upon forming cell contacts and that release from cellular stores or due to an increase in protein IQGAP1 switches localization from the ER to the plasma synthesis. Therefore, we measured protein synthesis rates in the membrane upon polarization of specific cell types. A similar stable cell lines by pulse-chase experiments. The labeled basal pattern was also observed for EXO70 and SEPT2 in these cells levels of insulin in cells expressing IQGAP1-N or IQGAP1-C were (not shown). higher compared with their vector-control cells (Fig. 7B, left To verify that these cell-cell contacts are the plasma membrane, panels), consistent with their basal secretion level (Fig. 7A), and in particular that these proteins are cytosolic (supplementary perhaps indicating a deregulation in the basal steady state. material Fig. S2Aii), we used the t-SNARE syntaxin 1A as a However, when these cells were chased for 20 minutes (not shown) membrane marker. IQGAP1 and syntaxin 1A overlapped in the or 1 hour and the labeled insulin levels measured by IP, IQGAP1- cell-cell contacts in pancreatic ␤-cells (supplementary material Fig. C cells retained higher levels of labeled proteins compared with S2B) and in HeLa cells (Fig. 8), indicating that IQGAP1 localizes their vector control and IQGAP1-N cells (Fig. 7B, right panels). at the plasma membranes of different cell types. Collectively, these The persistence of the labeled proteins in IQGAP1-C cells indicates data suggest that IQGAP1 co-localizes with and influences EXO70 a reduction in protein synthesis and exocytosis rates. localization and septin-filament organization, and that increasing The disappearance of labeled proteins in IQGAP1-N cells or decreasing the dosage of IQGAP1 has cellular effects. indicates higher rates of both protein synthesis and exocytosis, IQGAP1 regulates exocytosis 397

Fig. 6. Co-localization of IQGAP1 and SEPT2, and the effects of IQGAP1 depletion or overexpression on SEPT2 organization. (A) Stable expression of the IR- WW domain induces septin-filament disorganization. Immunofluorescence was performed in vector-control (upper panels) and IR-WW stable (lower panels) HeLa cells that were grown in dual-chamber slides with antibodies against SEPT2. (B) Localization of SEPT2 in IQGAP1-depleted cells. HeLa cells depleted for Journal of Cell Science IQGAP1 at 100 nM and 40 nM were double-stained with anti-SEPT2 (bottom, green) and anti-IQGAP1 (top, red) antibodies.

thereby replacing the labeled with new proteins. To verify whether 2003; Toikkanen et al., 2003) lends credence to such an this was the case, we measured the total insulin levels in the labeled idea. Furthermore, upon an ExPASy proteomic server cells by immunoblotting, revealing that total insulin levels were (http://br.expasy.org/) search for motifs on IQGAP1, we identified similar in N1 and C2 cells, and higher than in their vector-control an ER membrane-retention signal (KFYG) on the extreme C- cells (Fig. 7B, lower panel). In addition, immunofluorescence of terminus. Because IQGAP1 is a cytosolic protein and because an insulin in stable ␤TC-6 cells that were stimulated for 1 hour ER luminal signal (KDEL) is absent from IQGAP1, this confirmed these immunoblot data, showing more insulin in membrane-retention signal might be involved in retrieval events at IQGAP1-N and IQGAP1-C cells (Fig. 7C) caused by active protein the ER membrane. Therefore, we examined whether the synthesis in the former and defective synthesis/secretion rates in localization of IQGAP1 in the perinuclear meshwork observed in the latter. Taken together, these results indicate that both protein HeLa cells in Fig. 4 and Fig. 5B might be to the ER. This was synthesis and exocytosis are impaired in IQGAP1-C cells because tested by co-localization of IQGAP1 with two ER resident markers they accumulate labeled insulin and exhibit low secretion level. in a total of three cell types: with calnexin in HeLa cells (Fig. 8A), Thus, by three different measurement criteria – insulin-secretion and with the IP3R3 in NIH3T3 cells (Fig. 8B) and ␤-cells assays, pulse-chase for protein synthesis rate, and (supplementary material Fig. S2A). In the three cell types, IQGAP1 immunofluorescence – our data support the conclusion that the C- overlapped with each marker in the perinuclear area, whereas it terminus of IQGAP1 inhibits protein synthesis and exocytosis, localized alone to the leading edges of NIH3T3 and HeLa cells (in whereas its N-terminus enhances both of them. ~90% for n=200 of cells examined). However, we also observed non-overlapping spots of the proteins, perhaps reflecting IQGAP1 localizes and associates with sites of protein specialized functions of each protein. synthesis and exocytosis Thus, we investigated whether this ER localization indicates If IQGAP1 is involved in protein synthesis/translocation, then it an association with the ER translocon, as is the case with its would be expected to localize to the ER. The recent discovery exocyst partner SEC8 subunit (Lipschutz et al., 2003). Co- that the vesicle-tethering exocyst has a conserved role in immunoprecipitation of endogenous IQGAP1 with the ER communicating with the ER-translocon complex (Lipschutz et al., translocon subunit Sec61␤ from HEK293NT cells (and MD- 398 Journal of Cell Science 121 (3)

Fig. 7. IQGAP1 influences protein synthesis and exocytosis. (A) Differential effects of IQGAP1 domains on insulin exocytosis. Early passages of ␤TC-6 cells (Beta) stably expressing similar levels of the V5-IQGAP1 constructs (on the x-axis) were selected to measure insulin exocytosis (ng/ml) in duplicate. Means ± s.d. for n=6 are shown. Values were normalized to cells that incorporated the vector (Beta-V), which were used as a control. For each construct, the left column represents the basal and the right column the glucose-induced insulin exocytosis. Statistical significance: P<0.001. (B) IQGAP1 influences protein synthesis. Early passages of ␤TC-6 clones stably expressing the indicated IQGAP1 domains were pulsed and chased/stimulated with glucose, as described in the Materials and methods. Upper panels: insulin antibodies were used to immunoprecipitate insulin from the labeled (left) and the chased (right) sets, resolved on a 20% SDS- PAGE and evaluated with a phosphorimager. Lower panel: western blot (WB) of 5% of the lysate from the 1-hour chased cells. The autoradiograph and the blot represent three experiments with identical results. (C) Immunofluorescence of insulin in cells stably expressing the IQGAP1 domains. Fixed ␤TC-6 cells stably expressing equal amounts of the indicated IQGAP1 domains were stimulated for 1 hour, fixed and stained with insulin guinea-pig polyclonal antibodies and Alexa-Fluor-488 goat anti-guinea-pig secondary antibodies, and the images were acquired at a 500-msec exposure time without post-image-acquisition processing. Journal of Cell Science MB231 human breast cell lines, not shown) showed that Interplay between CDC42 and IQGAP1 regulates secretion each antibody conversely precipitated the other protein (Fig. 8D, Next, we examined whether CDC42 disruption of the association top panel). Thus, three lines of supporting evidence – an ER signal, of IQGAP1 with the exocyst-septin complex consequently disrupts localization with two ER markers and co-immunoprecipitation secretion. In such a case, we investigated whether expression of with the ER translocon – suggest that IQGAP1, like its exocyst IQGAP-C, which binds CDC42, would abrogate the disruption by partner, at least in part localizes to the ER and interacts with the sequestering CDC42, thereby allowing endogenous binding of translocation complex, providing additional support that it IQGAP1 and exocyst. Thus, we measured glucose-stimulated influences protein synthesis/translocation. insulin secretion in pancreatic ␤TC-6 cells stably expressing Furthermore, we examined whether IQGAP1 localization at the CDC42WT, in which the association is disrupted, comparing it plasma membrane indicates a connection with membrane-fusion with those stably co-expressing the V5-IQGAP1 domains (Fig. proteins in the secretory pathway. A likely candidate is the vesicle- 9A). First, our data affirmed a previous finding that stable fusion protein syntaxin 1A, which is a member of the t-SNARE expression of CDC42WT inhibits glucose-stimulated insulin complexes that are abundant in pancreatic ␤-cells. Syntaxin 1A has secretion (Fig. 9A, last two columns) (Daniel et al., 2002) and been shown to bind septins in order to regulate exocytosis (Beites further showed that expression of IQGAP1-F (F1) had no et al., 1999), to bind the exocyst to complete cytokinesis (Gromley measurable effect on the inhibition (Fig. 9A, first columns). By et al., 2005), and was recently shown to regulate docking and contrast, expression of IQGAP1-N or IQGAP1-IR-WW abrogated fusion of insulin granules in the first phase release (Ohara- the inhibition caused by CDC42WT, enhancing secretion Imaizumi et al., 2007). Our data show that IQGAP1 localized and significantly (Fig. 8A, second and fourth columns). Unexpectedly, co-precipitated with syntaxin 1A at polarized membrane regions in expression of IQGAP1-C (C2), which binds CDC42, did not HeLa (Fig. 8C, Fig. 8D, bottom panel) and ␤-cells (supplementary abrogate the secretion inhibition in CDC42WT stable cells (Fig. material Fig. S2B). Future sub-cellular fractionation and/or 8A, third columns). This finding is consistent with the result that immuno-electron-microscopy (immuno-EM) studies will be exogenous N-terminus and IR-WW domain can bind EXO70 in required to define exact points of overlap of these proteins. CDC42WT cells (supplementary material Fig. S1B), whereas However, these findings provide additional support for an IQGAP1 expression of IQGAP1-C depresses the level of endogenous bound role in exocytosis with the vesicle-tethering exocyst and vesicle- IQGAP1-EXO70 (supplementary material Fig. S1C). Therefore, fusion SNAREs. we investigated a possible mechanism that generates such an effect. IQGAP1 regulates exocytosis 399

Fig. 8. Localization and association of IQGAP1 with sites of protein synthesis and exocytosis. (A) Confocal slice (0.16 ␮m) showing the co-localization of IQGAP1 (red) with the ER marker calnexin (green) in HeLa cells. Arrows indicate localization spots. (B) Co-localization of IQGAP1 (red) with the ER resident marker IP3R3 (green) in NIH3T3 cells. (C) Co-localization of IQGAP1 (red) with the t-SNARE syntaxin 1A (green) in HeLa cells. No post-image- acquisition processing was performed. Arrows indicate plasma membrane leading edge or ruffles. (D) Top: co-immunoprecipitation of IQGAP1 with the ER- translocon subunit Sec61␤. Monoclonal antibodies for Sec61␤ or IQGAP1 were used conversely for IP. The immune complexes were resolved on a 15% SDS-PAGE and conversely blotted with anti-IQGAP1 or -Sec61␤ antibodies. Bottom: co-immunoprecipitation of IQGAP1 and syntaxin 1A (Synt1A). Anti- Synt1A or -IQGAP1 antibodies were used for IP, and the immune complexes resolved on a 10% SDS-PAGE and conversely blotted with the indicated antibodies.

Journal of Cell Science Several lines of evidence from yeast studies suggest that Discussion localized activation of Cdc42p occurs at membrane sites by IQGAP1 links the exocyst-septin complex with CDC42 positional cues, such as Iqg1p (reviewed in Osman and Cerione, signaling to regulate protein synthesis and exocytosis 2005). Given our finding that activation of CDC42 disrupts the In this study, we presented evidence for a novel role of IQGAP1, association of IQGAP1 with the exocyst-septin complex and a CDC42 target/effector, in regulating secretion by associating with inhibits secretion, we investigated whether expression of and influencing the localization of EXO70 and the organization of IQGAP1 activates endogenous CDC42, leading to such effects. SEPT2 (Figs 1-6), two subunits of protein complexes implicated Therefore, we measured the amounts of active CDC42 by using in cell polarization and directed exocytosis. The co-localization the CDC42/Rac interactive binding (CRIB) region of PAK, data agree with previously reported localization of the individual which specifically binds active CDC42 (GTP bound). Control ␤- proteins in other cell types. IQGAP1 and the exocyst each localize cells that incorporated the vector alone (V) did not have to the basolateral membrane in gastric parietal cells and to cell-cell measurable amounts of GTP-CDC42 (Fig. 9B, right panel), contacts in MDCK cells (Grindstaff et al., 1998; Katata et al., 2003; which also was the case with IQGAP1-N (N1) cells. However, Zhou et al., 2003). By contrast, the isoform IQGAP2 localizes to expression of either IQGAP1-F (F1) or IQGAP1-C (C2) the apical membranes (Yamashiro et al., 2003), explaining why it enhanced the level of GTP-CDC42. All cells had equal levels of did not associate with the exocyst-septin complex. Similarly, endogenous total CDC42, as demonstrated in the lower panel in septins localize to the plasma membrane, and with microtubule and Fig. 9, and these results are not unique to ␤-cells, because they actin cytoskeletons (Spiliotis and Nelson, 2006), and the effect of confirm similar findings from other cell types (Fukata et al., IQGAP1 on SEPT2 organization resembles that of the Borg3 2002; Mataraza et al., 2002; Grohmanova et al., 2004), affirming protein (Joberty et al., 2001), another effector of CDC42. Borg an apparently conserved function for IQGAP1 upstream of proteins are absent from yeast and other metazoans but IQGAP1 CDC42 (Osman et al., 2002). These data help explain why is widely conserved; thus, these two CDC42 effectors might IQGAP1-C disrupts binding, secretion and protein synthesis, represent different ‘flavors’ for fine-tuning septin functions/ confirming that active CDC42 negatively regulates IQGAP1 organization in different organisms and cell types, or for specificity function in secretion. They also indicate that IQGAP1 modulates in CDC42-divergent signaling pathways. CDC42 activity, in effect serving as a regulator of secretion. The data presented here suggest that IQGAP1 enhances protein Thus, interplay between IQGAP1 and CDC42 plays a role in synthesis and that it resides in the ER, associating with the regulating exocytosis. translocation complex (Fig. 8), and at the plasma membrane, 400 Journal of Cell Science 121 (3)

Fig. 9. Mechanism of the role of IQGAP1 in regulating secretion. (A) CDC42 negatively regulates IQGAP1 secretion and is abrogated by IQGAP1-N1 expression. Pancreatic ␤-cells stably co-expressing CDC42WT (depicted as 42) and a IQGAP1 domain (on the x-axis) were assayed for insulin exocytosis as described in the Materials and Methods. CDC42WT stable cells expressing the V5 vector were used for control (42-V). Means ± s.d. for n=6 are shown. Values were normalized to cells that incorporated the vector (42-V) as a control. (B) IQGAP1 increases cellular CDC42-GTP. Left: the expression level of the V5-IQGAP1 constructs the cells used for the pull-down. Middle: positive (GTP␥S) and negative (GDP) controls for the pull-down experiment. Right: GST construct expressing the CDC42-binding (CRIB) domain of PAK (PBD) was used to pull-down active CDC42 (GTP-CDC42) from ␤-cells expressing IQGAP1 (F1), IQGAP1-C (C2), IQGAP1-N (N1) or vector control (V). Bottom: western blot of 10% of the total protein used for IP demonstrating equal input of cellular CDC42. (C) A model for the mechanism of the role of IQGAP1 in secretion as a conformational switch. In protein synthesis/exocytosis, IQGAP1 operates in a closed form generated by folding of the C- terminus. External or internal signals lead to the phosphorylation of IQGAP1 at a C-terminus serine (pS) and to the binding and activation of CDC42, perhaps to switch effector molecules in order to regulate exocytosis (on or off) or influence alternate cellular functions, such as cell division. Journal of Cell Science associating with vesicle-tethering exocyst and vesicle-fusion By contrast, glucose-stimulated insulin secretion occurs via a Ca2+- SNAREs (Figs 4, 8 and supplementary material Fig. S2A), similar dependent pathway in response to nutrients (Kowluru, 2003), to mammalian and yeast exocysts (Lipschutz et al., 2003; supporting the involvement of IQGAP1 in the Ca2+-dependent Toikkanen et al., 2003). These findings support the idea of nutrient-stimulated pathway. Significantly, Ca2+ has been shown to involvement of these protein complexes in a positive-feedback dissociate IQGAP1 from CDC42 (Ho et al., 1999), lending support loop for exocytosis. In mammals, the Sec61␤ subunit that to our finding and to the model presented below that dissociation associates with IQGAP1 and the exocyst binds non-translating of CDC42 is necessary to allow the effects of IQGAP1 on ribosomes (Levy et al., 2001). By contrast, IQGAP1 and CDC42, secretion. but not other effectors such as WASP, were purified as binding- partners for the double-stranded RNA-binding polarity protein Interplay between CDC42 and IQGAP1 regulates the Staufen as part of RNA granules involved in the in vivo localization function(s) of IQGAP1 and translation of human mRNAs (Villace et al., 2004). Future Our results indicate that IQGAP1 serves both as an upstream investigation should reveal whether IQGAP1 tethers ribosomes to activator and downstream target for CDC42, in agreement with the mRNAs, serving as a scaffolding positional marker. previous finding that its yeast counterpart, Iqg1p, serves as an axial marker upstream of Cdc42p (Osman et al., 2002). Therefore, either Involvement of IQGAP1 in physiological secretion a sequestration or an activation model could account for the Roles for the exocyst in insulin secretion and septins in glucose- negative effects of CDC42 and IQGAP1-C on secretion. In the first stimulated growth-hormone release have been reported previously model, CDC42 binds and sequesters IQGAP1 from the exocyst- (Inoue et al., 2003; Beites et al., 1999), and the data presented here septin complex, thereby inhibiting secretion. In this scenario, further indicate that IQGAP1 associates with these protein ectopic expression of IQGAP1-C, which binds CDC42, would be complexes to regulate their effects on secretion in cooperation with expected to block the inhibition caused by CDC42. However, CDC42-GTPase (Figs 1 and 9). This agrees with the finding that expression of IQGAP1-C slightly exacerbated that inhibition (Fig. stable expression of CDC42, while enhancing mastoparan- 9) and depressed endogenous IQGAP1-EXO70 association activated CDC42 insulin secretion, inhibited glucose-stimulated (supplementary material Fig. S1C). Therefore, the sequestration secretion (Fig. 9) (Daniel et al., 2002). Mastoparan is a toxin from model cannot alone explain these effects, but they are consistent wasp venom that stimulates insulin release independent of Ca2+. with the activation model discussed below. IQGAP1 regulates exocytosis 401

Because CDC42 disrupts the association of IQGAP1 with the as cytokinesis, are positively influenced in this open, CDC42- exocyst-septin complex and expression of IQGAP1-F or IQGAP1- bound, state. C increases the cellular level of active CDC42 (Fig. 9B) and decreases the endogenous IQGAP1-EXO70 association, these data Materials and Methods provide a probable explanation as to why IQGAP1-F and IQGAP- Construction and expression of IQGAP1 domains C blocked secretion (Fig. 9A). IQGAP1 also binds nucleotide- Full-length IQGAP1, IQGAP-F1, IQGAP1-N (N1), IQGAP1-C (C2) and the IR- depleted CDC42 (ND-CDC42) and GDP-CDC42 (Grohmanova et WW domain (Fig. 3A) were generated by high-fidelity PCR, cloned into the expression vector TOPO pCDNA3.1 (Invitrogen) and confirmed by DNA sequencing al., 2004), confirming its GEF-like activity on CDC42 and (Cornell BRC facility) to create V5-6ϫHis double-tagged proteins. The providing an explanation as to why wild-type CDC42 blocked both QuickChange site-directed mutagenesis kit (Stratagene) was used to delete amino interaction and secretion (Figs 1 and 8) similar to its dominant acids M1054-K1077, comprising the CDC42-binding region (Mataraza et al., 2003), from the V5-tagged pcDNA3.1 plasmid encoding IQGAP1-F1, thus generating the active mutants, indicating that increasing the level of CDC42 FDMK construct. Three silent mutations (underlined) were introduced into the V5- enhances the pool of its active form, leading to the observed IR-WW plasmid at nucleotides 363-5Ј-TGCAATGGATGAAATTGGG-3Ј-381, inhibition. creating an RNAi-refractory [adapted from Watanabe et al. (Watanabe et al., 2004)] IR-WWR domain. Plasmid HA-pCDNA3-CDC42F28L was similarly mutated to add By contrast, IQGAP1-N, which binds the exocyst-septin C37A, which reduces the binding of IQGAP1 to CDC42. All constructs were complex (this study), was reported to self-associate and to inhibit confirmed by DNA sequencing and expression was further confirmed by recognition CDC42 activation, perhaps by associating with C-terminus of the expected sizes with V5-tag antibodies (Invitrogen) as well as with IQGAP1 antibodies specific for the N- or the C-halves of IQGAP1 (Upstate and Santa Cruz sequences of endogenous IQGAP1 (Mataraza et al., 2002; Le Biotech). Clainche et al., 2007), which would present a synergistic mechanism for enhancing secretion. Together, these data affirm the Cell culture, transfection, RNAi and IP ␤ idea that IQGAP1 modulates CDC42 activity, but, at present, it TC-6 cells were purchased from ATCC (CRL-11506) and mastoparan PTX was from Sigma-Aldrich. The transfection efficiency of ␤TC-6 cells was >70%, as remains unclear how, because purified recombinant IQGAP1 failed determined by GFP, and, like ␤HC-9 cells, they lost glucose sensitivity with higher to exhibit a direct GEF activity on CDC42. It is likely that IQGAP1 passage numbers. Therefore, early passages were used and the passage number was recruits a CDC42 GEF to facilitate an exchange activity. kept low. All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% calf serum (NIH3T3) or 10% fetal bovine serum Nevertheless, CDC42 activation impairs IQGAP1 function in (FBS; 15% for ␤TC-6 and 20% for ␤HC-9) and 100 units/ml penicillin, 100 ␮g/ml exocytosis by displacing IQGAP1 from its downstream effectors, streptomycin (Invitrogen) in a humidified 5% CO2 incubator at 37°C. HeLa cells such as the exocyst-septin complex (Fig. 1C, Fig. 2 and (ATCC) were cultured in MEM under the same conditions. Stable transfection of HeLa and ␤-TC6 cells was performed following the Invitrogen manual-selecting for supplementary material Fig. S1), by facilitating the binding of an clones with equal expression levels. For transient expression, cells from 100 mm inhibitor, or by dissociating the exocyst and/or septins from plates were transfected with 9 ␮g DNA with Lipofectamine (Invitrogen) following IQGAP1, leading to regulated inhibition of secretion in response the manufacturer’s instructions. After 48 hours, the cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed on ice for 20 minutes in buffer (25 mM to intrinsic or extrinsic stimuli. HEPES, pH 7.4, 15 mM MgCl2, 150 mM NaCl, 1% NP40, 10 ␮g/ml each of leupeptin and aprotinin and 0.2 mg/ml phenylmethylsulfonic chloride) prior to A mechanism for IQGAP1 in regulating protein synthesis and centrifugation at 13,000 g for 10 minutes. Protein concentrations were determined with the Bio-Rad Dc kit and equal amounts precleared with beads (15 ␮L) for 1 hour secretion at 4°C and used for IP. Briefly, the antibody was added to the precleared lysate, The finding that IQGAP1-C inhibits the function of IQGAP1 in incubated on ice for 1 hour and 40 ␮l of PBS-equilibrated protein A or G beads were Journal of Cell Science exocytosis suggests that this domain serves as a dominant-negative added and gently rocked overnight at 4°C. The beads were washed four times with 1 ml lysis buffer, boiled for 10 minutes in 40 ␮l 2ϫSDS sample buffer and loaded (Figs 1, 6 and 8). Apparently, this represents a common mechanism for SDS-PAGE. for IQGAP1, because this domain also inhibits the ability of Immunoblotting was performed with the antibodies indicated in the figures. IQGAP1 to dissociate cell-cell contacts (Fukata et al., 2001). How Antibodies for IQGAP1 and IQGAP2 were from Upstate Biotech, for IQGAP3 were this translates on the cellular level could be subject to many from Novus Biologicals, and those for EXO70 and SEPT2 were previously described (Vega and Hsu, 2001; Vega and Hsu, 2003). Mouse anti-rSec8 was from Stressgen interpretations; however, a likely mechanism is that IQGAP1 acts Biotechnology and HA from Covance. Insulin antibodies were from Abcam and as a conformational switch, operating in open or closed molecular secondary antibodies were from Jackson’s Laboratory or from Molecular Probe. The states (Fig. 9C). With regard to secretion, IQGAP1-C mimics immunoblot images were acquired with a Bio-Rad ChemiDoc XRS imager. The human IQGAP1 RNA 21-oligomers (5Ј-UGCCAUGGAUGAGAUUGGA- inactive (open) states and IQGAP1-N represents active (closed) 3Ј) were synthesized (Dharmacon) in both sense and antisense directions with dTdT states. This would explain why overexpression of full-length overhang at the 3Ј termini. The sequences were searched for in the GenBank database IQGAP1 had little effect on secretion and presents a mechanism against the , ensuring that only IQGAP1, and not its isoforms IQGAP2, IQGAP3 or other , was targeted. A scramble IQGAP1 sequence: 5Ј- whereby IQGAP1 could function positively or negatively with CAGUCGCGUUUGCGACUGG-3Ј and the siCONTROL non-targeting oligomer CDC42 in one state versus the other (Fig. 9C). In support of this from Dharmacon were used as control. The oligos were transfected at 100 nM with model is the finding by independent groups that two different Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. After 48 hours, the cells were fixed for immunofluorescence or lysed for western blotting. regions on the C-terminus form complexes in vitro (Fukata et al., The transfection efficiencies in HeLa cells were 70-90%, as monitored by 2001; Grohmanova et al., 2004) and that phosphorylation of serine fluorescent-label RNAi from Dharmacon. 1443 in the second region prevents this interaction, increasing the binding of nucleotide-depleted CDC42 (Grohmanova et al., 2004). In vitro pull-down assays The GST-EXO70 and GST-SEPT2 fusion proteins were previously described (Vega This model is generally consistent with that for DBL proteins and Hsu, 2001; Vega and Hsu, 2003). These were expressed in Escherichia coli BL21 (CDC42-GEFs) shown to exist in conformational states regulated (DE3) and sonicated in buffer S (20 mM Tris, pH 8, 2 mM EDTA, 2 mM DTT, 150 by tyrosine phosphorylation, which causes an opening of their mM NaCl, 0.1% Tween 20). The sonicates were centrifuged at 18,000 g for 15 minutes at 4°C, the supernatant was incubated with GST beads overnight with gentle structure leading to the activation of Rho GTPases (Aghazadeh et rocking at 4°C, and the beads were washed three times in buffer S and incubated al., 2000). In exocytosis, IQGAP1 operates in an inverse (closed) with equal amounts of proteins from COS7, precleared with GST beads, overnight state in which an opening of its structure leads to binding and at 4°C with gentle rocking. The beads were washed four times with excess buffer S, boiled in ~40 ␮l 2ϫSDS sample buffer and used for immunoblotting. For the activation of CDC42, resulting in the inhibition of secretion (Fig. reciprocal experiment, His-IQGAP1 proteins were purified using the BD TALON 9C). It is plausible, however, that other functions of IQGAP1, such cobalt-based resins (BD Biosciences), following the manufacturer’s instructions, 402 Journal of Cell Science 121 (3)

incubated with the bacterial extracts containing GST-SEPT2 or GST-EXO70 and Beites, C. L., Xie, H., Bowser, R. and Trimble, W. S. (1999). The septin CDCrel-1 binds processed as described above. The GST-PBD assay for CDC42 activity was syntaxin and inhibits exocytosis. Nat. Neurosci. 2, 434-439. performed using the CDC42 activation kit from Upstate following the manufacturer’s Bensenor, L., Kan, H.-M., Wang, N., Wallrabe, H., Davidson, L. A., Cai, Y., Schafer, instructions. The immunoblot images were acquired with a Bio-Rad ChemiDoc XRS D. A. and Bloom, G. S. (2007). IQGAP1 regulates cell by linking growth factor imager and exported as TIFF files. signaling to actin assembly. J. Cell Sci. 120, 658-669. Brill, S., Li, S., Lyman, C. W., Church, D. M., Wasmuth, J. J., Weissbach, L., Microscopy Bernards, A. and Snijders, A. J. (1996). The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with The cells were washed with PBS, fixed in –20°C methanol acetone for 10 minutes, calmodulin and Rho family GTPases. Mol. Cell. Biol. 16, 4869-4878. blocked with 1 mg/ml BSA in PBS and incubated with primary antibodies or IgG as Brown, D. and Sacks, D. B. (2006). IQGAP1 in cellular signaling: bridging the GAP. control followed by secondary antibodies (Texas red and Alexa-Fluor-488, Molecular Trends Cell Biol. 16, 242-249. Probes) for 1 hour each at room temperature. The nuclei were stained with DAPI Colosetti, P., Tunwell, P. E., Cruttwell, C., Arsanto, J. P., Mauger, J. P. and Cassio, D. (Sigma) and the images were captured with a Leica confocal or an Olympus (2003). The type 3 inositol 1,4,5-trisphosphate receptor is concentrated at the tight fluorescence microscope fitted with a CCD camera and Slide Book software with no junction level in polarized MDCK cells. J. Cell Sci. 116, 2791-2803. post-image-acquisition processing except for dark-field correction. Daniel, S., Noda, M., Cerione, R. and Sharp, G. W. (2002). A link between Cdc42 and syntaxin is involved in mastoparan-stimulated insulin release. Biochemistry 41, 9663- Insulin-secretion assays and immunofluorescence 9671. Pancreatic ␤TC-6 cells were stably transfected with the pCDNA3.1 vector alone or Epp, J. A. and Chant, J. (1997). An IQGAP-related protein controls actin-ring formation encoding either the V5-IQGAP1 domains and/or HA-tagged CDC42WT. Early and cytokinesis in yeast. Curr. Biol. 7, 921-929. Fukata, M., Kuroda, S., Nakagawa, M., Kawajiri, A., Itoh, N., Shoji, I., passages were used for insulin-secretion assays. Equal numbers of cells were grown Matsuura, Y., Yonehara, S., Kikuchi, A. and Kaibuchi, K. (1999). Cdc42 and and washed twice with Krebs-Ringer bicarbonate (KRB) buffer (129 mM NaCl, 5 Racl regulate the interaction of IQGAP1 with ␤-catenin. J. Biol. Chem. 274, 26044- mM NaHCO3, 4.8 mM KCL, 1.2 mM KH2PO2, 2.0 mM CaCl2, 1.2 mM MgSO4, 26050. 0.2% BSA, 10 mM HEPES, pH 7.4, and 0.1 mM glucose), incubated at 37°C in the Fukata, M., Nakagawa, M., Itoh, N., Kawajiri, A., Yamaga, M., Kuroda, S. and same buffer for 30 minutes and treated with buffer alone (basal) or with 30 mM Kaibuchi, K. (2001). Involvement of IQGAP1, an effector of Rac1 and Cdc42 GTPase, glucose (stimulated) as previously described (Daniel et al., 2002). Both sets were in cell-cell dissociation during cell scattering. Mol. Cell. Biol. 21, 2165-2183. incubated at 37°C for another 30 minutes. Aliquots of the buffer containing the Fukata, M., Watanabe, T., Noritake, J., Nakagawa, M., Yamaga, M., Kuroda, S., secreted insulin from basal or stimulated cells were collected and stored at –80°C, Matsuura, Y., Iwamatsu, A., Perez, F. and Kaibuchi, K. (2002). Rac1 and Cdc42 and the cells were washed twice with ice-cold PBS, lysed and the expression levels capture microtubules through IQGAP1 and CLIP-170. Cell 109, 873-885. of the constructs were determined by immunoblotting. Cells with equal levels of Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., Rodriguez-Boulan, domain expression were selected for comparison of insulin-secretion levels with the E., Scheller, R. H. and Nelson, W. J. (1998). Sec6/8 complex is recruited to cell-cell ultrasensitive mouse insulin enzyme-linked immunosorbent assay kit contacts and specifies transport vesicle delivery to the basal-lateral membrane in (Immunodiagnostic Systems) following the manufacturer’s instructions. For epithelial cells. Cell 93, 731-740. immunofluorescence, the cells were seeded on chamber slides treated as above and Grohmanova, K., Schlaepfer, D., Hess, D., Gutierrez, P., Beck, M. and Kroschewski, R. (2004). Phosphorylation of IQGAP1 modulates its binding to Cdc42, revealing a new stimulated with glucose, then washed and fixed. type of Rho-GTPase regulator. J. Biol. Chem. 279, 48495-48504. 35 Gromley, A., Yeaman, C., Rosa, J., Redick, S., Chen, C.-T., Mirabelle, S., Guha, M., S-labeling and pulse-chase experiments Sillibourne, J. and Doxsey, S. J. (2005). Centriolin anchoring of exocyst and SNARE Pancreatic ␤TC-6 cells expressing equal amounts of the indicated IQGAP1 domains complexes at midbody is required for secretory vesicle-mediated abscission. Cell 123, were seeded at 4ϫ104 in 100-mm plates, rinsed once in methionine- and cysteine- 75-87. free DMEM and incubated in the same medium containing 10% dialyzed FBS for Ho, Y. D., Joyal, J. L., Li, Z. and Sacks, D. B. (1999). IQGAP1 integrates 2-3 hours prior to labeling with 0.1 mCi/ml of 35S Met/Cys (Express Protein Labeling Ca2+/calmodulin and Cdc42 signaling. J. Biol. Chem. 274, 464-470. Mix, Perkin Elmer) for 30 minutes at 37°C in 3.5 ml of the same medium. The cells Hsu, S. C., Ting, A. E., Hazuka, C. D., Davanger, S., Kenny, J. W., Kee, Y. and Scheller, were washed once with 10 ml normal culture medium and incubated in fresh medium R. H. (1996). The mammalian brain rsec6/8 complex. Neuron 17, 1209-1219. containing glucose for the indicated time points, washed in cold PBS and lysed on Hsu, S. C., Hazuka, C. D., Roth, R., Foletti, D. L., Heuser, J. and Scheller, R. H. (1998). ice for 20 minutes in buffer A [40 mM HEPES, pH 7.5, 120 mM NaCl, 1 mM EDTA, Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron 20, 1111-1122. Journal of Cell Science 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 orthovanadate, Hsu, S. C., Hazuka, C. D., Foletti, D. L. and Scheller, R. H. (1999). Targeting vesicles EDTA-free protease inhibitors (Roche) and 1% Triton X-100]. The lysate was cleared to specific sites on the plasma membrane: the role of the sec6/8 complex. Trends Cell by centrifugation at 13,000 g for 10 minutes. The protein concentration was ␮ Biol. 9, 150-153. determined and 50 g was loaded for the total, or equal amounts were used for IP, Inoue, M., Chang, L., Hwang, J., Chiang, S. H. and Saltiel, A. R. (2003). The exocyst as described above, with insulin antibodies (Abcam) and resolved on 20% SDS- complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature PAGE. The images were acquired with a phosphoimager. 422, 629-633. Joberty, G., Perlungher, R. R., Sheffield, P. J., Kinoshita, M., Noda, M., Haystead, T. Cell fractionation and Macara, I. G. (2001). Borg proteins control septin organization and are negatively ␤HC-9 cells were washed with cold PBS, homogenized in buffer H (150 mM NaCl, regulated by Cdc42. Nat. Cell Biol. 3, 861-866. 100 mM Tris, pH 7.4, 10 mM MgCl2 and protease inhibitors) and centrifuged for 5 Johnson, K. and Wodarz, A. (2003). A genetic hierarchy controlling cell polarity. Nat. minutes at 3024 g. The supernatant was centrifuged at 27,216 g for 15 minutes prior Cell Biol. 5, 12-14. to ultra-centrifugation at 100,000 X g for 90 minutes. The supernatant was saved as Katata, T., Irie, K., Fukuhara, A., Kawakatsu, T., Yamada, A., Shimizu, K. and Takai, the cytosol fraction and the pellet suspended in the same buffer containing 1% NP40, Y. (2003). Involvement of nectin in the localization of IQGAP1 at cell-cell ahesion sites incubated on ice for 30 minutes with regular mixing then centrifuged at 27,216 g for through the actin in Madin-Darby canine kidney cells. 22, 2097- 2109. 30 minutes and the supernatant saved as the solubilized membrane fraction. Equal Kinoshita, M., Kumar, S., Mizoguchi, A., Ide, C., Kinoshita, A., Haraguchi, T., amounts of proteins in the cytosol and the membrane fractions were used for Hiraoka, Y. and Noda, M. (1997). Nedd5, a mammalian septin, is a novel cytoskeletal immunoblotting. component interacting with actin-based structures. Genes Dev. 15, 1535-1547. Kowluru, A. (2003). Regulatory roles for small G proteins in the pancreatic ␤-cell: lessons This work was supported by grants to M.A.O. from NIH-National from models of impaired insulin secretion. Am. J. Physiol. Endocrinol. Metab. 285, Cancer Institute (#K22CA104285), the American Cancer Society and E669-E684. Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A. and research funds from Cornell University. We thank John Hellmann and Kaibuchi, K. (1996). Identification of IQGAP as a putative target for the small Ahmad Gaballa for facilitating the radiolabel experiments. We thank GTPases, Cdc42 and Rac1. J. Biol. Chem. 271, 23363-23367. Richard Cerione for the CDC42 plasmids and comments on a draft; Le Clainche, C., Schlaepfer, D., Ferrari, A., Klingauf, M., Grohmanova, K., Geoffrey Sharp and Troitza (Trisha) Bratanova-Tochkova for ␤HC-9 Veligodskiy, A., Dirdy, D., Le, D., Egile, C., Carlier, M.-F. et al. (2007). IQGAP1 stimulates actin assembly through the N-Wasp-Arp2/3 pathway. J. Biol. Chem. 282, 426- cells; and Carmen Hassan and Joanne (Chung-Un) Kim for technical 435. assistance. Finally, we appreciate the insights of the anonymous Levy, R., Wiedmann, M. and Kreibich, G. (2001). In vitro binding of ribosomes to the reviewers of this manuscript. beta subunit of the Sec61p protein translocation complex. J. Biol. Chem. 276, 2340- 2346. Lin R., Cerione, R.A. and Manor, D. (1999). Specific contribution of the small GTPases References Rho, Rac and Cdc42 to Dbl transformation. J. Biol. Chem. 274, 23633-23641. Aghazadeh, B., Lowry, W. E., Xin, Y. H. and Rosen, M. K. (2000). Structural basis for Lippincott, J. and Li, R. (1998). Sequential assembly of myosin II, an IQGAP-like relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine protein, and filamentous actin to a ring structure involved in budding yeast cytokinesis. phosphorylation. Cell 102, 625-633. J. Cell Biol. 140, 355-366. IQGAP1 regulates exocytosis 403

Lipschutz, J. H. and Mostov, K. E. (2002). Exocytosis: the many masters of the exocyst. Sakai, K., Kurimoto, M., Tsugu, A., Hubbard, S. L., Trimble, W. S. and Rutka, J. T. Curr. Biol. 12, R212-R214. (2002). Expression of Nedd5, a mammalian septin, in human brain tumors. J. Lipschutz, J. H., Lingappa, V. R. and Mostov, K. E. (2003). The exocyst affects protein Neurooncol. 57, 169-177. synthesis by acting on the translocation machinery of the endoplasmic reticulum. J. Biol. Spiliotis, E. T. and Nelson, W. J. (2006). Here come the septins: novel polymers that Chem. 278, 20954-20960. coordinate intracellular functions and organization. J. Cell Sci. 119, 4-10. Mataraza, J. M., Li, Z. and Sacks, D. B. (2002). IQGAP1 is a component of Cdc42 Stinchcombe, J. C., Majorovits, E., Bossi, G., Fuller, S. and Griffths, G. M. (2006). signaling to the cytoskeleton. J. Biol. Chem. 277, 24753-24763. Centrosome polarization delivers secretory granules to the immunological synapse. Mataraza, J. M., Briggs, M. W., Li, Z., Frank, R. and Sacks, D. B. (2003). Identification Nature 443, 462-465. and characterization of the Cdc42-binding site of IQGAP1. Biochem. Biophys. Res. Toikkanen, J. H., Miller, K. J., Soderlund, H., Jantti, J. and Keranen, S. (2003). The Commun. 305, 315-321. ␤ subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst Mateer, S. C., Wang, N. and Bloom, G. S. (2003). IQGAPs: integrators of the complex in Saccharomyces cerevisiae. J. Biol. Chem. 278, 20946-20953. cytoskeleton, machinery, and signaling networks. Cell Motil. Cytoskeleton Trimble, W. S. (1999). Septins: a highly conserved family of membrane-associated 55, 147-155. GTPases with functions in cell division and beyond. J. Membr. Biol. 169, 75-81. McCallum, S. J., Wu, W. J. and Cerione, R. A. (1996). Identification of a putative Vega, I. E. and Hsu, S. C. (2001). The exocyst complex associates with microtubules to effector for Cdc42Hs with high sequence similarity to the RasGAP-related mediate vesicle targeting and neurite outgrowth. J. Neurosci. 21, 3839-3848. protein IQGAP1 and a Cdc42Hs binding partner IQGAP2. J. Biol. Chem. 271, Vega, I. E. and Hsu, S. C. (2003). The septin protein Nedd5 associates with both the 21732-21737. exocyst complex and microtubules and disruption of its GTPase activity promotes Nelson, W. J. (2003). Adaptation of core mechanisms to generate cell polarity. Nature 422, aberrant neurite sprouting in PC12 cells. NeuroReport 14, 31-37. 766-774. Villace, P., Marion, R. M. and Ortin, J. (2004). The composition of Staufen-containing Novick, P. and Guo, W. (2002). Ras family therapy: Rab, Rho and Ral talk to the exocyst. RNA granules from human cells indicates their role in the regulated transport and Trends Cell Biol. 12, 247-249. translation of messenger RNAs. Nucleic Acids Res. 32, 2411-2420. Ohara-Imaizumi, M., Fujiwara, T., Nakamichi, Y., Okamura, T., Akimoto, Y., Kawai, Wang, S., Watanabe, T., Noritake, J., Fukata, M., Yoshimura, T., Itoh, N., Harada, J., Matsushima, S., Kawakami, H., Watanabe, T., Akagawa, K. et al. (2007). T., Nakagawa, M., Matsuura, Y., Arimura, N. et al. (2007). IQGAP3, a novel effector Imaging analysis reveals mechanistic differences between first- and second-phase of Rac1 and Cdc42, regulates neurite outgrowth. J. Cell Sci. 120, 567-577. insulin exocytosis. J. Cell Biol. 177, 695-705. Watanabe, T., Wang, S., Noritake, J., Sato, K., Fukata, M., Takefuji, M., Nakagawa, Osman, M. A. and Cerione, R. A. (1998). Iqg1p, a yeast homologue of the mammalian M., Izumi, N., Akiyama, T. and Kaibuci, K. (2004). Interaction with IQGAP1 links IQGAPs, mediates cdc42p effects on the actin cytoskeleton. J. Cell Biol. 142, 443-455. APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. Dev. Osman, M. A. and Cerione, R. A. (2005). Actin doesn’t do the locomotion: secretion Cell 7, 871-883. drives cell polarization. In Protein Trafficking: Mechanisms and Regulation (ed. N. Yamashiro, S., Noguchi, T. and Mabuchi, I. (2003). Localization of two IQGAPs in Segev). Georgetown, TX: Landes Bioscience. cultured cells and early embryos of Xenopus laevis. Cell Motil. Cytoskeleton 55, 36-50. Osman, M. A., Konopka, J. B. and Cerione, R. A. (2002). Iqg1p links spatial and Zhou, R., Guo, Z., Watson, C., Chen, E., Kong, R., Wang, W. and Yao, X. (2003). secretion landmarks to polarity and cytokinesis. J. Cell Biol. 159, 601-611. Polarized distribution of IQGAP proteins in gastric parietal cells and their roles in Psatha, M. I., Razi, M., Koffer, A., Moss, S. E., Sacks, D. B. and Bolsover, S. R. (2007). regulated epithelial cell secretion. Mol. Biol. Cell 14, 1097-1108. Targeting of calcium:calmodulin signals to the cytoskeleton by IQGAP1. Cell Calcium Zuo, X., Zhang, J., Zhang, Y., Hsu, S.-C., Zhou, D. and Guo, W. (2006). Exo70 interacts 41, 593-605. with the Arp2/3 complex and regulates . Nat. Cell Biol. 8, 1383-1388 Journal of Cell Science