Inhibition of RhoGAP activity is sufficient for the induction of Rho-mediated actin reorganization

doi:10.1016/S0171-9335(99)80019-3

European Journal of Cell Biology

Volume 78, Issue 8, August 1999, Pages 539-548

https://doi.org/10.1016/S0171-9335(99)80019-3 Get rights and content

Abstract

It is generally believed that the induction of actin cytoskeleton rearrangements by extracellular stimuli results from the activation of guanine nucleotide exchange factors for the Rho GTPases. Here, we present evidence that the inactivation of RhoGAP (GTPase activating protein) activity is an equally effective means of promoting Rho-mediated cellular processes. We observed that exposure of cultured fibroblasts to sodium fluoride (NaF) results in a rapid and potent stimulation of actin stress fiber formation. This effect is mediated by the Rho GTPase and is associated with the inactivation of cellular RhoGAP activity. Specifically, NaF promotes formation of a high-affinity complex between Rho and the two cellular p190 RhoGAPs in vivo, apparently sequestering limiting amounts of RhoGAP activity, thereby resulting in Rho activation. p190 RhoGAP activity was found to account for approximately 60 % of the total RhoGAP activity detected in whole cell extracts, indicating that relatively small changes in cellular RhoGAP activity can have potent effects on Rho activatiOn. We also found that sub-effective concentrations of NaF combined with sub-effective concentrations of the Rho pathway activator, lysophosphatidic acid, which stimulates guanine nucleotide exchange activity on the Rho GTPase, results in the rapid induction of actin stress fibers. Together, these results suggest that the Rho GTPase is regulated by a fine balance of nucleotide exchange and RhoGAP activities, and that inactivation of RhoGAP activity may be a physiologically important regulatory mechanism for activating the Rho GTPase.

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Citation Excerpt :
Similarly to AlF4−, albeit to a smaller extend, NaF inhibited the cleavage of the bond Gly42-Val43 in F-Mg-actin (Fig. 3B) which is consistent with the NaF-induced tightening of the intermonomer contacts involving the DNase-I-binding loop. In cultured fibroblasts treated with NaF, polymerized actin was shown to be rapidly reorganized into prominent stress fibers apparently due to its interference in the RhoA pathway [26]. To determine whether NaF can directly stimulate actin assembly, we examined the organization of the actin cytoskeleton in serum-starved fibroblasts exposed to NaF.
Dynamic instability of actin filaments can be inhibited by Pi analogs beryllium fluoride and aluminium fluoride that mimic the intermediate ADP-Pi state and stabilize actin filaments. On the other hand, the phosphoryl transfer enzymes can be activated in the absence of aluminium by magnesium fluoride if magnesium ions and sodium fluoride (NaF) were present in the solution. Whether magnesium fluoride promotes functional activities of actin is not known. Here we show, for the first time, that sodium fluoride strongly accelerates polymerization of highly dynamic Mg-F-actin assembled from the monomers proteolytically cleaved between Gly42 and Val43 within the D-loop with actin-specific protease protealysin (Pln-actin), apparently due to stabilization of nuclei formed at the initial step of actin polymerization. Thereby, NaF did not inhibit the ATPase activity (subunit exchange) on Pln-F-actin, did not increase the amount of Pln-F-actin sedimented by ultracentrifugation, and did not stabilize the inter-strand contacts of Pln-F-actin. On the other hand, NaF diminished accessibility of the nucleotide binding cleft of Mg-G-actin to trypsin, pointing to an additional cleft closure, and additionally protected the D-loop from the protealysin cleavage in Mg-F-actin, thus indicating that the longitudinal contacts are stabilized. We also demonstrate that in cultured cells NaF can directly promote assembly of F-actin structures under conditions when the corresponding activity of the RhoA pathway is inhibited. These data suggest that the NaF-induced assembly of actin filaments is promoted by magnesium fluoride that can be formed by the NaF-originating fluoride and the actin tightly bound magnesium.
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¹: Dr. Jeffrey Settleman, Massachusetts General Hospital Cancer Center and Harvard Medical School, Building 149, 13th Street, Charlestown, MA 02129/USA, Fax: ++61772 67808.

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Inhibition of RhoGAP activity is sufficient for the induction of Rho-mediated actin reorganization

Abstract

FEBS Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Cell

J. Biol. Chem.

J. Biol. Chem.

Cell

Eur. J. Pharmacol.

Exp. Cell Res.

Curr. Opin. Genet. Dev.

Cell

Cell

Cell. Signal.

Cell

J. Biol. Chem.

Proteins regulating Ras and its relatives

Nature

The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells

EMBO J.

The role of the PH domain in the signal-dependent membrane targeting of Sos

EMBO J.

Regulation of Sos activity by intramolecular interactions

Mol. Cell. Biol.

Control of Ras activation

Cancer Surv.

Stimulation of p21ras upon T-cell activation

Nature

Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases

Nature

Nonsense mutations in the C-terminal SH2 region of the GTPase activating protein (GAP) gene in human tumours

Nature Genet.

Stimulation of p21^ras upon T-cell activation