The J domain of hTid-1 selectively interacts with kinase-inactive MetR. (a) A schema depicting the GST constructs used in the GST pull-down experiments: full-length Tid-1 construct, a J-domain construct, a G/F/Cys-rich and C-terminal construct. (b) 786-0 RCC cells were serum-starved and either left untreated (−) or treated with 40ng/ml HGF (+). Lysates were incubated with the indicated GST proteins and the resulting complexes were subjected to immunoblotting with specific antibodies to MetR, and a phosphotyrosine specific antibody (pTyr 4G10). To ensure equal amounts of GST proteins used, the SDS–PAGE gel was stained with Coomassie blue (bottom panel). (c) 786-0 RCC cells were serum-starved and either left untreated (−) or treated with HGF (+). Cell lysates were immunoprecipitated with Met or hTid-1 antibodies and immune complexes were resolved by SDS-PAGE and analyzed by western blotting using antibodies specific to phospho-Met (Tyr1234/1235 in activation loop, pMet), total Met and hTid-1. (d) Cell lysates processed as shown in panel c were immunoprecipated with pMet antibodies and immune complexes probed with Tid-1 and pMet antibodies. (e) 786-0 Tid1 null cells were infected with adenoviruses expressing either Ad-GFP, Ad-hTid-1_S or Ad-hTid-1_L and stimulated with HGF as indicated. Cell lysates were immunoprecipitated with Met antibodies and immunoblotted with either total Met, Tid-1 or pMet antibodies.

hTid-1_S enhances HGF-mediated migration. (a) hTid-1_S (Ad-hTid-1_S) and hTid-1_L (Ad-hTid-1_L) were overexpressed in 786-0 RCC cells via adenoviral infection. An adenovirus expressing GFP alone was used as a control (Ad-GFP). The cells were serum-starved overnight and plated in the upper chamber of a transwell chamber, using HGF as a chemoattractant in the lower chamber. After 24 h of incubation, the cells that had migrated were fixed and stained with Diff-Quik. Representative pictures from each filter are shown. (b) Lysates from cells infected simultaneously as those used for migration were immunoblotted with anti-hTid-1 to confirm efficient hTid-1 overexpression. (c) Migrated cells were counted in five individual fields and averaged. The results are pooled from three independent experiments. An unpaired t-test was used to determine significance between GFP and hTid-1_S or hTid-1_L overexpressing cells. *, a P-value of <0.05 was considered significant.

hTid-1 overexpression does not affect 786-0 cellular proliferation. (a) 786-0 cells were infected with adenoviruses expressing either GFP, hTid-1_S or hTid-1_L, and serum-starved overnight. A total of 10 000 cells/well were plated in 24-well plates on day 0. Triplicate wells were counted with a haemocytometer on the indicated days post-infection. The mean and s.d. of each triplicate time point was determined. (b) Experiment was repeated as shown in panel a except 2500 cells/well were plated in each well of a 24-well plate and cells were treated with 100 ng/ml HGF. (c) Lysates from adenoviral infected 786-0 cells from panels a and b were subjected to immunoblot analysis using anti-hTid-1 antibodies.

hTid-1 knockdown inhibits Met activation and HGF-mediated migration. (a) Left panel, 786-0 RCC cells that had been stably infected with either a control lentivirus or a si-hTid-1 expressing lentivirus were serum-starved overnight and treated with HGF for the indicated time points. Whole-cell lysates were collected and resolved by SDS–PAGE. Western blotting was used to analyze phospho-Met (Tyr1234/Tyr1235, pMet), total Met and hTid-1 levels. Right panel, densitometric analysis was used to determine the degree of Met phosphorylation relative to total Met levels and is shown as the relative pixel density of pMet:Met compared with untreated cells. (b) Left panel, 786-0 cells that have been stably infected with either a control lentivirus or a si-hTid-1 expressing lentivirus were serum-starved overnight and used in a transwell migration assay. A total of 7.5 × 10⁵ cells were plated in the upper chamber with HGF in the lower chamber. Cells were allowed to migrate for 24 h, and representative pictures for each filter is shown. Right panel, The number of cells that migrated was counted in five individual fields/filter and the average number of cells/field was calculated, and is shown as a ratio to the number of cells that migrated in the control condition. The data shown represent pooled data from three independent experiments. An unpaired t-test was used to determine significance. *, a P-value of <0.05 was considered significant. (c) hTid-1 knockdown inhibits signaling through Erk1/2 and STAT3. 786-0 RCC cells that had been stably infected with either a control lentivirus or a si-hTid-1 expressing lentivirus were serum-starved overnight and treated with 10 ng/ml HGF for the indicated time points. Whole-cell lysates were prepared and subjected to immunoblot analysis using antibodies specific for phospho-Met (Tyr1234/1235, pMet), total Met, pSTAT3, total STAT3, phospho-Erk1/2 (pErk1/2), hTid-1 and β-actin.

hTid-1 modulates MetR tyrosine phosphorylation. (a) 786-0 parental RCC cells (786-0–VHL) and 786-0 cells in which VHL has been reintroduced (786-0+VHL) were infected with adenoviruses expressing either GFP alone (Ad-GFP), hTid-1_S (Ad-hTid-1_S) or hTid-1_L (Ad-hTid-1_L) as indicated. Cells were serum-starved overnight and either left untreated (−) or treated with 40ng/ml HGF (+). Whole-cell lysates were immunoblotted with antibodies specific to phospho-Met (Tyr1234/1235 in the activation loop; pMet), total Met, hTid-1, VHL and β-actin. Met antibodies detect Met molecules of 170 kDa (precursor) and 140 kDa (mature β-chain). (b) MDA-MB-231 cells were infected and treated as in panel a, except that cells were treated with HGF over the indicated time course. (c) Experiment was repeated as in panel b using A549 lung cancer cells.

Localization of MetR with hTid-1. 786-0 cells were seeded on coverslips and either left untreated (-HGF) or stimulated with HGF (+HGF) at 37 °C for 10 min. Coverslips were fixed in 2% PFA and stained with MetR and Tid-1 antibodies and images captured with a Zeiss LSM 410 Laser Scanning Confocal Microscope. Yellow staining represents colocalization between endogenous hTid-1 (red) and MetR (green).

hTid-1_S enhances HGF-mediated wound closure. (a) 786-0 RCC cells were infected with adenoviruses expressing either hTid-1_S (Ad-hTid-1_S) or hTid-1_L (Ad-hTid-1_L). A GFP expressing adenovirus (Ad-GFP) was used as a control. At 24-h post-infection, the cells were serum-starved for an additional 24 h. A uniform wound was made in the confluent cell layer at 0 h. The cells were then incubated with fresh serum-free media. (b) The same experiment was performed as shown in panel a, except that 5ng/ml HGF was added to the serum-free media at 0 h. Pictures of the wound at specific locations were taken at the indicated time points. (c, d) The wound width was calculated using Simple PCI software, and the relative wound closure was calculated setting wound closure of the control condition at 24 h as 1. The data presented is a representative experiment from three independent experiments. An unpaired t-test was used to calculate significance of either hTid-1_S or hTid-1_L overexpressing cells compared with the control at each time point. *, a P-value of <0.05 was considered significant.

Enhanced motility is associated with increased Erk1/2 activation. 786-0 RCC cells were infected with adenoviruses expressing hTid-1_S (Ad-hTid-1_S) or GFP as a control (Ad-GFP). The cells were serum-starved overnight and treated with HGF for the indicated time periods. Whole-cell lysates were resolved by SDS–PAGE and analyzed by western blotting with antibodies specific to phospho-Met (Tyr1234/1235; pMet), total Met, phospho-Erk1/2, total Erk1/2, pAkt, total Akt and hTid-1.