Ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the Rho GTPase CDC42

Abstract

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.

Keywords: CDC42; GAP-related domain; IQGAP1; Ras-related C3 botulinum toxin substrate 1 (Rac1); cell signaling; scaffold protein; small GTPase; ubiquitylation (ubiquitination).

Figure 1.

Ubiquitination of IQGAP1. A, HEK293 cells were transfected with (+) or without (−) Myc-IQGAP1 and/or His-ubiquitin (Ub). Where indicated, cells were incubated with MG132 (+) or DMSO (−) (vehicle) for 4 h. Equal amounts of protein lysate were loaded directly onto gels or immunoprecipitated (IP) with anti-Myc beads. Proteins were analyzed by SDS-PAGE and immunoblotting (IB) using anti-ubiquitin and anti-IQGAP1 antibodies. B, HEK293 cells were transfected with (+) or without (−) His-ubiquitin and incubated with MG132 (+) or DMSO (−). His-ubiquitin was pulled-down (PD) by incubating equal amounts of protein lysate with TALON beads. Lysates and complexes were resolved by Western blotting. The PVDF membranes were probed with anti-ubiquitin and anti-IQGAP1 antibodies. All data are representative of at least three independent experiments.

Figure 2.

Identification of ubiquitination sites on IQGAP1. A, HEK293 cells were transfected with (+) or without (−) His-ubiquitin (Ub) and incubated with MG132 (+) or DMSO (−). Lysates were IP with anti-IQGAP1 polyclonal antibodies and resolved by SDS-PAGE. Three gel sections corresponding to IQGAP1 and the regions above IQGAP1 (1–3; red rectangles) were excised, digested with trypsin, and analyzed by LC-MS/MS as described under “Experimental procedures.” A portion of the immunoprecipitated lysate was analyzed by Western blotting and probed with anti-IQGAP1 and anti-ubiquitin antibodies. Aliquots of lysate not subjected to immunoprecipitation were processed in parallel (lysate). B, LC-MS/MS analysis of the in-gel tryptic digested samples allowed the identification of ubiquitinated lysine residues. A representative MS/MS spectrum of a ubiquitinated peptide is shown for peptide IQGAP1 1517–1532 containing ubiquitinated Lys-1528. C, schematic representation of IQGAP1 showing the six ubiquitination sites identified by LC-MS/MS and the tryptic peptides in which each is located. CC, coiled-coil; WW, tryptophan-containing domain; IQ, IQ domain.

Figure 3.

Mutation of selected lysine residues of IQGAP1 reduces its ubiquitination. A, schematic representation of IQGAP1 mutant constructs. Individual lysine residues are replaced with arginine for each construct: CC-1K (Lys-556 is replaced with Arg), GRD-2K (K1155R; K1230R), RGCT-3K (K1465R; K1475R; K1528R), and 6K (K556R; K1155R; K1230R; K1465R; K1475R; K1528R). All plasmids contain a Myc-tag. B, equal amounts of protein lysate from HEK293 control (+/+) and IQGAP1-knockdown (−/−) cells generated with the CRISPR/Cas9 system were resolved by Western blotting. PVDF membranes were probed with anti-IQGAP1 and anti-actin (loading control) antibodies. A representative blot is shown. C, WT Myc-IQGAP1 was expressed in IQGAP1-knockdown HEK293 cells (−/−). Equal amounts of protein lysate from control (+/+) and knockdown cells were resolved by Western blotting using anti-IQGAP1, anti-Myc, and anti-actin antibodies. A representative blot is shown. D, IQGAP1-knockdown HEK293 cells were transfected with vector (V), WT IQGAP1 or the indicated IQGAP1 mutant constructs. Samples were IP using anti-Myc beads. Lysates and immunopurified proteins were resolved by Western blotting. Membranes were probed with anti-IQGAP1 and anti-ubiquitin antibodies. Data are representative of at least four independent experiments. E, the ubiquitin bands were quantified with Image Studio 2.0 and corrected for the amount of immunoprecipitated IQGAP1 in the corresponding sample. Data are expressed as mean ± S.D. (n = 5 to 6), with cells transfected with WT IQGAP1 set as 1. Each mutant protein was compared with WT IQGAP1 using Welch's t test. *, p < 0.05.

Figure 4.

Mutation of ubiquitination sites in the GRD of IQGAP1 enhances its binding to CDC42 and RAC1. A, IQGAP1-knockdown HEK293 cells were transfected with WT IQGAP1 or the indicated mutant IQGAP1 proteins and incubated with MG132. GST-CDC42(Q61L) or GST alone was incubated with equal amounts of protein from cell lysates. Complexes were isolated with GSH-Sepharose. Lysates and pulled down (PD) proteins were analyzed by Western blotting using anti-Myc and/or anti-GST antibodies. B, Myc-IQGAP1 in GST pulldowns was quantified with Image Studio 2.0 (LI-COR) and corrected for the amount of GST-CDC42 in the same sample. Data are expressed as mean ± S.D. (n = 5), with cells transfected with WT IQGAP1 set as 1. Each mutant protein was compared with WT IQGAP1 using Welch's t test. *, p < 0.05. C, RAC1 pulldown was performed as described above for CDC42, except GST-RAC1(Q61L) was used. D, Myc-IQGAP1 was quantified as described for panel B, except data were corrected for the amount of GST-RAC1 in the same sample. Data were analyzed as described for panel B (n = 3, except for IQGAP1–6K with n = 2). *, p < 0.05.

Figure 5.

Mutation of ubiquitination sites in the IQGAP1 GRD enhances activation of CDC42. A, IQGAP1-knockdown HEK293 cells were transfected with vector (V), WT IQGAP1, or IQGAP1 GRD-2K. Cells were starved of serum overnight and stimulated with (+) or without (−) 100 ng/ml of EGF for 10 min. Equal amounts of protein lysate were analyzed by Western blotting using anti-Myc, anti-pERK, anti-ERK, and anti-actin (loading control) antibodies. B, equal amounts of protein lysate were used to quantify active CDC42 using GTPase-specific ELISA (G-LISA) as described under “Experimental procedures.” The amounts of GTP-CDC42 are expressed as mean ± S.D. (n = 4, each performed in triplicate), with unstimulated cells expressing WT IQGAP1 set as 1. *, p < 0.05; **, p < 0.001; Welch's t test.

Figure 6.

Mutation of ubiquitination sites in the IQGAP1 GRD enhances cell migration. A, expression of GFP-tagged WT IQGAP1 (WT) or IQGAP1 GRD-2K (GRD-2K) was induced in IQGAP1-null MEF cells (−/−) by adding doxycycline (Dox). Equal amounts of protein lysate from IQGAP1-null (−/−), control (+/+), and reconstituted MEFs were resolved by Western blotting. Membranes were probed with anti-IQGAP1 and anti-tubulin antibodies. A representative blot is shown. B, wound healing assays were performed using IQGAP1^−/− MEFs reconstituted with WT IQGAP1 or IQGAP1 GRD-2K. After serum starvation, cells were incubated with medium containing 0.1% FBS, 100 ng/ml of doxycycline, and 25 ng/ml of EGF, and a wound was generated. Images were taken every 30 min for 24 h. Representative wounds are shown at 0, 4, 8, 16, and 24 h (scale bar, 100 μm). A video of the migration is provided in the supporting information (Video S1). C, open wound areas were analyzed with Fiji/ImageJ as described under “Experimental procedures.” Data are expressed as mean ± S.D. (1 or 2 fields from at least 6 independent wells were analyzed; WT IQGAP1 n = 9; IQGAP1 GRD-2K n = 14). Means between the two groups are significantly different at 3 h (p < 0.05) and reached p < 0.001 at 4.5 h (Welch's t test).

Figure 7.

Ubiquitination of Lys-1155 and Lys-1230 in the GRD of IQGAP1 would impair CDC42 binding. A, two CDC42 molecules (green with red switch regions) bind to the GRD (yellow). One CDC42 molecule binds to the extra subdomain (Ex mode binding), whereas the other CDC42 molecule binds the middle region of GRD (RasGAP mode binding). B, ubiquitin (orange) conjugation at Lys-1155 tethers a large molecule very close to the CDC42 Ex mode-binding site; shown here occupied by CDC42. Inset, a magnified view of the interaction, with Gly-75 and Gly-76 of ubiquitin shown in stick representation. C, conjugation of ubiquitin at Lys-1230 within the GRD of IQGAP1. D, the same orientation of the GRD with CDC42 occupying the RasGAP-binding site. E, significant steric overlap would prevent simultaneous ubiquitin conjugation and CDC42 binding to the GRD.