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. 2012 Apr 20;287(17):13752-60.
doi: 10.1074/jbc.M111.337469. Epub 2012 Mar 5.

RhoGDI SUMOylation at Lys-138 increases its binding activity to Rho GTPase and its inhibiting cancer cell motility

Jianxiu Yu  1 , Dongyun Zhang, Jinyi Liu, Jingxia Li, Yonghui Yu, Xue-Ru Wu, Chuanshu Huang
Affiliations

RhoGDI SUMOylation at Lys-138 increases its binding activity to Rho GTPase and its inhibiting cancer cell motility

Jianxiu Yu et al. J Biol Chem. .

Abstract

The Rho GDP dissociation inhibitor (RhoGDI) can bind to small GTPases and keep them in a biologically inactive state in cytoplasm, through which it affects actin polymerization and cell motility. However, mechanisms underlying how RhoGDI regulates Rho GTPase complex formation/membrane extraction/GTPase dissociation remain largely unexplored. Our previous studies reported that X-linked inhibitor of apoptosis protein (XIAP) interacted with RhoGDI via its RING domain and negatively modulated RhoGDI SUMOylation and HCT116 cancer cell migration. Here, we identified that RhoGDI SUMOylation specifically occurred at Lys-138, which was inhibited by XIAP domain. We further demonstrated that RhoGDI SUMOylation at Lys-138 was crucial for inhibiting actin polymerization and cytoskeleton formation as well as cancer cell motility. Moreover, SUMO-RhoGDI had a much higher binding affinity to small Rho GTPase compared with the un-SUMOylated form of RhoGDI. Taken together, our study demonstrated a novel modification of RhoGDI, SUMOylation at Lys-138, which played a key role in regulating Rho GTPase activation in cancer cells. The physiological regulation of RhoGDI SUMOylation by the RING domain of XIAP may account for modulation of cancer cell invasion and metastasis by XIAP.

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Figures

FIGURE 1.
FIGURE 1.
RhoGDI SUMOylation occurred at lysine 138. A, SUMOplot prediction of human RhoGDI protein. SUMO consensus contains the sequences ΨKX(E/D) (where Ψ is a large hydrophobic amino acid, K is the target lysine, X is any amino acid, and D or E is an acidic residue). SUMOylation sites of human RhoGDI protein were predicted by the program of Abgent SUMOplotTM. Three lysine residues at Lys-199, Lys-138, and Lys-105 with a high score are the putative SUMOylation sites (marked in bold and underlined). B, identification of SUMOylated site(s) of human RhoGDI. 293T cells were transiently transfected with constructs of His-SUMO1 and FLAG-Ubc9, together with either WT or mutants (K105R, K138R, or K199R) of GFP-RhoGDI. Cell extracts were used for nickel-nitrilotriacetic acid (Ni-NTA) precipitation, and pulled down proteins were identified by Western blotting. The data for input are shown in the lower five panels. IB, immunoblot; Endo, endogenous.
FIGURE 2.
FIGURE 2.
RhoGDI SUMOylation at Lys-138 was crucial for RhoGDI inhibition of cancer cell migration, invasion, and actin polymerization. A, stable transfectants of GFP-RhoGDI-WT, GFP-RhoGDI-K138R, GFP-RhoGDI-K105R, and the empty vector in HCT116 cells were identified by Western blotting assay. B, wound healing capabilities were compared among the indicated transfectants of HCT116 cells. C, proliferation rates were determined in the indicated cell lines by a CellTiter-Glo® luminescent cell viability assay kit. Results were represented as the means ± S.D. of the triplicate wells. The asterisk indicates a significant decrease in proliferation rate in GFP-RhoGDI-WT transfectants compared with that in the vector transfectant (p < 0.05). D, wound area on the 4th day was quantified using Cell Migration Analysis software and normalized over the proliferation rate on 4th day as shown in C. The quantitative data were shown as relative wound area (%, error bar represent S.D., n = 3). The asterisk indicates a significant increase in wound area in GFP-RhoGDI-WT transfectants compared with that in the vector transfectant (p < 0.05). E, indicated transfectants were used for comparison of their invasive capabilities using BD BioCoatTM MatrigelTM invasion chamber. Results were represented as the means ± S.D. of invasive cells from at least three independent experiments with duplicate wells for each experiment. The asterisk indicates a significant decrease in comparison with HCT116(vector) cells (p < 0.01), and the (♣) indicates a significant increase in comparison with HCT116(vector) cells (p < 0.01). F, indicated transfectants were used to evaluate cell actin polymerization under confocal microscopy as indicated: GFP-RhoGDI, F-actin, and DAPI.
FIGURE 3.
FIGURE 3.
XIAP RING domain was crucial for XIAP inhibiting RhoGDI SUMOylation. WT, XIAP−/−, and XIAP−/−(HA-XIAP) HCT116 cells (A) and 293T cells (B) were transfected with GFP-RhoGDI, together with His-SUMO1 and FLAG-Ubc9 (A) and various constructs as indicated (B). Cell extracts were used for nickel-nitrilotriacetic acid (Ni-NTA) precipitation, and pulled down proteins were identified by Western blotting. IB, immunoblot.
FIGURE 4.
FIGURE 4.
XIAP RING domain played an important role in inhibiting Rho GTPase activity. A–C, lysates from HCT116 cells stably transfected with GFP-RhoGDI-K138R or GFP-RhoGDI-WT or the empty vector (A) or from WT, XIAP−/−, and XIAP−/−(HA-XIAP) (B), or from XIAP−/−(HA-ΔRING) and XIAP−/−(HA-ΔBIRs) HCT116 cells (C) were used for small GTPase activity assay following the manufacturer's instructions. GTPγS and GDP were used for positive and negative controls, respectively. The densitometric analyses of the active (RBD/PBD-bound) proteins are determined as relative ratio to the GTPγS-bound form proteins. Results shown were representative of three independent experiments. D and E, XIAP−/− cells were transfected with Rac1 expression vector and then infected with SUMO1 overexpression virus (Ad-SUMO1 Q94P) or control virus (Ad-LacZ). Immunoprecipitation was carried out using anti-Rac antibody-conjugated agarose beads. The immune complex was examined by Western blotting assay (D), and the densitometric analyses were carried to show the ratio of SUMO-RhoGDI protein to un-SUMOylated RhoGDI in the immunocomplex and in Input (E).
FIGURE 5.
FIGURE 5.
A model for the XIAP-regulated RhoGDI SUMOylation signaling pathway in modulation of cell motility. SUMO-RhoGDI down-regulates Rho GTPase (RhoA, Rac1, and Cdc42) activity. XIAP binds to RhoGDI through its RING domain and inhibits RhoGDI SUMOylation at Lys-138, resulting in activation of GTPases, through which XIAP promotes actin polymerization and facilitates cell migration, invasion, and metastasis.
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