RON mediates signaling and induces cell migration in response to MSP. ZR75.1 cells (A) or A2780 and A2780-Ron cells (B) were stimulated with MSP for 1 h, and lysates were analyzed for activation of signaling pathways as described under “Experimental Procedures.” RON migrates as two separate bands of molecular mass 175 kDa (unprocessed (U)) and 150 kDa (processed (P)), as indicated. RON phosphorylation was analyzed by immunoprecipitating RON from lysates using anti-RON antibody coupled to agarose beads and immunoblotting with anti-phosphotyrosine antibody Tyr(P)-100. Migration assays were performed as described under “Experimental Procedures.” Cell migration was analyzed by microscopy (C) and quantified (D). Data in D are depicted as mean ± S.D. (error bars) of two separate experiments performed in triplicate.

Docking site tyrosine Tyr-1353 mediates RON-induced signaling and cell migration. A, sequence homology surrounding the two docking site tyrosines of MET and RON. B, surface expression of wild-type or mutant RON variants by stably transfected A2780 cells was analyzed by FACS. C, morphology of transfected A2780 cells stably expressing RON variants. D, pathway activation in response to 30 min of MSP treatment in A2780 cells stably expressing wild type or the indicated docking-site mutants of RON. E, phosphorylation of p44/42 MAPK (Thr-202/Tyr-204), AKT (Ser-473), and S6 (Ser-235/236) was quantified using LI-COR based on the fluorescent intensity of the protein bands and normalized to the intensity of either total MAPK, AKT, and S6 proteins (from two independent experiments) or actin (from two additional independent experiments). Data represent mean ± S.D. (error bars) of four independent experiments. F and G, migration of A2780 cells stably expressing wild-type or mutant RON variants in response to MSP. Data in G are depicted as mean ± S.D. of two independent experiments performed in triplicates.

Docking site tyrosines of RON and MET bind the Gab1 and Grb2 adaptor proteins differentially. A, A2780-RON cells were left unstimulated or stimulated with MSP for 30 min. Cells were lysed, and RON was immunoprecipitated. Total and phosphorylated RON, Gab1, and Grb2 proteins present in RON immunoprecipitates were detected by IB with the indicated antibodies (IP-RON panels). Expression of RON, Gab1, and Grb2 in cell lysates is shown (bottom panel). B, RON or RON-KD was co-expressed with FLAG-tagged Gab1 or FLAG-tagged Grb2 in 293-T cells by transient transfection as described under “Experimental Procedures.” Cells were lysed and subjected to RON IP, followed by detection of total or phosphorylated RON (B, top). Binding of RON or RON-KD to the indicated adaptors was detected in anti-FLAG (M2) immunoprecipitates by IB (B, middle). Expression of RON, Gab1, and Grb2 in the cell lysates is shown (B, bottom). C and D, wild-type RON or the indicated docking site mutants were co-expressed with FLAG-Gab1 or FLAG-Grb2. Binding between RON and each adaptor was detected by IP of the adaptor using anti-FLAG (M2) antibody, followed by IB as indicated (C and D, top panels; input is shown in the bottom panels). E, interaction between MET and Gab1 or Grb2 was analyzed as in C and D.

Differential requirement of Gab1 and Grb2 adaptor proteins in RON and MET signaling. Gab1 and Grb2 were depleted by siRNA transfection of A2780-RON cells (A and B) or A549 cells (C and D), as described under “Experimental Procedures.” Activation of intracellular signaling pathways in response to MSP (A) or hepatocyte growth factor (HGF) (C) was quantified using LI-COR, and the phosphorylated p42/44 MAPK, AKT, and S6 proteins were normalized to the expression of total MAPK, AKT, and S6 proteins, respectively. -Fold change shown in B for MSP and D for hepatocyte growth factor indicates the response of cells depleted of Gab1 or Grb2 individually or together relative to undepleted control cells. Data represent mean ± S.D. (error bars) from three independent experiments.

Phosphorylated RON is enriched in Gab1 immunoprecipitates compared with Grb2 immunoprecipitates. A and B, RON was transfected into 293-T cells singly or with FLAG-tagged Gab1 or FLAG-tagged Grb2. The amount of phosphorylated and non-phosphorylated RON in anti-RON immunoprecipitates (A) and anti-FLAG IPs (B) is shown, with the input of RON, Gab1, and Grb2 proteins in B (bottom). C, RON was co-transfected with FLAG-tagged Gab1. The FLAG (M2) immunoprecipitate was treated with buffer or calf intestinal phosphatase (CIP) for 1 h at 37 °C. Phosphorylated proteins were detected by IB as indicated. D, MET was transfected singly or with FLAG-tagged Gab1 or FLAG-tagged Grb2. The amount of phosphorylated and non-phosphorylated MET in anti-MET immunoprecipitates (D) and anti-FLAG immunoprecipitates (E) is shown.

Grb2 attenuates binding of Gab1 to RON by reducing RON autophosphorylation. A, RON was co-expressed with FLAG-Gab1 or with FLAG-Gab1 and HA-Grb2, and binding to Gab1 was analyzed. Phosphorylated and non-phosphorylated RON in the presence of Gab1 alone or Gab1 plus Grb2 is shown (A, middle panel, input in lower panel). B, autophosphorylation of RON by in vitro kinase assay. RON immunoprecipitate was incubated in kinase buffer in the presence of ATP for 30 min as described under “Experimental Procedures.” C, a time course of RON autophosphorylation by in vitro kinase assay. RON was transfected singly or with Gab1 or Grb2. RON immunoprecipitate from each transfection was divided into four equal parts and incubated in the absence or presence of ATP for the indicated times. D, MET was co-transfected with FLAG-Gab1 or FLAG-Grb2 or FLAG-Gab1 plus HA-Grb2. Phosphorylated proteins in Gab1 immunoprecipitates (lanes 2 and 3) and Grb2 immunoprecipitates (lane 4) are shown (B, top). Expression of receptor and adaptor proteins in Gab1 immunoprecipitates (lanes 2 and 3) or Grb2 immunoprecipitates (lane 4) is shown (B, middle panel; input in bottom panel).

Grb2 negatively regulates RON signaling. A, Gab1 and Grb2 were depleted in A2780 cells expressing the Y1360F RON mutant by siRNA as described under “Experimental Procedures.” Phosphorylated AKT (Ser-473) and p42/44 MAPK (Thr-202/Tyr-204) proteins were quantified and normalized to total AKT and MAPK proteins from three independent experiments using LI-COR. MSP response for each siRNA transfection was determined by comparing AKT and MAPK phosphorylation in the absence or presence of MSP. -Fold change shown in A represents response of cells depleted of Gab1 or Grb2 individually or together relative to undepleted control cells. Data represent mean ± S.D. (error bars) from three independent experiments. B, RON or RON Y1360F mutant was expressed in 293-T cells in the absence or presence of Grb2 knockdown. RON immunoprecipitates were analyzed by IB for bound Gab1 and phosphorylated Gab1 proteins (top panel, IP-RON). Expression of RON, Gab1, and Grb2 proteins in cell lysates is shown (bottom panel). C, time course of signaling pathway activation in NIH3T3 cells stably expressing wild-type RON and the docking site mutants of RON in response to MSP. D, a model depicting how Gab1 and Grb2 adaptor proteins regulate RON signaling. Activated RON dimers phosphorylate docking site tyrosines to recruit Gab1 and Grb2. Binding of Gab1 to Tyr-1353 enhances receptor autophosphorylation and Gab1 phosphorylation, driving a strong signal. Binding of Grb2 to Tyr-1360 results in incomplete autophosphorylation of RON, abrogates Gab1 binding, and attenuates signaling.