Journal: Journal of Cell Science

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

doi: 10.1242/jcs.197434

Figure Lengend Snippet: Domain organization, phylogeny, substrate specificity and subcellular localization of ARHGAP42. (A) Domain organization of ARHGAP42 in comparison to the three other mammalian members of the BAR-PH RhoGAP family. For ARHGAP42, the position of the major site of Src-mediated phosphorylation, Tyr-376, is indicated. OPHN1, oligophrenin-1. (B) Phylogram showing evolutionary relationships among the mammalian BAR-PH RhoGAP family members and to more distant relatives predicted from C. elegans (Ce T04C9.1A) and Drosophila (Dm Graf) genomes. The phylogram was generated using Multalin software (Corpet, 1988). (C) ARHGAP42 is a GAP for RhoA and Cdc42, but not Rac1. The GAP domain of ARHGAP42 was bacterially expressed, recovered as a GST fusion protein, and assessed for its activity toward the Rho GTPases RhoA, Rac1 and Cdc42 by measuring the amount of phosphate released by GTP hydrolysis using an in vitro assay. Ras was included as a negative control. Values are mean±s.d. from triplicate assays. (D,E) MEFs were transfected with GFP-ARHGAP42 expression plasmid and viewed 24 h later by fluorescence microscopy of fixed cells. The cells were either immunostained with an antibody against paxillin to mark focal adhesions (D, red) or with phalloidin to mark F-actin (E, red). In the representative cell shown in D, GFP-ARHGAP42 is most prominently localized at the focal adhesions and actin stress fibers. In the representative cell shown in E, GFP-ARHGAP42 is more prominently observed in association with actin stress fibers, as well as in apparent focal adhesions. Scale bar: 30 μm.

Article Snippet: Rabbit monoclonal antibody against RhoA (cat. no. 2117, 1:1,000), and phosphospecific antibodies against Src-pTyr416 (cat. no. 6943, 1:1,000) and ARHGAP42-pTyr376 (cat. no. 5617, 1:1,000) were all obtained from Cell Signaling Technology.

Techniques: Generated, Software, Activity Assay, In Vitro, Negative Control, Transfection, Expressing, Plasmid Preparation, Fluorescence, Microscopy

Journal: Journal of Cell Science

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

doi: 10.1242/jcs.197434

Figure Lengend Snippet: Src promotes phosphorylation of ARHGAP42 Tyr-376. (A,B) Immunoblot analysis of GFP-ARHGAP42 phosphorylation. GFP-ARHGAP42 variants were transiently expressed in (A) COS-7 cells or (B) MEFs, either with or without constitutively active Src-F529, and ARHGAP42 tyrosine phosphorylation was assessed by immunoprecipitation (IP) with anti-GFP antibody followed by immunoblotting (IB) with general anti-pTyr (pTyr) or anti-pTyr376 (pY376) antibody. (B) Prior to immunoprecipitation in MEFs, Src activity was further manipulated by incubating the cells for 2 h in the presence or absence of 5 µM saracatinib. Src-F529 expression was confirmed by immunoblot analysis of total cell lysates with antibody against the Src autophosphorylation site (pSrc). (C) In vitro kinase assay. GFP-ARHGAP42 variants were individually expressed in MEFs and immunoprecipitated using anti-GFP antibody, eluted, and incubated with immunoprecipitated Src-F529. After the kinase reaction had been carried out for 1.5 h, levels of GFP-ARHGAP42 phosphorylation were assessed using a pY376 antibody. The numbers on the right indicate the positions of molecular size markers (kDa).

Article Snippet: Rabbit monoclonal antibody against RhoA (cat. no. 2117, 1:1,000), and phosphospecific antibodies against Src-pTyr416 (cat. no. 6943, 1:1,000) and ARHGAP42-pTyr376 (cat. no. 5617, 1:1,000) were all obtained from Cell Signaling Technology.

Techniques: Western Blot, Immunoprecipitation, Activity Assay, Expressing, In Vitro, Kinase Assay, Incubation

Journal: Journal of Cell Science

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

doi: 10.1242/jcs.197434

Figure Lengend Snippet: ARHGAP42 with deleted BAR domain has enhanced RhoGAP activity. (A) MEFs were transfected with plasmids expressing GFP-ARHGAP42 variants WT, ΔBAR or ΔGAP, and 24 h later the cell lysates were analyzed by immunoblotting with an antibody raised against mouse ARHGAP42. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. Actin was detected as an additional loading control (bottom panel). The numbers indicate the positions of molecular size markers (kDa). (B) Example of a highly arborized MEF cell expressing GFP-ARHGAP42-ΔBAR. 24 h after transfection, the cell was fixed and visualized for GFP fluorescence. Scale bar: 30 µm. (C) Quantification of arborized morphology in MEFs expressing GFP-ARHGAP42 variants. Values are mean±s.d. from four independent transfections, with 500 fluorescent cells scored per transfection. (D) Deletion of the BAR domain significantly enhances the RhoGAP activity of ARHGAP42. Lysates from MEFs stably expressing ARHGAP42 variants were analyzed using a G-LISA assay to detect GTP-bound RhoA. Values are mean±s.d. RhoGTP signal compared to the signal from ARHGAP42-WT cells from three independent experiments. P-values indicate statistical significance determined by one-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: Rabbit monoclonal antibody against RhoA (cat. no. 2117, 1:1,000), and phosphospecific antibodies against Src-pTyr416 (cat. no. 6943, 1:1,000) and ARHGAP42-pTyr376 (cat. no. 5617, 1:1,000) were all obtained from Cell Signaling Technology.

Techniques: Activity Assay, Transfection, Expressing, Western Blot, Fluorescence, Stable Transfection

Journal: Journal of Cell Science

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

doi: 10.1242/jcs.197434

Figure Lengend Snippet: Expression of ARHGAP42 promotes membrane tubulation that is enhanced by deletion of the GAP domain. Plasmids expressing GFP-ARHGAP42-WT versus -ΔGAP (or the empty vector) were transfected into COS-7 cells and the cells were analyzed 48 h later. (A) Immunoblot analysis of whole cell lysates shows expression levels of the ARHGAP42 variants, with actin as a control for equal loading. (B) Representative cells expressing GFP-ARHGAP42-WT (left) and GFP-ARHGAP42-ΔGAP (middle and right, exhibiting membrane tabulation). The cells were fixed and visualized for GFP fluorescence. The boxed regions in the upper panels are enlarged in the lower panels. Scale bars: 30 μm. (C) Quantitative analysis of membrane tubulation induced by ARHGAP42 variants. Values are mean±s.d. from four independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: Rabbit monoclonal antibody against RhoA (cat. no. 2117, 1:1,000), and phosphospecific antibodies against Src-pTyr416 (cat. no. 6943, 1:1,000) and ARHGAP42-pTyr376 (cat. no. 5617, 1:1,000) were all obtained from Cell Signaling Technology.

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Fluorescence

Journal: Journal of Cell Science

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

doi: 10.1242/jcs.197434

Figure Lengend Snippet: Expression of ARHGAP42-ΔGAP increases focal adhesion size; expression of ARHGAP42-ΔBAR promotes focal adhesion turnover and cellular motility. (A) Quantification of focal adhesion size. MEFs expressing ARHGAP42 variants (WT, ΔBAR and ΔGAP) were grown on fibronectin-coated cover slips, fixed, immunostained with an antibody against paxillin, and focal adhesion size was analyzed by fluorescence microscopy. The box-and-whisker plot shows the range of size of focal adhesions. Center line shows the median, box limits indicate the first and third quartiles, whiskers extend to the minimum and maximum values. (B,C) MEFs were cotransfected with GFP-ARHGAP42 expression plasmids (WT, ΔBAR, ΔGAP) and (B) mCherry-zyxin or (C) mCherry-vinculin to mark focal adhesions. After transfection, cells were plated on fibronectin-coated glass bottom dishes and analyzed 48 h later by confocal live-cell microscopy. (B) The percentage of focal adhesions that either assembled or disassembled during a 20 min time interval. (C) FRAP analysis of mCherry-vinculin dynamics in focal adhesions, showing mean recovery halftimes. (A-C) The numbers in the histogram bars indicate the number of focal adhesions analyzed. (D) MEFs stably expressing GFP-ARHGAP42 variants (WT, ΔBAR, ΔGAP) were allowed to migrate for 24 h on a Petri dish and the healed area was subsequently determined by light microscopy followed by analysis using ImageJ software. Values are mean±s.d. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

Article Snippet: Rabbit monoclonal antibody against RhoA (cat. no. 2117, 1:1,000), and phosphospecific antibodies against Src-pTyr416 (cat. no. 6943, 1:1,000) and ARHGAP42-pTyr376 (cat. no. 5617, 1:1,000) were all obtained from Cell Signaling Technology.

Techniques: Expressing, Fluorescence, Microscopy, Whisker Assay, Transfection, Stable Transfection, Light Microscopy, Software

Journal: Journal of Cell Science

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

doi: 10.1242/jcs.197434

Figure Lengend Snippet: Src activates the GAP activity of ARHGAP42, requiring the Tyr-376 phosphorylation site. GFP-ARHGAP42 variants, -WT versus -Y376F, were expressed in either nontransformed or v-Src-transformed NIH-3T3 fibroblasts and analyzed 24 h after transfection. (A) Expression and tyrosine phosphorylation of GFP-ARHGAP42 variants was assessed by IP using anti-GFP antibody, and IB with ARHGAP42 antibody (top panel) or anti-pTyr antibody (middle panel). Src activity is indicated by IB of whole cell lysates with antibody against the Src autophosphorylation site (bottom panel). (B) Quantitative analysis of the arborized morphology characteristic of RhoA inhibition in NIH-3T3 cells. Values are mean±s.d. from five independent transfection experiments, with 500 cells scored per experiment. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test. (C) Subcellular localization of GFP-ARHGAP42 variants and cellular morphology were assessed by fluorescence microscopy of fixed cells. Representative nontransformed cells (left two panels) and v-Src-transformed cells (right two panels) are shown. The podosomal-like structures in the central region of a GFP-ARHGAP42-Y376F-expressing cell are shown in the inset. Scale bars: 30 μm.

Article Snippet: Rabbit monoclonal antibody against RhoA (cat. no. 2117, 1:1,000), and phosphospecific antibodies against Src-pTyr416 (cat. no. 6943, 1:1,000) and ARHGAP42-pTyr376 (cat. no. 5617, 1:1,000) were all obtained from Cell Signaling Technology.

Techniques: Activity Assay, Transformation Assay, Transfection, Expressing, Inhibition, Fluorescence, Microscopy

Journal: Journal of Cell Science

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

doi: 10.1242/jcs.197434

Figure Lengend Snippet: Src phosphorylation on Tyr376 regulates ARHGAP42 activity. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were transfected to stably express GFP-ARHGAP42-WT versus -Y376F. (A) Expression of ARHGAP42 and Src was confirmed by IB, with actin (bottom) as a control for equal loading. The ARHGAP42 antibody detects the GFP-tagged variants as well as an additional band of expected size for the endogenous protein. (B) Relative RhoA-GTP levels were determined in cell lysates using the G-LISA assay. Values are mean±s.d. RhoA-GTP signal compared to the signal from ARHGAP42-WT-expressing cells, from three independent experiments performed in triplicate. (C) The ability of ARHGAP42 to bind RhoA-CA was analyzed using a GST-RhoA-CA pull-down assay and (D) quantified as a ratio of the amount of indicated GFP-ARHGAP42 variant pulled down with GST-RhoA-CA and the corresponding amount of GFP-ARHGAP42 in the input cell lysate. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

Article Snippet: Rabbit monoclonal antibody against RhoA (cat. no. 2117, 1:1,000), and phosphospecific antibodies against Src-pTyr416 (cat. no. 6943, 1:1,000) and ARHGAP42-pTyr376 (cat. no. 5617, 1:1,000) were all obtained from Cell Signaling Technology.

Techniques: Activity Assay, Expressing, Transfection, Stable Transfection, Pull Down Assay, Variant Assay

Journal: Journal of Cell Science

Article Title: ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility

doi: 10.1242/jcs.197434

Figure Lengend Snippet: Src phosphorylation of ARHGAP42 on Tyr376 regulates focal adhesion size and dynamics of lamellipodia and focal adhesions. SYF cells or SYF cells expressing constitutively active Src-F529 (SrcF) were cotransfected with GFP-ARHGAP42 expression plasmids (WT, Y376F and ΔBAR), and plated on fibronectin-covered glass bottom dishes. At 24 h, cells showing similar levels of GFP-ARHGAP42 fluorescence, judged using an integrated intensity value of the GFP signal per cell and acquired with same settings (exposure, laser power, detector gain, etc.) of the microscope, were analyzed by confocal cell microscopy. (A) Quantitative analysis of focal adhesion size. The box-and-whisker plot shows the range in size of focal adhesions marked by paxillin staining in fixed cells. (B) Quantitative analysis of focal adhesion dynamics in live cells. Values are mean±s.e.m. percentage of dynamic focal adhesions during a 10 min time interval. (A,B) The numbers in the indicate the number of focal adhesions analyzed. (C) Quantitative analysis of lamellipodia velocities. Values are mean±s.e.m. velocities of protruding lamellipodia. The numbers in the histogram bars indicate the number of cells analyzed. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test.

Article Snippet: Rabbit monoclonal antibody against RhoA (cat. no. 2117, 1:1,000), and phosphospecific antibodies against Src-pTyr416 (cat. no. 6943, 1:1,000) and ARHGAP42-pTyr376 (cat. no. 5617, 1:1,000) were all obtained from Cell Signaling Technology.

Techniques: Expressing, Fluorescence, Microscopy, Whisker Assay, Staining