Met is hyperphosphorylated in response to HGF in the absence of either PTP1B or TCPTP. A, HeLa cells were transfected with scrambled, PTP1B, TCPTP, or TCPTP + PTP1B siRNA and stimulated with HGF for the times indicated. Proteins from lysates were immunoprecipitated with the anti-human Met antibody and immunoblotted for either Met p-Tyr-1234/1235 or total Met. Protein lysates were also probed for TCPTP and PTP1B. B, phosphorylated Met/total Met quantitation of three independent replicates. The error bars represent S.E.

Loss of PTP1B or TCPTP increases the invasive potential of cells in response to HGF. PTP1B, TCPTP, and TCPTP and PTP1B or scrambled siRNA-treated cells were seeded on Matrigel in the absence or presence of HGF. A, representative images of invading cells post-fixing and staining, treated as indicated. B, quantitative representation of number of invading cells. The experiment was performed a minimum of three times, and statistical analysis was performed using Students t test. Error bars represent S.E. NS, not significant; UT, untransfected.

Dephosphorylation precedes Met degradation. A, HeLa cells were stimulated with HGF at 4 °C for 60 min and chased at 37 °C for times indicated. Protein extracts were subjected to immunoprecipitation using the human anti-Met antibody and probed with an anti-p-Tyr-20 and anti-Met antibody simultaneously. Western blots were analyzed using the Odyssey imaging system (Licor) and then probed for actin as loading control. B, densitometric analysis of data presented in Fig. 2A measured by NIH ImageJ program.

Localization of Met with TC48 D/A. A, HeLa cells were plated on coverslips and transfected with GFP-TC48 D/A. Yellow staining represents colocalization between endogenous Met (red) and TC48 (green). B, 16 h post-transfection cells were treated with cycloheximide (0.1 mg/ml) for 2 h and stimulated with 1.5 nm HGF at 37 °C for the indicated times. A and B, coverslips were fixed in 2% PFA and stained with Met AF276 and EEA1 antibody (B only). Confocal images were taken with a 100× objective and 1× zoom. Yellow staining represents colocalization between endogenous Met (red) and TC48 (green) or EEA1 (green or purple). The Scale bar is indicated in the figure.

Localization of Met with PTP1B D/A. A, HeLa cells were seeded on coverslips and transfected with GFP-PTP1B D/A. B, 16 h post-transfection, cells were treated with cycloheximide (0.1 mg/ml) for 2 h and stimulated with 1.5 nm HGF at 37 °C for the indicated times. A and B, coverslips were fixed in 2% PFA and stained with Met AF276 and EEA1 antibodies (B only). Confocal images were taken with a 100× objective and 1× zoom. Yellow staining represents colocalization between endogenous Met (red) and TC48 (green) or EEA1 (green or purple, as shown). The scale bar is as indicated in the figure.

Met activation causes nuclear exit of TC45. A, HeLa cells were seeded on coverslips and transfected with Myc-TC45 D/A. 16 h post-transfection, cells were stimulated with 1.5 nm HGF at 37 °C for the indicated times. Coverslips were fixed in 2% PFA and stained with anti-Met AF276 or anti-Myc 9E10 antibodies. Confocal images were taken with a 100× objective and 1× zoom. Yellow staining represents colocalization between endogenous Met (red) and Myc-TC45 D/A (green). B, T47D cells were plated on coverslips and transiently transfected with Myc-TC45 and either WT Met or Met K1110A (kinase dead). 16 h post-transfection, coverslips were fixed in 2% PFA and stained with Met AF276 or Myc 9E10 antibodies. Confocal images were taken with a 100× objective and 1× zoom. Yellow staining represents colocalization between Met (red) and Myc-TC45 D/A (green). The scale bar is indicated in the figure.

Met stably interacts with substrate trapping mutants of PTP1B and TCPTP. Extracts prepared from HEK 293 cells co-expressing Tpr-Met and either GFP-tagged WT (WT) PTP1B (1B), and TCPTP (TC45 or TC48) or their respective trapping mutants (DA) were immunoprecipitated using an anti-GFP antibody. A, immunoprecipitates of either PTP1B, TC45, or TC48 were probed for the presence of Met, phosphotyrosine (p-Tyr-100), and GFP. Immunoblot analysis of whole cell lysates (WCL) was performed with antibodies against Met and GFP to examine levels of Met, PTP1B, TC45, and TC48. B, immunoprecipitates of either PTP1B, TC45, or TC48 from cells lysed in the presence or absence of 1 mm sodium orthovanadate (NaV) were probed for the presence of Met, GFP, and overall phosphotyrosine (p-Tyr-100). Immunoblot analysis of WCL was performed with antibodies against Met, GFP, and p-Tyr-100 to examine levels of Met, phosphatases (PTP1B, TC45, TC48), and phosphotyrosine. Immunoblot analysis of WCL with actin was used as the loading control.

Interaction of Met with PTP1B or TCPTP requires tyrosines Tyr-1234 and Tyr-1235 of the Met receptor. A, protein lysates from HEK 293 cells co-expressing a combination of either GFP-tagged trapping mutants of PTP1B or TC48 or Myc-tagged TC45 and 1234 and/or 1235 twin tyrosine substitutions of Tpr-Met were subjected to immunoprecipitation using an anti-GFP or anti-Myc antibody as indicated, and immunoblot analysis was performed on both the immunoprecipitates and the corresponding WCL with antibodies as indicated. B, immunoprecipitation was performed with anti-GFP antibody, and blots were probed with antibodies specific for p-Tyr-100, Met, Met p-Tyr-1349, and GFP in lysates of cells expressing GFP-tagged PTP1B, TC45 or TC48, and Met mutants as indicated. Met receptor was immunoprecipitated, and immunoblot analysis was performed with antibodies raised against p-Tyr-100, Met, or Met p-Tyr-1349. WCL were probed with an antibody against GFP to examine levels of phosphatase present in each lane. C, immunoprecipitations were performed with anti-GFP and anti-Met antibodies in lysates of cells expressing GFP-tagged phosphatases and Met mutants where indicated, and immunoblot analysis was performed with antibodies against Met, Met p-Tyr-1234/1235, Met p-Tyr-1003, and GFP. D, densitometric analysis of amount of Met trapped by PTP1B, TC45, or TC48. Analysis was performed using NIH ImageJ. Results shown are the average of three independent experiments. The error bars in D represent S.E. Immunoblot analysis of WCL with actin was used as a loading control.

In response to HGF, Met is hyperphosphorylated in livers from PTP1B-null mice versus WT mice. A, PTP1B WT or null mice were injected with 15 nm HGF via the hepatic portal vein at the times indicated. Protein extracts prepared from livers of the injected mice were subjected to immunoprecipitation using an anti-mouse Met antibody and probed with the anti-phosphotyrosine antibody, p-Tyr-100, or phosphotyrosine antibodies directed against specific tyrosines of the Met receptor (p-Tyr-1003, p-Tyr-1365, or p-Tyr-1234/1235). Each time point represents an individual mouse. B, densitometric analysis of the data presented in A measured by NIH ImageJ and normalized against total Met levels from three independent replicates. C, protein lysates from livers of mice injected with HGF for times indicated probed with anti-pJNK, JNK, pERK, or total ERK antibodies. D, densitometric analysis of the p54 JNK blot shown in C compared against total protein levels. The error bars represent S.E. Each experiment was performed a minimum of three times.