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. 1998 Sep;18(9):5567-78.
doi: 10.1128/MCB.18.9.5567.

Ras-GAP controls Rho-mediated cytoskeletal reorganization through its SH3 domain

V Leblanc  1 , B Tocque, I Delumeau
Affiliations

Ras-GAP controls Rho-mediated cytoskeletal reorganization through its SH3 domain

V Leblanc et al. Mol Cell Biol. 1998 Sep.

Abstract

Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton following growth factor stimulation. A major regulator of Ras, Ras-GAP, contains several structural motifs, including an SH3 domain and two SH2 domains, and there is evidence that they harbor a signalling function. We have previously described a monoclonal antibody to the SH3 domain of Ras-GAP which blocks Ras signalling in Xenopus oocytes. We now show that microinjection of this antibody into Swiss 3T3 cells prevents the formation of actin stress fibers stimulated by growth factors or activated Ras, but not membrane ruffling. This inhibition is bypassed by coinjection of activated Rho, suggesting that the Ras-GAP SH3 domain is necessary for endogenous Rho activation. In agreement, the antibody blocks lysophosphatidic acid-induced neurite retraction in differentiated PC12 cells. Furthermore, we demonstrate that microinjection of full-length Ras-GAP triggers stress fiber polymerization in fibroblasts in an SH3-dependent manner, strongly suggesting an effector function besides its role as a Ras downregulator. These results support the idea that Ras-GAP connects the Ras and Rho pathways and, therefore, regulates the actin cytoskeleton through a mechanism which probably does not involve p190 Rho-GAP.

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Figures

FIG. 1
FIG. 1
MAb 200, an anti-Ras-GAP SH3 antibody, inhibits Ras- and PDGF-induced stress fiber formation, but not membrane ruffles, in Swiss 3T3 cells. Actin structures are shown in serum-deprived cells, quiescent and uninjected (A) and 3 h (B) or 5 h (C) after coinjection of Ha-RasK12 (1 mg/ml) with a control mouse IgG (8 mg/ml) and 3 h (D) or 5 h (E) after coinjection of Ha-RasK12 (1 mg/ml) with MAb 200 (8 mg/ml). (F and G) Cells were stimulated for 30 min with 3 ng of PDGF per ml 2 h after injection of a control IgG or MAb 200, respectively. Actin filaments were detected with FITC-conjugated phalloidin. The white stars indicate noninjected cells.
FIG. 2
FIG. 2
Inhibition of LPA-induced stress fibers by MAb 200 but not by Y13-259. Actin filaments are shown in serum-starved Swiss 3T3 cells without injection (A) or stimulated with 0.115 μM LPA for 10 min 2 h after injection of a control mouse IgG (B), MAb 200 (C), or Y13-259 (D). Proteins were injected at a concentration of 8 mg/ml. Actin filaments were detected with FITC-conjugated phalloidin. All cells in each field have been injected.
FIG. 3
FIG. 3
Inhibition of LPA-induced focal complex assembly by GST-GAPSH2-SH3-SH2. Focal complexes are shown in serum-deprived Swiss 3T3 cells without injection or stimulation (A) and in cells stimulated for 10 min with 0.115 μM LPA 2 h after injection of GST (B) or GST-GAPSH2-SH3-SH2 (D). (C and E) Injected cells corresponding to those in panels B and D, respectively. Proteins were injected at 1.5 mg/ml. Cells were double stained with an antiphosphotyrosine antibody, revealed by an FITC-labelled secondary antibody to detect focal complexes (left panel), and with an anti-GST antibody, revealed by a Texas red-labelled secondary antibody to detect injected cells (right panel). The white star indicates noninjected cells.
FIG. 4
FIG. 4
MAb 200 does not inhibit RhoAV14-induced stress fibers in Swiss 3T3 cells. The photographs show phalloidin staining of uninjected cells (A) and cells 2 h after coinjection of RhoAV14 with a control IgG (B) or with MAb 200 (C). RhoAV14 was injected at a concentration of 0.3 mg/ml, and the antibodies were injected at a concentration of 8 mg/ml. F-actin was detected with FITC-conjugated phalloidin. All cells in each field have been injected.
FIG. 5
FIG. 5
Full-length Ras-GAP induces stress fiber polymerization through its SH3 domain and requires Rho but not Ras activity. Actin structures are shown in serum-deprived Swiss 3T3 cells 4 h after microinjection of GST (A), GST-Ras-GAP plus a control mouse IgG (B), GST plus C3 toxin (C), GST-Ras-GAP plus C3 toxin (D), GST-Ras-GAP plus Y13-259 (E), and GST-Ras-GAP plus MAb 200 (F). GST and GST-Ras-GAP were injected at a concentration of 1.5 mg/ml, C3 toxin was injected at 70 μg/ml, and MAb 200, the control IgG, and Y13-259 were injected at 8 mg/ml. F-actin was detected with FITC-conjugated phalloidin. The white stars indicate noninjected cells.
FIG. 6
FIG. 6
Effect of MAb 200 on DNA synthesis induced by FCS, LPA, and Ras. For Ras induction of DNA synthesis, MAb 200 (8 mg/ml) or the control IgG (8 mg/ml) was coinjected with Ha-RasK12 (2 mg/ml) in serum-deprived cells. After injection, cells were incubated in the presence of BrdU-FdU for 40 h. For LPA or FCS induction of S-phase entry, cells were injected with MAb 200 or the control IgG and incubated in the presence of 100 μM LPA or 10% FCS and BrdU-FdU for 40 h. Cells were then processed for anti-BrdU staining as described in Materials and Methods. The percentage of MAb 200-injected cells which incorporated BrdU is compared to that of control IgG-injected cells, which was taken as 100% for each condition of stimulation. These control values represent 98% of the injected cells in the case of FCS stimulation, 80 to 60% in the case of LPA stimulation, and 50% in the case of Ras injection. The results are the average ± standard error of three experiments.
FIG. 7
FIG. 7
Effect of MAb 200, GST-GAPSH2-SH3-SH2, and GST-Ras-GAP on the morphology of PC12 cells. PC12 cells are shown 3 days after injection of a control mouse IgG (A) or MAb 200 (B). Note the presence of short neurites in MAb 200-injected cells, which are absent in control cells. In panel C are shown cells 6 days after coinjection of Ha-RasK12 with GST, while cells injected with Ha-RasK12 and GST-GAPSH2-SH3-SH2 are shown in panel D. Note the difference in neurite length between panels C and D. Cells coinjected with Ha-RasK12 plus GST are fully differentiated after 4 days (E), whereas those injected with Ha-RasK12 plus GST-Ras-GAP are not (F). The antibodies were injected at a concentration of 8 mg/ml, Ha-RasK12 was injected at 1.5 mg/ml, and GST, GST-GAPSH2-SH3-SH2, and GST-Ras-GAP were injected at 2 mg/ml. The cell clumps in panels E (bottom right corner) and F (bottom left corner) were not injected; in the other fields, all cells have been injected or stem from injected cells.
FIG. 8
FIG. 8
MAb 200 protects NGF-differentiated PC12 cells from LPA-induced neurite retraction and cell rounding. IgG- or MAb 200-injected cells (upper and lower panels, respectively) are shown 2 h postinjection without addition (A and D) or 30 min (B and E) or 3 h (C and F) after stimulation with 10 μM LPA. All cells in these fields have been injected.
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