Journal: The Journal of Biological Chemistry

Article Title: HGF-induced formation of the MET–AXL–ELMO2–DOCK180 complex promotes RAC1 activation, receptor clustering, and cancer cell migration and invasion

doi: 10.1074/jbc.RA118.003063

Figure Lengend Snippet: Regulation of actin cytoskeleton by ELMO2 and DOCK180. A, MET and AXL kinase activities are required for the HGF-induced cytoskeleton reorganization. Serum-starved U373-MG cells were pretreated with DMSO, 1 μm crizotinib or 1 μm R428 for 3 h and then treated with or without HGF for 7 min. Cells were processed for co-immunostaining with anti-p-MET (Tyr-1234/1235–phosphorylated) (red) and anti-ELMO2 (green) antibodies as indicated. Cells were also counterstained with phalloidin (magenta). B, ELMO2, but not ELMO1, regulates cytoskeleton reorganization in response to HGF. U373-MG cells were transfected with control (luciferase; Luc), ELMO2, or ELMO1 siRNAs for 72 h. Cells were then serum-starved and stimulated with HGF as in A. Cells were co-immunostained with antibodies for p-MET (Tyr-1234/1235–phosphorylated) (red) and MET (total Met) (green) and then counterstained with phalloidin (magenta) as indicated. C, cells were transfected with control (luciferase) or DOCK180 siRNAs for 72 h and processed as in B. Cells were co-immunostained with antibodies for p-MET (Tyr-1234/1235–phosphorylated) (red) and ELMO2 (green) and then counterstained with phalloidin (magenta) as indicated.

Article Snippet: Monoclonal mouse antibodies against ELMO2 (C-12), ELMO1 (B-7), or DOCK180 (H-4) and polyclonal goat antibody against DOCK180 (C-19) were from Santa Cruz Biotechnology, Inc. Rabbit antibody against DOCK 180 (A301-287A) was from Bethyl Laboratories.

Techniques: Immunostaining, Transfection, Control, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: HGF-induced formation of the MET–AXL–ELMO2–DOCK180 complex promotes RAC1 activation, receptor clustering, and cancer cell migration and invasion

doi: 10.1074/jbc.RA118.003063

Figure Lengend Snippet: HGF-dependent interaction and activation of MET and AXL in melanoma and breast cancer cells. A, serum-starved A375 or MDA-MB-231 cells were treated with or without HGF (50 ng/ml) for 7 min. Western blotting analyses were conducted using antibodies specific for the indicated proteins with actin as a loading control. B, serum-starved A375 cells were treated with or without HGF for 7 min. Cell lysates were used for immunoprecipitation (IP) with anti-MET antibodies and then blotted with anti-p-AXL (Tyr-779–phosphorylated), AXL, p-MET (Tyr-1234/1235–phosphorylated), MET, and ELMO2 antibodies as indicated. C, same as in B except that MDA-MB-231 cells were used for immunoprecipitation and Western blotting analyses. D, serum-starved MDA-MB-231 cells were pretreated with 1 μm crizotinib or 1 μm R428 for 3 h. Cells were assayed for cell migration using the Oris cell migration chamber as that described in the legend to Fig. 9A, either in the absence or presence of HGF (50 ng/ml). After 24 h, cells were fixed, stained with DAPI, and imaged with a Nikon fluorescence microscope. Quantitation of the HGF-dependent cell migration was conducted similarly as in Fig. 9A and shown on the right (shown are the means with S.D.). Statistical analysis was conducted for each of the drug-treated samples versus the DMSO control (***, p < 0.001). E, quantitation of the HGF-dependent cell migration of A375 cells. Cell migration assay of A375 cells was conducted similarly to that described for MDA-MB-231 cells in D and quantified. F and G, A375 and MDA-MB-231 cells were subjected to the cell invasion assays using a Boyden chamber precoated with Matrigel like that described as in the legend to Fig. 9C. Serum-starved A375 cells (top of F) or MDA-MB-231 cells (bottom of F) were pretreated with 1 μm crizotinib or 1 μm R428 for 3 h. Cells were then seeded to the top chamber of the Boyden chamber, and the bottom chamber contained HGF (50 ng/ml). After 24 h, cells that migrated to the bottom chamber (invaded through Matrigel) were stained and imaged. Assays were performed in triplicates, and quantification results are shown in G (means with S.D.). Statistical analysis was conducted (compared with the control) (***, p < 0.001). H, schematic model summarizing our results. HGF binds to the MET RTK to form the MET–AXL heterodimer on the plasma membrane, promoting the phosphorylation of Tyr-779 on the p140 isoform of AXL. The MET–AXL RTK signaling complex recruits ELMO2 and DOCK180 to activate the RAC1-dependent cell migration and invasion. This pathway is different from the HGF-dependent activation of MET homodimer, which leads to the activation of AKT and S6 kinases. It is also distinct from the GAS6-mediated AXL homodimerization that leads to the phosphorylation of Tyr-702 on the p120 isoform of the AXL RTK, leading to the ELMO1/RAC1-dependent cell migration.

Article Snippet: Monoclonal mouse antibodies against ELMO2 (C-12), ELMO1 (B-7), or DOCK180 (H-4) and polyclonal goat antibody against DOCK180 (C-19) were from Santa Cruz Biotechnology, Inc. Rabbit antibody against DOCK 180 (A301-287A) was from Bethyl Laboratories.

Techniques: Activation Assay, Western Blot, Control, Immunoprecipitation, Migration, Staining, Fluorescence, Microscopy, Quantitation Assay, Cell Migration Assay, Clinical Proteomics, Membrane, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Cell adhesion controlled by adhesion G protein–coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo–Dock

doi: 10.1074/jbc.M117.780304

Figure Lengend Snippet: GPR124-dependent cell adhesion is mediated by its interaction with ITSN, which directly interacts with Elmo–Dock180, forming a signaling complex that co-localizes with GPR124. A, working model. GPR124 interacts with the Gβγ–Elmo–Dock complex as well as with ITSN1/2. These atypical and conventional guanine nucleotide exchange factors also directly interact with each other, constituting a novel signaling complex that mediates GPR124-dependent cell adhesion. B, Elmo interacts with ITSN during cell adhesion. COS7 cells expressing full-length HA–ITSN1 and FLAG–GPR124 (as indicated) were left in suspension or adhering for 30 min. Then endogenous Elmo was immunoprecipitated, and interacting HA-ITSN1 was revealed by Western blotting. Preimmune rabbit IgG was used as a negative control for immunoprecipitation (first lane for suspension and adhesion conditions). C, endogenous Elmo directly interacts with ITSN1-SH3A, ITSN1-SH3C, and ITSN1-SH3E domains. HEK293T cell lysates were incubated for 3 h at 4 °C with individual recombinant GST–ITSN1-SH3 domains (A–E). GST pulldown assays were performed as described under “Experimental procedures.” Endogenous Elmo specifically binds to SH3A, SH3C, and SH3E domains of ITSN1 (PD, Western blotting, anti-Elmo). Recombinant GST–ITSN-SH3 domains are shown stained with Ponceau. D, the proline-rich (PXXP) motif in Elmo is required to bind the ITSN1-SH3C domain. Cell lysates from HEK293T cells expressing either wild-type Myc–Elmo or PXXP* mutant Myc–Elmo were incubated for 3 h at 4 °C with the recombinant GST–ITSN1-SH3C domain, and the interacting Elmo was revealed by Western blotting with anti-Myc (top). The expression of Elmo in total cell lysates (TCL) is shown at the bottom. GST was used as a negative control in the pulldown assays. The interaction between Elmo and ITSN1-SH3C domain was lost when the proline-rich region of Elmo was mutated. E, GPR124-dependent cell adhesion is inhibited by the ITSN1-SH3A–E module. COS7 cells were transfected with GPR124–GFP either with or without the FLAG–ITSN1-SH3A–E module. Cell adhesion assays were performed for 30 min. GPR124-dependent cell adhesion was decreased in FLAG–ITSN1-SH3A–E module-expressing cells. Basal adhesion of EGFP–CAAX–expressing cells was used as a reference. Bars, mean ± S.E. (error bars). Statistics were performed by one-way ANOVA followed by Tukey's multiple-comparison post hoc test (*, p < 0.05; n = 3). The middle panel shows the expression of FLAG–ITSN1-SH3A–E module in total cell lysates, and actin was used as a loading control. Representative images showing adherent cells are shown at the bottom; insets show all fluorescent cells in the field before washing out non-adherent cells. F, GPR124, Gβγ, Elmo, and ITSN are detected in the isolated adhesion complex. Control and GPR124-transfected COS7 cells were left to adhere for 30 min on collagen IV or fibronectin-coated plates. Adherent cells were lysed, and proteins that remained attached to the plates were washed and recovered with Laemmli sample buffer. GPR124, Gβγ, Elmo, and ITSN were detected by Western blotting in the isolated adhesion complex as well as in total cell lysates. AKT was used to confirm that isolated adhesion complexes were not contaminated by nonspecifically bound cytosolic proteins and as a loading control in TCL. G, endogenous interaction between GPR124 and Elmo2 as well as between GPR124 and intersectin 1 in endothelial cells. Proximity ligation assays were performed in endothelial cells (HUVECs) using the indicated pairs of antibodies to detect endogenous GPR124 interacting with endogenous Elmo or with endogenous ITSN1. Individual antibodies were used alone as a control. The PLA signal per cell was quantified by ImageJ software. At least 30 cells were analyzed. The graph represents three independent experiments (mean ± S.E., n = 3). Representative pictures of PLA signals, depicted as red dots, are shown in the top and middle panels. Cell nuclei were stained with DAPI. H, endogenous GPR124 co-localizes with Elmo and ITSN at cell protrusions of adhering endothelial cells. Cell adhesion assays were performed on gelatin-coated glass coverslips for 30 min, followed by immunostaining to determine the localization of endogenous GPR124, Elmo, and ITSN proteins in HUVECs (white arrows). A representative cell observed by confocal microscopy is shown. Similar results were observed in 29 of 61 cells.

Article Snippet: Antibodies Anti-HA (H-3663), FLAG M2 (F-3165), AKT (P-2482), and c-Myc (clone 9E10, M-5546) were purchased from Sigma; GST (B-14, sc-138), GFP (B-2, sc-9996), Cdc42 (B-8, sc-8401), Gβ (M-14, sc-261), and ERK2 (C-14, sc-154) from Santa Cruz Biotechnology, Inc.; and Elmo2 from Novus Biologicals (catalog no. 100-879).

Techniques: Expressing, Suspension, Immunoprecipitation, Western Blot, Negative Control, Incubation, Recombinant, Staining, Mutagenesis, Transfection, Comparison, Control, Isolation, Ligation, Software, Immunostaining, Confocal Microscopy