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. 2014 Mar 7;289(10):6839-6849.
doi: 10.1074/jbc.M113.527655. Epub 2014 Jan 17.

Functional cross-talk between ras and rho pathways: a Ras-specific GTPase-activating protein (p120RasGAP) competitively inhibits the RhoGAP activity of deleted in liver cancer (DLC) tumor suppressor by masking the catalytic arginine finger

Mamta Jaiswal  1 , Radovan Dvorsky  1 , Ehsan Amin  1 , Sarah L Risse  1 , Eyad K Fansa  1 , Si-Cai Zhang  1 , Mohamed S Taha  1 , Aziz R Gauhar  1 , Saeideh Nakhaei-Rad  1 , Claus Kordes  2 , Katja T Koessmeier  1 , Ion C Cirstea  3 , Monilola A Olayioye  4 , Dieter Häussinger  2 , Mohammad R Ahmadian  5
Affiliations

Functional cross-talk between ras and rho pathways: a Ras-specific GTPase-activating protein (p120RasGAP) competitively inhibits the RhoGAP activity of deleted in liver cancer (DLC) tumor suppressor by masking the catalytic arginine finger

Mamta Jaiswal et al. J Biol Chem. .

Abstract

The three deleted in liver cancer genes (DLC1-3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins.

Keywords: Arginine Finger; DLC1; Deleted in Liver Cancer; GTPase; Kinetics; Molecular Modeling; RhoGAP; SH3 Domains; Structural Biology; p120RasGAP.

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Figures

FIGURE 1.
FIGURE 1.
Schematic representation of domain organization and designed fragments of GAP (A) and SH3 domain-containing proteins (B) used in this study. The numbers indicate the N and C termini of the amino acids of the respective fragments. BAR, Bin/Amphiphysin/Rvs; C1, cysteine-rich region; CC, coiled coil; DH, Dbl homology domain; FF, double phenylalanine; P, proline-rich; PH, pleckstrin homology; PSET, proline, serine, glutamic acid, and threonine; RGS, regulator of G-protein signaling; Sec14, secretion and cell surface growth 14.
FIGURE 2.
FIGURE 2.
Inefficient GAP activities of the DLC isoforms. A, Cdc42-tamraGTP (0.2 μm) was rapidly mixed with 5 μm DLC1GAP to monitor the GAP-stimulated tamraGTP hydrolysis reaction of Cdc42 in real time. Note the very slow intrinsic GTPase reaction of Cdc42 (inset) that was measured in the absence of GAP. Rate constants (kobs) were obtained by single exponential fitting of the data. B, the kobs values of GTP hydrolysis of Rho proteins (0.2 μm) measured in the presence of DLC1GAP (5 μm) are represented as a column chart. Calculated -fold activation values were obtained by dividing the kobs values of GAP-stimulated reactions by the kobs values of the intrinsic reactions of respective GTPases. For convenience, the kobs values are given above the bar charts. C, measured GAP activities of DLC1, DLC2, and DLC3 (5 μm, respectively) toward Cdc42 (0.2 μm) were very low as compared with p150 and p190. D, the GTP hydrolysis of Cdc42 (0.2 μm) was measured in the presence of increasing concentrations of the respective GAP domains of DLC1 and GRAF1 (inset). The dependence of the kobs values of the GAP-stimulated GTP hydrolysis plotted on the concentrations of DLC1GAP and GRAF1 was fitted by a hyperbolic curve to obtain the kinetic parameters (kcat and Kd).
FIGURE 3.
FIGURE 3.
cis-Acting regulation of DLC1GAP activity. A, kinetics of the tamraGTP hydrolysis reaction of Cdc42 (0.2 μm) stimulated by DLC1fl (5 μm) was much slower (inset) than that stimulated by DLCGAP (5 μm). B, the kobs values, illustrated as a bar chart, showed that the GAP activity of DLC1fl is reduced by 23.5-fold as compared with that of the DLC1GAP but not completely inhibited as compared with the intrinsic GTPase reaction. For convenience, the kobs values are given above the bar charts. C, the activity of DLC1GAP (10 μm) on tamraGTP hydrolysis of Cdc42 (0.2 μm) was not significantly changed in the presence of a 100-fold excess of SAM, START, or both domains (1 mm, respectively).
FIGURE 4.
FIGURE 4.
p120SH3 as a potent inhibitor of the DLC GAP function. A, kinetics of the tamraGTP hydrolysis reaction of Cdc42 (0.2 μm) stimulated by DLC1GAP (5 μm) was reduced in the presence of a 10-fold excess of p120SH3 (50 μm). The complete reaction is shown in the inset. B, DLC1GAP activities toward Cdc42, RhoA, and Rac1, measured under the same conditions as in A, are strongly inhibited by p120SH3. For convenience, the kobs values are given above the bar charts. C, DLC3GAP (5 μm) was not inhibited by p120SH3 (50 and 500 μm) as efficiently as DLC1GAP and DLC2GAP (5 μm, respectively). D, p120SH2-3-2 and p120Δn128 (40 μm) inhibited the activity of DLCGAP (10 μm) but not as efficiently as p120SH3 (40 μm).
FIGURE 5.
FIGURE 5.
Highly selective interaction between p120SH3 and DLC1GAP. A, p120SH3-inhibiting effect on seven additional RhoGAPs (2 μm, respectively) was measured using the tamraGTP hydrolysis reaction of Cdc42 (0.2 μm) and p120SH3 (20 and 200 μm, respectively). p120SH3 inhibited only DLC1GAP but not the other RhoGAPs. For convenience, the kobs values are given above the bar charts. B, the effect of seven additional SH3 proteins (100 μm, respectively) on inhibiting DLC1GAP (10 μm) was measured. Only p120SH3 inhibited DLC1GAP but not the other SH3 domains.
FIGURE 6.
FIGURE 6.
High affinity interaction between p120SH3 and DLC1GAP. A, co-elution of a mixture of DLC1GAP (10 μm) and p120SH3 (15 μm) (open circles) from a Superdex 75 (10/300) as shown by SDS-PAGE (15%) and Coomassie Brilliant Blue (CBB) staining (inset) indicates their complex formation. B, the activity of DLC1GAP (20 μm) toward Cdc42 (0.2 μm) was measured at increasing concentrations of p120SH3, and the obtained kobs values were plotted against increasing concentrations of the inhibitor p120SH3. The Ki value was obtained by non-linear regression based on the Morrison equation for tight binding inhibitors (58). C, ITC analysis was performed by titrating DLC1GAP (20 μm) with p120SH3 (400 μm). Kd is the dissociation constant, and n is the stoichiometry.
FIGURE 7.
FIGURE 7.
Structural insight into a putative binding mode between p120SH3 and DLC1GAP. A, molecular docking analyses were performed between the available crystal structures of p120SH3 (Protein Data Bank code 2J05) (53) and DLC1GAP (Protein Data Bank code 3KUQ) using the program PatchDock (54). In the best ranked and refined model, p120SH3 was located in close proximity of the catalytic arginine finger (Arg-677; magenta) of DLC1GAP. In this model, p120SH3 supplied three amino acids (Asn-311, Leu-313, and Trp-319) to directly contact the catalytic core of DLC1GAP, especially Arg-677, and mask its accessibility to the Rho proteins. B, p50GAP provides an arginine finger (Arg-282; red) in the active site of RhoA to stabilize the transition state of the GTP hydrolysis reaction (Protein Data Bank code 1TX4) (60). GDP-AlF4− mimics the transition state of the GTP hydrolysis reaction.
FIGURE 8.
FIGURE 8.
Loss of p120-DLC1 interaction by mutational analysis. No interaction was observed between DLC1GAP(R677A) and p120SH3(WT) (A and B) and DLC1GAP(WT) and p120SH3(N311R,L313A,W319G) (C and D). Loss of interaction and of inhibition was measured by ITC (A and C) and aSEC (B and D) as compared with the p120SH3(WT)-DLC1GAP(WT) interaction shown in Fig. 6. E, the activity of DLC1GAP (25 μm) in stimulating tamraGTP hydrolysis of Cdc42 (0.2 μm) was measured in the presence of p120SH3 variants (125 μm), respectively. For convenience, the kobs values are given above the bar charts.
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