Fig 1.
Identification of a 10 kDa amino-terminal RhoA fragment.
A) Western blot analysis of COS-7 cell lysates transfected with Flag-tagged WT-RhoA or with 2Xmyc-tagged WT-RhoA using an anti-Flag M2 or anti-c-myc antibody reveals the presence of FL-RhoA and RhoA-NTF bands. B, C) Western blot of cell lysates following treatment with the proteosome inhibitors MG132 (B) or epoxomicin (C). D) Expression of wild-type (WT), constitutively active (Q63L and G14V), dominant-negative (T19N) as well as non-prenylated wild-type (C190A), active (Q63L/C190A) and inactive (T19N/C190A) RhoA constructs in COS-7 cells analyzed by western blot using the Flag M2 antibody. Western blot panels illustrating FL-RhoA only are exposed to evaluate loading of full-length protein while panels with FL-RhoA and RhoA-NTF are a longer exposure of the same blot to visualize RhoA-NTF.
Fig 2.
Endogenous RhoA proteolysis is enhanced by oxidative stress.
A, B) Lysates from COS-7 cells were transfected with Flag-tagged WT-RhoA (A) or T19N-RhoA (B) and western blotted with anti-Flag M2 antibody. Cells were exposed to H2O2 for 1 hour, 24 hours prior to lysis. C) COS-7 cells transfected with Flag-tagged WT-RhoA were immunoprecipitated with the Rho Y486 antibody from Abcam. Lysates and immunoprecipitates were probed with anti-RhoA Y486 or anti-FLAG M2 antibodies. D) Immunoprecipitation of RhoA-NTF from untransfected COS-7 cells treated with H2O2 for 1 hour at 24 hour prior to lysis or from various healthy adult rat tissues, including the heart, brain and lungs, using the Rho Y486 antibody. Arrow: RhoA-NTF. E-F) Analysis of the relative abundance of FL-RhoA and RhoA-NTF in cell lysates compared to a dose curve of recombinant WT-RhoA by Western blot analysis with the Rho Y486 antibody. The graph in F quantifies the average concentration of FL-RhoA as well as RhoA-NTF upon treatment with 1000 μM H2O2.
Fig 3.
Serine proteases, caspases and calpain regulate RhoA proteolysis.
(A-F). Lysates from COS-7 cells transfected with Flag-tagged WT-RhoA were analyzed by western blot with anti-Flag M2 antibody following treatment with protease inhibitors. Transfected cells were treated for 3h with the serine protease inhibitor AEBSF (A), 24h hours with the pan-caspase inhibitor z-VAD-fmk (C), or 3 hours with the calpain inhibitor calpeptin (E) and the levels of RhoA-NTF were quantified by densitometry (B, D, F). Water and DMSO were vehicle controls. (G-H). 2Xmyc WT-RhoA (G) or Flag RhoA 1–56 (H) were immunoprecipitated from transfected COS-7 cells and treated for 45 minutes with recombinant μ-calpain in the presence or absence of 14 μM calpeptin. I) Diagram illustrating the mechanism underlying RhoA proteolysis.
Fig 4.
Mapping of the RhoA cleavage site.
A) Coomassie brilliant blue-stained SDS-Page gel of FL-RhoA and RhoA-NTF showing the bands excised (boxes) for tandem mass spectrometry. B) Alignment of the peptides obtained by mass spectrometry of RhoA-NTF following trypsin (red) or chymotrypsin (blue) digest onto the theoretical sequence of Q63L-RhoA. C) Mutations were introduced in Q63L-RhoA to localize the cleavage site. D) Rhotekin-binding-domain (RBD) pull-down of COS-7 cell lysates transfected with the various Q63L-RhoA mutants evaluated the activation of each construct. E) Diagram of the FL-RhoA as well as both amino (NTF)- and carboxy (CTF)- terminal RhoA fragments. F) Expression of the constructs encoding the Flag-tagged RhoA fragments illustrated in E by western blot with Flag M2 antibody. G) Western blot analysis of COS-7 cells lysates transfected with the dual tag Flag Q63L-RhoA V5-His to evaluate the presence of RhoA-CTF using anti-V5 antibody.
Fig 5.
RhoA fragments and cleavage-resistant promote the formation of actin stress fibres.
A) Serum-starved Swiss 3T3 fibroblasts were transfected with Flag-tagged RhoA 1–56 and 57–193, which correspond to RhoA-NTF and -CTF respectively, as well as WT-, Q63L- and the cleavage-resistant L57A/Q63L-RhoA. Cells were stained with anti-Flag M2 antibody (green), Alexa-Fluor 546 phalloidin (red) and Hoechst (blue) to label RhoA, the actin stress fibres and the nucleus respectively. Scale bar, 20 μm. B) Classification of the actin phenotype in transfected Swiss 3T3 cells. Significance was determined by the Chi-square test. *, p < 0.05; ** p < 0.005; ***, p < 0.0005; ****, p < 0.0001. n > 40 cells from 7 independent experiments. C) Quantification of the ratio of actin-covered area divided by the total surface of the transfected cells represented as the mean +/- S.E.M. Significance was established by one-way ANOVA from n > 40 cells collected from 7 independent experiments. *, p < 0.05; ** p < 0.005; ***, p < 0.0005; ****, p < 0.0001. D) Representative side view of a z-stack of a Swiss 3T3 cell overexpressing RhoA-CTF showing the localization of nuclear actin rods (red) relative to the nucleus (blue) and RhoA 57–193 (green). Scale bar, 10 μm.
Fig 6.
Both RhoA fragments, but not cleavage-resistant RhoA, interfere with the organization of actin stress fibres induced by activated endogenous full-length RhoA.
A) Serum-starved Swiss 3T3 fibroblasts were transfected the Flag-tagged RhoA 1–56 and 57–193 fragments as well as WT-, Q63L- and L57A/Q63L-RhoA. The endogenous full-length RhoA was activated by treating the cells with 10% FBS for 30 min prior to fixation. The cells were stained with anti-Flag M2 antibody (green), Alexa-Fluor 546 phalloidin (red) and Hoechst (blue) to label RhoA, the actin stress fibres and the nucleus respectively. Scale bar, 20 μm. B) Classification of the actin phenotype in the transfected Swiss 3T3 cells. n > 20 cells from 3 independent experiments. Significance was determined by the Chi-square test. *, p < 0.1; ****, p < 0.0001. C) Quantification of the ratio of the actin-covered area divided by the total area of the transfected cell represented as the mean +/- S.E.M. Significance was established by one-way ANOVA from n > 20 cells from 3 independent experiments. *, p < 0.05; ** p < 0.005; ***, p < 0.0005; ****, p < 0.0001. D) Frequency distribution of the angles’ deviation from the mode of the actin stress fibres present in the transfected Swiss 3T3 cells as measured with AngleJ. The area under the curve contained within 20° of the mode is representative of the proportion of segments with a high degree of organization.