SmgGDS is a guanine nucleotide exchange factor that specifically activates RhoA and RhoC

Abstract

SmgGDS is an atypical guanine nucleotide exchange factor (GEF) that promotes both cell proliferation and migration and is up-regulated in several types of cancer. SmgGDS has been previously shown to activate a wide variety of small GTPases, including the Ras family members Rap1a, Rap1b, and K-Ras, as well as the Rho family members Cdc42, Rac1, Rac2, RhoA, and RhoB. In contrast, here we show that SmgGDS exclusively activates RhoA and RhoC among a large panel of purified GTPases. Consistent with the well known properties of GEFs, this activation is catalytic, and SmgGDS preferentially binds to nucleotide-depleted RhoA relative to either GDP- or GTPγS-bound forms. However, mutational analyses indicate that SmgGDS utilizes a distinct exchange mechanism compared with canonical GEFs and in contrast to known GEFs requires RhoA to retain a polybasic region for activation. A homology model of SmgGDS highlights an electronegative surface patch and a highly conserved binding groove. Mutation of either area ablates the ability of SmgGDS to activate RhoA. Finally, the in vitro specificity of SmgGDS for RhoA and RhoC is retained in cells. Together, these results indicate that SmgGDS is a bona fide GEF that specifically activates RhoA and RhoC through a unique mechanism not used by other Rho family exchange factors.

FIGURE 1.

Purified SmgGDS specifically activates RhoA and RhoC in vitro. The intrinsic exchange of the indicated GTPase (1 μm) was measured for 600 s in exchange buffer. At the indicated time (arrow), SmgGDS (50 μm) or EDTA (25 μm) was added to stimulate nucleotide exchange.

FIGURE 2.

Activation of RhoA by SmgGDS is catalytic and independent of SmgGDS isoform. A, domain architecture of SmgGDS isoforms is shown with conserved ARM repeats (blue boxes) and inserted repeat (red). B, intrinsic exchange of RhoA (1 μm) was measured for 600 s in exchange buffer before stimulation of nucleotide exchange with the indicated concentration of SmgGDS or SmgGDS-558. C, MANT-GDP-loaded RhoA (1 μm) was incubated in unloading exchange buffer for 600 s before addition of SmgGDS at the indicated concentrations to stimulate nucleotide exchange. D, RhoA (1 μm) was incubated in unloading exchange buffer with 400 nm MANT-GDP in place of the free GDP for 600 s before addition of SmgGDS at the indicated concentrations to stimulate nucleotide exchange.

FIGURE 3.

SmgGDS preferentially forms a high affinity complex with nucleotide-depleted RhoA possessing a polybasic region. SmgGDS was incubated on ice for 30 min with a 2-fold molar excess of RhoA loaded with GDP (A), GTPγS (B), or in the presence of EDTA (C), separated over size-exclusion chromatography, and fractions were analyzed via SDS-PAGE.

FIGURE 4.

SmgGDS utilizes a novel exchange mechanism to activate RhoA. A, B, E–G, intrinsic exchange of 1 μm GTPase was measured for 600 s in exchange buffer, and at the indicated time (arrow) SmgGDS (50 μm) or Dbs (200 nm) was added to stimulate nucleotide exchange. A, RhoA A61G. B, RhoA E64A. C, sequence comparison of Rho family member polybasic regions with basic residues highlighted in blue. D, SmgGDS incubated on ice for 30 min with a 2-fold molar excess of RhoA Δpbr in the presence of EDTA, separated over size-exclusion chromatography, and fractions analyzed via SDS-PAGE. E, RhoA Δpbr. F, RhoA R182A+R183A. G, RhoA K185A+K186A+K187A. H, intrinsic exchange of the indicated GTPase construct (1 μm) measured for 600 s in exchange buffer. At the indicated time (arrow), SmgGDS (50 μm) was added to stimulate nucleotide exchange.

FIGURE 5.

Electronegative patch and highly conserved binding groove on SmgGDS facilitate activation of RhoA. A, electrostatic surface potential of SmgGDS homology model with acidic regions in red and basic regions in blue. B, homology model of SmgGDS with completely (red) and highly (pink) conserved acidic residues. C, intrinsic exchange of RhoA (1 μm) measured for 600 s in exchange buffer. At the indicated time (arrow), wild-type or mutant SmgGS (50 μm) was added to stimulate nucleotide exchange. D, homology model of SmgGDS colored according to ClustalX consensus score from a multiple sequence alignment of full-length SmgGDS isoforms. E, intrinsic exchange of RhoA (1 μm) measured for 600 s in exchange buffer. At the indicated time (arrow), wild-type or mutant SmgGDS (50 μm) was added to stimulate nucleotide exchange.

FIGURE 6.

SmgGDS specifically activates RhoA in cells. HEK293 cells were transfected with the indicated GEF expression vectors and then lysed. Active GTPase was precipitated from lysate with GST-RBD (Rho) or GST-PBD (Rac) beads. Pulldown (GTP-bound GTPase) and lysates (total GTPase) were detected by Western blotting. Expression of GEF was confirmed with immunoblotting for HA antibody. Experiments were performed in triplicate, and mean band intensity relative to the control is graphed with S.E. (error bars). A, active Rho detected with a pan-Rho antibody. B, active Rac detected with a pan-Rac antibody. C, active RhoA, RhoB, and RhoC detected with RhoA-, RhoB-, and RhoC-specific antibodies.