Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue

Rittinger, Katrin; Walker, P.A.; Eccleston, John F.; Smerdon, Stephen J.; Gamblin, Steven J.

doi:10.1038/39651

Cited by 405 publications

(358 citation statements)

References 25 publications

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“…However, these GTPases can be stimulated by GAPs [38,39] that donate a catalytic arginine in trans to the active site [9,10]. These catalytic arginine residues of trimeric GTPases and GAPs stimulate the GTPase activity by 100-fold [33,34] and 1000-fold [11] respectively, by stabilizing a γ-phosphoryl oxygen and\or the β-γ bridge oxygen in the transition state [9,10,12,13]. Modelling an arginine into the Gly"$ position of the Ras structure in the Ras-GAP complex [10] (Figure 4) has revealed that its guanidinium group can occupy a similar position to that of the catalytic Arg()* of the GAP, with no steric clashes.…”

Section: Figure 4 Molecular Modelling Of An Arg 13 Substitution (Equimentioning

confidence: 99%

“…Structural and biochemical data indicate that the conserved residue Gln'" functions in GTP hydrolysis [5] by positioning the hydrolytic water molecule in alignment with the γ-phosphoryl group of GTP [6][7][8]. The transition-state alignment is stabilized further by interaction with a GTPase-activating protein (GAP) [9,10]. Another important catalytic group is the so-called ' arginine finger ' [11].…”

Section: Introductionmentioning

confidence: 99%

“…In the latter case, however, GAPs fulfil this role by providing the arginine residue in trans [9,10]. The phosphate-binding loop (i.e.…”

Section: Introductionmentioning

confidence: 99%

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GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

Liang

Mather

2000

Biochemical Journal

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Structural and biochemical data indicate the importance of the phosphate-binding loop residues Gly"# and Gly"$ of Ras both in the GTP hydrolysis reaction and in biological activity, but these two residues are not conserved in other Ras-related GTPases. To gain a better understanding of this region in GTP hydrolysis and GTPase function, we used the Ras-related Rab5 GTPase as a model for comparison, and substituted the Ala$! residue (the equivalent of Gly"$ of Ras) with all the other 19 amino acids. The resulting mutants were analysed for GTP hydrolysis, GTP binding, GTP dissociation and biological activity. Only the substitution of alanine with proline reduced the GTPase activity by an order of magnitude. This effect is in sharp contrast with the observation that a proline substitution at the neighbouring position (Gly"# of Ras) has little effect on the GTPase activity. Whereas most other substitutions showed either a small negative effect or no effect on the GTPase activity, the arginine substitution

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Section: Figure 4 Molecular Modelling Of An Arg 13 Substitution (Equimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

Liang

Mather

2000

Biochemical Journal

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“…Indeed, studies performed in fibroblasts have established that upon activation, the three main members of the Rho family remodel the cytoskeleton, each giving rise to a distinct pattern of F-actin structures. Constitutively active forms of Cdc42 (cell-provided conditions suitable for the elucidation of three-dimensional structures of small GTPases (Rittinger et al, 1997;Nassar et al, 1998). Other studies then revealed that formation of GAPRhoGTPase complexes is independent of aluminium and of the guanine nucleotide, suggesting a role for fluoride distinct from that of a γ-phosphatemimic, formulated in the initial hypothesis (Vincent et al, 1998).…”

Section: Introductionmentioning

confidence: 98%

Sodium fluoride induces podosome formation in endothelial cells

Tatin

Grise

Reuzeau

et al. 2010

Biology of the Cell

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Background information. Fluoride is a well‐known G‐protein activator. Exposure of cultured cells to its derivatives results in actin cytoskeleton remodelling. Podosomes are actin‐based structures endowed with adhesion and matrix‐degradation functions. This study investigates actin cytoskeleton reorganization induced by fluoride in endothelial cells.Results. Treatment of cultured endothelial cells with sodium fluoride (NaF) results in a rapid and potent stimulation of podosome formation. Furthermore, we show that Cdc42 (cell‐division cycle 42), Rac1 and RhoA activities are stimulated in NaF‐treated cells. However, podosome assembly is dependent on Cdc42 and Rac1, but not RhoA. Although the sole activation of Cdc42 is sufficient to induce individual podosomes, a balance between RhoGTPase activities regulates podosome formation in response to NaF, which in this case are often found in groups or rosettes. As in other models, podosome formation in endothelial cells exposed to NaF also involves Src. Finally, we demonstrate that NaF‐induced podosomes are fully competent for matrix protein degradation.Conclusions. Taken together, our findings establish NaF as a novel inducer of podosomes in endothelial cells in vitro.

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“…The active form is located on the plasma membrane, and the inactive form, in the cytosol. 44 RhoA activation has been shown to affect cell fusion and syncytia formation after infection with paramyxoviurses such as respiratory syncytia virus 45 and Hendra virus. 25 RhoA is regulated by heat shock proteins: HSP90 is required for RhoA activation in thrombin-induced signaling to cytoskeleton, and the activation of RhoA could be specifically inhibited by GA in this reaction.…”

Section: Combining Measles Virus With Heat Shock Protein Inhibitors Cmentioning

confidence: 99%

Heat shock protein inhibitors increase the efficacy of measles virotherapy

Liu

Erlichman

et al. 2008

Gene Ther

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Oncolytic measles virus strains have activity against multiple tumor types and are currently in phase I clinical testing. Induction of the heat shock protein 70 (HSP70) constitutes one of the earliest changes in cellular gene expression following infection with RNA viruses including measles virus, and HSP70 upregulation induced by heat shock has been shown to result in increased measles virus cytotoxicity. HSP90 inhibitors such as geldanamycin (GA) or 17-allylaminogeldanamycin result in pharmacologic upregulation of HSP70 and they are currently in clinical testing as cancer therapeutics. We therefore investigated the hypothesis that heat shock protein inhibitors could augment the measles virus-induced cytopathic effect. We tested the combination of a measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) and GA in MDA-MB-231 (breast), SKOV3.IP (ovarian) and TE671 (rhabdomyosarcoma) cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated 6-24 h following MV infection. Western immunoblotting confirmed HSP70 upregulation in combination-treated cells. Combination treatment resulted in statistically significant increase in syncytia formation as compared to MV-CEA infection alone. Clonogenic assays demonstrated significant decrease in tumor colony formation in MV-CEA/GA combination-treated cells. In addition there was increase in apoptosis by 4,6-diamidino-2-phenylindole staining. Western immunoblotting for caspase-9, caspase-8, caspase-3 and poly(ADP-ribose) polymerase (PARP) demonstrated increase in cleaved caspase-8 and PARP. The pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected tumor cells from MV-CEA/GA-induced PARP activation, indicating that apoptosis in combinationtreated cells occurs mainly via the extrinsic caspase pathway. Treatment of normal cells, such as normal human fibroblasts, however, with the MV-CEA/GA combination, did not result in cytopathic effect, indicating that GA did not alter the MV-CEA specificity for tumor cells. One-step viral growth curves, western immunoblotting for MV-N protein expression, QRT-PCR quantitation of MV-genome copy number and CEA levels showed comparable proliferation of MV-CEA in GAtreated vs -untreated tumor cells. Rho activation assays and western blot for total RhoA, a GTPase associated with the actin cytoskeleton, demonstrated decrease in RhoA activation in combination-treated cells, a change previously shown to be associated with increase in paramyxovirus-induced cell-cell fusion. The enhanced cytopathic effect resulting from measles virus/GA combination supports the translational potential of this approach in the treatment of cancer.

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Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue

Cited by 405 publications

References 25 publications

GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

Sodium fluoride induces podosome formation in endothelial cells

Heat shock protein inhibitors increase the efficacy of measles virotherapy

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