Abstract
RhoA plays a significant role in actin stress fibers formation. However, silencing RhoA alone or RhoA and RhoC did not completely suppress the stress fibers suggesting a residual "Rho-like" activity. RhoB, the third member of the Rho subclass, is a shortlived protein barely detectable in basal conditions. In various cell types, the silencing of RhoA induced a strong up-regulation of both total and active RhoB protein levels that were rescued by re-expressing RhoA and related to an enhanced half-life of the protein. The RhoA-dependent regulation of RhoB does not depend on the activity of RhoA but is mediated by its GDP-bound form. The stabilization of RhoB was not dependent on isoprenoid biosynthesis, Rho kinase, extracellular signal-regulated kinase, p38 mitogen-activated kinase, or phosphatidylinositol 3'-OH kinase pathways but required RhoGDIalpha. The forced expression of RhoGDIalpha increased RhoB half-life, whereas its knock-down antagonized the induction of RhoB following RhoA silencing. Moreover, a RhoA mutant (RhoAR68E) unable to bind RhoGDIalpha was significantly less efficient as compared with wild-type RhoA in reversing RhoB up-regulation upon RhoA silencing. These results suggest that, in basal conditions, RhoGDIalpha is rate-limiting and the suppression of RhoA makes it available to stabilize RhoB. Our results highlight RhoGDIalpha-dependent cross-talks that regulate the stability of RhoGTPases.
Highlights
The small GTPases of the Rho family are at the cross-roads of signaling pathways initiated by receptors to diffusible biological mediators and those depending on cell-adhesion receptors
A RhoA mutant (RhoAR68E) unable to bind RhoGDI␣ was significantly less efficient as compared with wild-type RhoA in reversing RhoB up-regulation upon RhoA silencing. These results suggest that, in basal conditions, RhoGDI␣ is rate-limiting and the suppression of RhoA makesitavailabletostabilizeRhoB.OurresultshighlightRhoGDI␣dependent cross-talks that regulate the stability of RhoGTPases
Phosphorylation of RhoGDI␣ by various kinases that decreases its affinity for RhoGTPases is one mechanism used by receptors to activate specific RhoGTPases [3, 4]
Summary
Reagents and Cells—The antibodies were purchased from the following manufacturers: mouse anti-RhoA (sc-418), rabbit anti-RhoB (number sc-180), goat anti-RhoC (number sc-26480), rabbit anti-RhoGDI␣ (number sc-360), and normal rabbit IgG (number sc-2027) were from Santa Cruz Biotechnology; mouse anti-Rac (23A8) from Upstate Biotechnology; mouse anti-Cdc from BD Biosciences; rabbit anti-ERK1,2 (number M-5670) was from Sigma; rabbit monoclonal anti-actin (13E5, number 4970), rabbit polyclonal anti-AKT (number 9272), anti-phospho-AKT (Ser-473, number 9271), and anti-phospho ERK1,2 (number 4370X) were from Cell Signaling; mouse monoclonal anti-human p38 (AHO0782) was from BioSource International; rabbit anti-phospho-p38 was from Zymed Laboratories Inc. (number 36-8500); rabbit polyclonal anti-Tet-repressor antibody was from MoBiTec (number TET01); rat anti-HA (clone 3F10, number 11867423001) was from Roche Diagnostics. To generate the HA-tagged RhoBR68E, 3 mutations were introduced in the HA-RhoB sequence by a PCR-based approach These three cDNAs were cloned into pcDNA4/TO. RhoA-dependent Regulation of siScr[2X] RhoB—RhoB is a short-lived and inducible protein that is barely detectable as observed by Western blot analysis of whole lysate of mock-transfected cells or of cells transfected with a control siRNA siRhoA+C (Fig. 2). A minimum of 100 cells in each condition tested S1, the use of these controls confirmed the negative regulations were analyzed to determine the percentage of cells displaying operated by RhoA and RhoC on RhoB and the specificity of stress fibers. Silencing of Rac or Cdc with our previously published siRNA sequences [18] did not affect RhoB protein level (Fig. Silencing RhoA and RhoC Does Not Completely Suppress the 3A). Its specific To definitively ascertain the specificity of the negative regularepression, up to 95%, in various cell types with two different tion operated by RhoA on RhoB expression, the silencing of sets of siRNA did not alter the cell morphology of either actin RhoA was rescued by re-expressing a RhoA mRNA resistant to HS578T HT1080
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