Rho isoform-specific interaction with IQGAP1 promotes breast cancer cell proliferation and migration

Abstract

We performed a proteomics screen for Rho isoform-specific binding proteins to clarify the tumor-promoting effects of RhoA and C that contrast with the tumor-suppressive effects of RhoB. We found that the IQ-motif-containing GTPase-activating protein IQGAP1 interacts directly with GTP-bound, prenylated RhoA and RhoC, but not with RhoB. Co-immunoprecipitation of IQGAP1 with endogenous RhoA/C was enhanced when RhoA/C were activated by epidermal growth factor (EGF) or transfection of a constitutively active guanine nucleotide exchange factor (GEF). Overexpression of IQGAP1 increased GTP-loading of RhoA/C, while siRNA-mediated depletion of IQGAP1 prevented endogenous RhoA/C activation by growth factors. IQGAP1 knockdown also reduced the amount of GTP bound to GTPase-deficient RhoA/C mutants, suggesting that IQGAP enhances Rho activation by GEF(s) or stabilizes Rho-GTP. IQGAP1 depletion in MDA-MB-231 breast cancer cells blocked EGF- and RhoA-induced stimulation of DNA synthesis. Infecting cells with adenovirus encoding constitutively active RhoA(L63) and measuring absolute amounts of RhoA-GTP in infected cells demonstrated that the lack of RhoA(L63)-induced DNA synthesis in IQGAP1-depleted cells was not due to reduced GTP-bound RhoA. These data suggested that IQGAP1 functions downstream of RhoA. Overexpression of IQGAP1 in MDA-MB-231 cells increased DNA synthesis irrespective of siRNA-mediated RhoA knockdown. Breast cancer cell motility was increased by expressing a constitutively-active RhoC(V14) mutant or overexpressing IQGAP1. EGF- or RhoC-induced migration required IQGAP1, but IQGAP1-stimulated migration independently of RhoC, placing IQGAP1 downstream of RhoC. We conclude that IQGAP1 acts both upstream of RhoA/C, regulating their activation state, and downstream of RhoA/C, mediating their effects on breast cancer cell proliferation and migration, respectively.

FIGURE 1.

IQGAP1 interacts with RhoA/C but not RhoB. A, 293T cells were transfected with expression vectors encoding Flag epitope-tagged, constitutively active RhoA, RhoB, and RhoC (each with a glycine to valine substitution at position 14), or empty vector (E.V.), and cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Precipitated proteins were eluted with Flag peptide and analyzed by SDS-PAGE/silver staining. Bands specifically associated with Rho A and C were excised, subjected to trypsin digestion, and analyzed by mass spectrometry. Bands migrating at ∼190 kDa were identified as IQGAP1. B, 293T cells were transfected with the same Rho constructs as in panel A, and anti-Flag immunoprecipitates were analyzed by Western blotting for the presence of endogenous IQGAP1 (top panel). A fraction of the immunoprecipitates was analyzed by blotting with anti-Flag antibody (bottom panel). C, N-terminal or C-terminal halves of IQGAP1 (IQGAP-N (amino acids 1–862) or IQGAP-C (amino acids 863–1657)) were expressed as GST-fusion proteins and purified from bacteria. Beads loaded with GST-IQGAP-N, IQGAP-C, or GST were incubated with Flag-tagged RhoA^V14, RhoB^V14, or RhoC^V14 purified from 293T cells; beads were washed, and analyzed for the presence of RhoA, RhoB, or RhoC, respectively, with 10% input of each Rho protein shown in the last lane.

FIGURE 2.

The IQGAP1/RhoA interaction requires GTP binding and prenylation of RhoA. A, 293T cells were co-transfected with Myc epitope-tagged IQGAP1 and Flag-tagged RhoA constructs encoding wild type (Wt), constitutively active (V14 or L63), dominant-negative (N19) RhoA proteins, or empty vector. Anti-Flag immunoprecipitates were analyzed by Western blotting with anti-Myc antibody for the presence of IQGAP1 (top panel), or with anti-Flag antibody for the amounts of RhoA (bottom panel). Input lysates (2.5%) are shown in the right lanes. B, cells were transfected with IQGAP1 and the Rho constructs described in panel A; absolute amounts of GTP bound to anti-Flag immunoprecipitates were measured using a luciferase-based assay, as described under “Experimental Procedures.” C and D, cells were transfected with IQGAP1 and Flag-tagged wild type RhoA as in panel A, but cells were serum-deprived for 8 h and treated with EGF (20 ng/ml) for 5 min. In panel C, anti-Flag immunoprecipitates were analyzed by Western blotting for the presence of IQGAP1, and in panel D, GTP bound to anti-Flag immunoprecipitates was measured enzymatically as in panel B. E and F, 293T cells were co-transfected with Myc-tagged IQGAP1 and vectors encoding Flag-tagged RhoA(V14) in its normal prenylated form (C190 is the cysteine modified by geranyl-geranylation), or with a mutation in the prenylation signal (S190); anti-Flag immunoprecipitates were probed for the association of IQGAP1 (E) or for the amount of GTP bound to RhoA(V14/C190) and RhoA(V14/S190) (F).

FIGURE 3.

Co-immunoprecipitation of endogenous RhoA and RhoC with IQGAP1. A, 293T cells were co-transfected with Flag-tagged IQGAP1 and either empty vector, constitutively active ΔNp115 Rho-GEF, or Gα13QL as indicated. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody; precipitated proteins were eluted with Flag peptide and analyzed by Western blotting with antibody specific for RhoA (top two panels) or anti-Flag antibody (bottom panel). B, cells were transfected as in panel A, and GTP-bound endogenous Rho was isolated from cell lysates using the RBD of Rhotekin as described under “Experimental Procedures”; endogenous RhoA bound to the beads was analyzed by Western blotting with a RhoA-specific antibody (top two panels). Cell lysates were also directly analyzed by Western blotting for total RhoA (lower panel). C, MDA-MB-231 cells were serum-starved for 24 h prior to stimulation with EGF for 20 min. Cell lysates were subjected to immunoprecipitation with IQGAP1-specific antibody or control IgG (cIg), and precipitates were analyzed by Western blotting with antibodies specific for RhoC (top panel) or IQGAP1 (bottom panel). 1% input lysates was analyzed in parallel. D, MDA-MB-231 cells were treated with EGF for the indicated times; GTP-bound endogenous Rho was isolated by RBD-pulldown, and RhoC-GTP bound to the beads or total RhoC were detected by Western blotting as described in panel B for RhoA.

FIGURE 4.

IQGAP1 regulates RhoA activation. A, 293T cells were co-transfected with expression vector encoding HA epitope-tagged wild type RhoA or RhoC and either empty vector or vector encoding Flag-tagged IQGAP1. Cell lysates were subjected to immunoprecipitation with anti-epitope antibody, and GTP bound to the immunoprecipitates was quantified as described in Fig. 2B. The amount of Rho-GTP found in cells transfected with empty vector was assigned a value of one; *, p < 0.05 for the comparison between the presence and absence of IQGAP1. B, MDA-MB-231 cells were transfected with siRNA specific for IQGAP1 or GFP (control) as described under “Experimental Procedures”; cells were serum-starved for 24 h, and stimulated with 20% FBS for 5 min to stimulate Rho activation. GTP-bound Rho was isolated from cell lysates as described in Fig. 3B. RhoA-GTP bound to Rhotekin-RBD was assessed by Western blotting with a RhoA-specific antibody (top panel). Cell lysates were also directly analyzed by Western blotting for total RhoA (middle panel, 1% of input) and IQGAP1 (lower panel). C, Western blots from three independent experiments performed as in panel B were analyzed by densitometry, and the amount of endogenous RhoA-GTP (normalized to total RhoA) present in serum-starved, GFP siRNA-transfected cells was assigned a value of 1. *, p < 0.05 for the comparison to serum-starved, GFP siRNA-transfected cells; #, p < 0.05 for the comparison to serum-stimulated, GFP siRNA-transfected cells.

FIGURE 5.

RhoA-induced MDA-MB-231 cell proliferation requires IQGAP1. A, MDA-MB-231 cells were transfected with siRNAs specific for GFP (GFPsi) or IQGAP1 (IQGAPsi) as described in Fig. 4B; some cells were treated with EGF (20 ng/ml). Cell proliferation was assessed by BrdU uptake, identified by immunofluorescence staining as described under “Experimental Procedures,” and DNA was counter-stained with Hoechst 33342. The percentage of BrdU-positive nuclei found in GFP siRNA-transfected, control cells was assigned a value of one (*, p < 0.05 for the comparison to control cells; ns, non-significant). Below: IQGAP1 expression was analyzed by Western blotting in cells that were transfected in parallel, with β-actin serving as a loading control. B, MDA-MB-231 cell were transfected as in panel A, but cells were additionally infected with adenoviral vectors encoding LacZ (vAd-LacZ, control) or constitutively active RhoA^L63 (vAd-RhoA^L63), as indicated. Immunofluorescence staining for BrdU uptake is shown in red. C, siRNA-transfected cells were infected with two different concentrations of virus encoding HA-tagged RhoA^L63 (1 or 3×) or LacZ virus (“0”). The percentage of BrdU-positive nuclei found in control cells (GFP siRNA-transfected cells infected with LacZ virus) was assigned a value of one (*, p < 0.05 for the comparison to GFP siRNA-transfected cells infected with LacZ virus; #, p < 0.05 for the comparison to GFP siRNA-transfected cells infected with the same amount of RhoA^L63 virus). D, cells were siRNA-transfected and Rho virus infected as in panel C, and the amount of GTP bound to RhoA^L63 was measured in anti-HA immunoprecipitates as described under “Experimental Procedures” ( #, p < 0.05 for the comparison to GFP siRNA-transfected cells infected with the same amount of RhoA^L63 virus). E, cell lysates from cells treated as in panel C were analyzed by Western blotting using antibodies specific for RhoA (top), IQGAP1 (middle), or β-actin (bottom panel). Note that the HA epitope-tagged RhoA ^L63 migrates with a higher apparent molecular weight than endogenous RhoA.

FIGURE 6.

IQGAP1-induced DNA synthesis in MDA-MB-231 cells is independent of RhoA. A, MDA-MB-231 cells were transfected with siRNA specific for GFP (GFPsi) or RhoA (RhoAsi); cells were additionally infected with adenoviral vectors encoding LacZ (control, −) or IQGAP1 (vAd-IQGAP1). Cells were serum-deprived for 24 h and received EGF as indicated; cell proliferation was assessed as in Fig. 5 (**, p < 0.01 for the comparison to GFP siRNA-transfected control cells infected with LacZ virus; ###, p < 0.001 for the comparison between cells infected with LacZ virus versus IQGAP1 virus; ns, non-significant). B, cells were treated as in A, and cell lysates were analyzed by Western blotting for IQGAP1 (top), RhoA (middle), or α-tubulin (bottom panel). C and D, MDA-MB-231 cells were siRNA transfected and infected with control or IQGAP1 virus as in panel A, but were not treated with EGF. BrdU immunofluorescence staining is shown in red (C), and three independent experiments are summarized in D (*,# p < 0.05 for the comparison between cells infected with LacZ virus versus IQGAP1 virus).

FIGURE 7.

RhoC-induced MDA-MB231 cell migration requires IQGAP1. A, MDA-MB-231 cells were transfected with siRNAs specific for GFP or IQGAP1; cell migration was assessed in a transwell assay with some cells receiving EGF in the lower chamber, as described under “Experimental Procedures.” The number of cells migrating under control conditions (GFP siRNA-transfected, no EGF) was assigned a value of one (*, p < 0.05 for the comparison to control; ns, non-significant). IQGAP knock-down was assessed by Western blotting (below). B, cells were transfected as in panel A, and some cells were stimulated with EGF for 20 min. Endogenous, GPT-bound Rho proteins were isolated by RBD-pulldown assay and absolute amounts of GTP bound to the beads were measured using the luciferase-based assay described under “Experimental Procedures” (**, p < 0.01 for the comparison to control). C, MDA-MB-231 cells were siRNA transfected as in panel A, but were infected with LacZ virus (0) or with two different concentrations of adenovirus encoding HA-tagged RhoC^V14 (1 and 3×). Cell migration was assessed by transwell assay with all cells receiving FBS in the lower chamber. The number of cells migrating under control conditions (GFP siRNA-transfected and LacZ virus-infected) was assigned a value of one (*, p < 0.05 for the comparison to control; #, p < 0.05 for the comparison to GFP siRNA-transfected cells infected with the same amount of RhoC^V14 virus). D, cells were treated as in panel C, and the amount of GTP bound to RhoC^V14 was measured in anti-HA immunoprecipitates as described under “Experimental Procedures” (#, p < 0.05 for the comparison to GFP siRNA-transfected cells infected with the same amount of RhoA^L63 virus). E, cell lysates from cells treated as in panel C were analyzed by Western blotting with antibodies specific for RhoC (top panel, note that the HA epitope-tagged RhoC^V14 migrates with a higher apparent molecular weight than endogenous RhoC), IQGAP1 (middle panel), and β-actin (bottom panel).

FIGURE 8.

IQGAP1 can restore migration in RhoC-depleted cells; IQGAP1 functions both upstream and downstream of RhoA and C. A, MDA-MB-231 cells were transfected with siRNAs specific for GFP or RhoC, and infected with adenovirus encoding LacZ or IQGAP1; cell migration was measured as in Fig. 7A, with some cells receiving EGF in the lower chamber (*, p < 0.05 or **, p < 0.01 for the comparison to control; #, p < 0.05 or ##, p < 0.01 for the comparison between LacZ and IQGAP1 virus in RhoC siRNA-transfected cells). B, cells were treated as in A, and cell lysates were analyzed by Western blotting with antibodies specific for IQGAP1 (top), RhoC (middle), or RhoA (bottom panel). C, cells were siRNA transfected and infected with virus encoding LacZ (vLacZ) or IQGAP1 (vIQGAP1) as in panel A, but migration was measured in a wound-closure assay, with photographs taken immediately, and 40 h after cells were scratched off the plate. D, summary of three experiments performed as in C; the number of cells migrating into the wound in the control culture (GFP siRNA-transfected and LacZ virus-infected) was assigned a value of one (*, p < 0.05 for the comparison to control; #, p < 0.05 for the comparison between LacZ and IQGAP1 virus in RhoC siRNA-transfected cells). E, model depicting IQGAP1 binding GTP-bound RhoA/C and mediating RhoA and RhoC-induced DNA synthesis and migration, respectively. The activation state of RhoA/C is regulated by GEFs and GAPs; growth factors stimulate GEFs, and IQGAP1 appears to enhance Rho activation by GEFs. Interaction of RhoA/C-GDP with Rho guanine nucleotide dissociation inhibitors is not shown in this figure. IQGAP1 has no intrinsic GAP activity, and IQGAP1 overexpression leads to increased cell proliferation and migration through RhoA/C-independent mechanisms.