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First published online 30 October 2007
doi: 10.1242/jcs.003806

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A novel role for Lsc/p115 RhoGEF and LARG in regulating RhoA activity downstream of adhesion to fibronectin

¹ Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill NC 27599, USA
² Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill NC 27599, USA

View larger version (61K): [in this window] [in a new window]   Fig. 1. Adhesion to FN causes activation of RhoA and the RhoA-specific GEFs Lsc/p115 RhoGEF and LARG. Mouse fibroblasts were starved in serum-free media and then held in suspension in the same media for 2 hours. (A) Cells were plated onto FN-coated coverslips for the times indicated, then fixed and stained with phalloidin to visualize F-actin, and antibodies against phosphotyrosine to visualize focal adhesions. Bar, 40 µm. (B,C) Cells were plated onto FN-coated dishes for the times indicated, lysed and (B) pulldowns performed with GST-RBD and samples blotted with an antibody against RhoA, or (C) pulldowns performed with GST-RhoA(17A) and samples blotted with antibodies against the indicated GEFs. Quantification of all blots was performed as described in Materials and Methods.

View larger version (74K): [in this window] [in a new window]   Fig. 2. Lsc/p115 RhoGEF and LARG increase stress fibers and localizes to focal adhesions on FN. (A) Domain structure of full-length p115 RhoGEF [p115(FL)] and the different mutants used in this study. The DH-dead p115(4A) mutant contains alanine point substitutions (*) of four residues (E423, K567, L570, N603) in the DH domain that are important for the catalytic exchange reaction. The p115(N) mutant lacks the N-terminus of the protein containing the RGS domain. All constructs were cloned into N-terminal GFP- or V5-tagged vectors. (B,C) REF52 fibroblasts were transfected with vector encoding either GFP-p115(FL) or GFP-LARG(FL). 24 hours post transfection, cells were serum-starved, held in suspension for 2 hours and plated onto FN-coated coverslips for the times indicated. (B) The cells were then fixed and stained with phalloidin to visualize F-actin. Arrows in the top panels point to the tight cortical actin bundles known as arcs. Arrows in the bottom panels point to the discrete patches of p115 RhoGEF or LARG localization. Bar, 40 µm. (C) The cells were fixed and stained with antibody against paxillin to visualize focal adhesions. The images represent 0.3 µm confocal sections at the ventral surface of the cells. Arrows point to areas of colocalization between paxillin-containing focal adhesions and the discrete patches of p115 RhoGEF or LARG localization.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Knockdown of Lsc/p115 RhoGEF and LARG decreases formation of stress fibers and focal adhesions and RhoA activity downstream of FN. (A) REF52 cells were transfected with either control or siRNA oligonucleotides against Lsc and LARG as described in Materials and Methods. 72 hours post transfection, the cells were serum starved, held in suspension for 2 hours and plated onto FN-coated coverslips for 90 minutes. The cells were fixed and stained with phalloidin and antibody against phosphotyrosine to visualize stress fibers and focal adhesions. Bar, 40 µm. (B) Control cells or cells transfected with siRNA against Lsc and LARG were lysed and samples blotted with antibodies against Lsc and LARG to demonstrate the efficiency of knockdown. Identical blots with an antibody against Lfc show that protein levels of the closely related GEF Lfc are unaffected, demonstrating the specificity of the knockdown. (C) Fibroblasts were transfected with either control or siRNA oligonucleotides against Lsc and LARG. 72 hours post transfection, the cells were serum-starved, held in suspension for 2 hours and plated onto FN-coated dishes for 60 minutes. The cells were then lysed, GST-RBD pulldowns performed and samples blotted with an antibody against RhoA to visualize the levels of RhoA activity. (D) Fibroblasts were transfected with siRNA oligonucleotides against Lsc and LARG. 48 hours post transfection, the cells were re-transfected with either a vector control or a V5-tagged full-length p115 RhoGEF construct. 72 hours post transfection, the cells were processed for Rho activity assays, as described in (C) above. Lysates were also blotted with an antibody against V5 to show the expression levels of V5-p115(FL).

View larger version (46K): [in this window] [in a new window]   Fig. 4. DH-dead p115 RhoGEF inhibits formation of stress fibers and focal adhesions and RhoA activity on FN. (A) REF52 fibroblasts were transfected with vector encoding GFP-p115(FL) or GFP-p115(4A). 24 hours post transfection, the cells were lysed, pulldowns performed with RhoA(17A) and samples blotted with an antibody against GFP. (B) REF52 cells transfected with vector encoding GFP-p115(4A) were serum-starved, held in suspension for 2 hours and plated onto FN-coated coverslips. The cells were then fixed and stained with phalloidin to visualize F-actin and an antibody against phosphotyrosine to visualize focal adhesions. Bar, 40 µm. (C) REF52 cells overexpressing p115(4A) were plated onto FN-coated coverslips for 60 minutes. The cells were then treated with either DMSO or 10 µM nocodazole for 30 minutes and fixed and stained with phalloidin to visualize stress fibers. Cells were scored according to whether they had prominent stress fibers versus few to no stress fibers. (D) Cells were transfected with GFP-p115(4A), serum-starved and held in suspension for 2 hours. The cells were then plated onto FN-coated dishes, and GST-RBD pulldowns performed, and samples blotted with an antibody against RhoA to visualize the levels of RhoA activity. Lysates were also blotted with an antibody against GFP to show the levels of expression of GFP-p115(4A).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Activation of Lsc/p115 RhoGEF by adhesion to FN involves integrins but is independent of GPCRs. (A) Fibroblasts were transfected with constructs expressing either V5-p115(FL) or V5-p115(N). 24 hours post transfection, the cells were serum starved for 16 hours, treated with 5% fetal bovine serum for the times indicated and pulldowns performed with RhoA(17A). (B) V5-p115(N)-transfected fibroblasts were serum starved, held in suspension for 2 hours, plan ted onto FN-coated dishes and RhoA(17A) pulldowns performed. (C) Fibroblasts were transfected with siRNA oligonucleotides against Lsc and LARG. 48 hours post transfection, the cells were re-transfected with either a vector control or V5-p115(N). The next day, all the cells were serum starved, held in suspension for 2 hours and plated onto FN-coated dishes for 60 minutes. The cells were then lysed, GST-RBD pulldowns performed and samples blotted with an antibody against RhoA to visualize the levels of RhoA activity. Lysates were also blotted with an antibody against V5 to show the levels of expression of V5-p115(N). (D) Fibroblasts were serum starved and held in suspension for 2 hours. To prevent the production and secretion of endogenous FN by the fibroblasts, 25 µg/ml cycloheximide was included in the media during starvation and suspension. The cells were then plated onto dishes coated with FN, CBD or FN plus heparin. Samples were lysed, incubated with RhoA(17A) and processed for SDS-PAGE and blotting with an antibody against Lsc.