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First published online January 23, 2008
doi: 10.1242/10.1242/jcs.016881

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A dual role for IQGAP1 in regulating exocytosis

¹ Department of Microbiology, Cornell University, Ithaca, NY 14853-2703, USA
² Department of Molecular Medicine, Cornell University, Ithaca, NY 14853-2703, USA
³ Division of Endocrinology, Diabetes and Bone Disease, Mount Sinai School of Medicine, New York, NY 10029-6574, USA
⁴ Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA

View larger version (38K): [in this window] [in a new window]   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 βTC-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. βTC-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, calponin 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.

View larger version (35K): [in this window] [in a new window]   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 β-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-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-FMK, 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-FMK and their parental constructs transiently expressed in βTC-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.

View larger version (44K): [in this window] [in a new window]   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 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 6xHis-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.

View larger version (81K): [in this window] [in a new window]   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 µm) of the perinuclear area. Middle, thin confocal slice (0.18 µm). 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.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Effects of IQGAP1 depletion on EXO70. (A) Depletion of IQGAP1 with targeted RNAi. A representative western blot with the indicated antibodies from equal amounts of total protein 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-localization of IQGAP1 with EXO70 in control HeLa cells. Fixed cells were double-stained with antibodies for IQGAP1 (Texas red) and EXO70 (Alexa-Fluor-488, green). Lower panels: localization of EXO70 (right panel) in HeLa cells that were depleted of IQGAP1 (left panel).

View larger version (42K): [in this window] [in a new window]   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 IQGAP1 at 100 nM and 40 nM were double-stained with anti-SEPT2 (bottom, green) and anti-IQGAP1 (top, red) antibodies.

View larger version (28K): [in this window] [in a new window]   Fig. 7. IQGAP1 influences protein synthesis and exocytosis. (A) Differential effects of IQGAP1 domains on insulin exocytosis. Early passages of βTC-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 βTC-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 βTC-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.

View larger version (73K): [in this window] [in a new window]   Fig. 8. Localization and association of IQGAP1 with sites of protein synthesis and exocytosis. (A) Confocal slice (0.16 µm) 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 Sec61β. Monoclonal antibodies for Sec61β or IQGAP1 were used conversely for IP. The immune complexes were resolved on a 15% SDS-PAGE and conversely blotted with anti-IQGAP1 or -Sec61β 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.

View larger version (25K): [in this window] [in a new window]   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 β-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 (GTPS) 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 β-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.

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