PMC

HGF/SF-dependent phosphorylation profiles of ERK and MEK are insensitive to lactacystin. HeLa cells were preincubated for 2 h with (+) or without (−) lactacystin and then stimulated with HGF/SF for the times indicated. Cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phospho-ERK1/2 (top) and anti-phospho-MEK1/2 (bottom).

HRS knockdown by siRNA retards HGF-dependent degradation of Met and inhibits dephosphorylation. HeLa cells were treated with siRNA, targeted against Hrs for 72 h, or underwent a dummy transfection protocol as described in MATERIALS AND METHODS. Cells were then stimulated with HGF for the indicated times. Cell lysates were blotted with anti-Met, anti–PY1349-Met (which selectively recognizes a phosphotyrosine containing peptide that provides a docking site for proteins downstream of Met activation), anti-Hrs, and anti-tubulin, as indicated.

Ligand-induced ubiquitination of Met can occur in the presence of proteasome inhibitors, although subtle differences in the pattern of ubiquitination are apparent. HeLa cells were cultured in medium with or without HGF/SF as indicated. Equal quantities of lysates from cells harvested at the indicated times after HGF/SF addition were immunoprecipitated, resolved by SDS-PAGE on 5% or 8% gels, and examined by Western analysis under reducing conditions by using the indicated antibody combinations. Molecular mass markers are shown on the left. Similar results were obtained in five experiments.

Proteasome activity and clathrin-mediated endocytosis are required for the dephosphorylation of Met that occurs after an HGF/SF-induced phosphorylation. (A) HeLa cells were preincubated for 2 h with (+) or without (−) lactacystin and then stimulated with HGF/SF for the times indicated. The cells were then lysed and proteins immunoprecipitated with anti-Met antibody. Immunoprecipitated proteins were analyzed by immunoblotting with PY20 antibody (top) or anti-Met (bottom). (B) Intensity of phospho-Met bands (top) and total Met bands (bottom) was quantitated using NIH Image 1.62 software and a ratio of phospho-Met:Met was calculated for each time point. Phospho-Met:Met after 30-min stimulation was expressed as a percentage of phospho-Met:Met after 10-min stimulation. Each data point represents mean ± SEM of five different experiments. *p < 0.005. (C) K44A cells were cultured for 48 h with (+) or without (−) tetracycline and then stimulated with HGF/SF for the times indicated. Analysis of Met phosphorylation was performed as in A.

Proteasome activity and clathrin-mediated endocytosis modulate HGF/SF and EGF-induced Hrs phosphorylation. (A) K44A cells were cultured for 48 h with (+) or without (−) tetracycline and then stimulated with HGF/SF for the times indicated. The cells were then lysed and proteins immunoprecipitated with anti-Hrs antibody. Immunoprecipitated proteins were analyzed by immunoblotting with PY20 antibody (top) or anti-Hrs (bottom). (B and C) HeLa cells were preincubated for 2 h with (+) or without (−) lactacystin and then stimulated with HGF/SF or EGF for the times indicated. Analysis of Hrs phosphorylation was performed as in A. (D) Intensity of phospho-Hrs bands (top) and total Hrs bands (bottom) from C were quantitated using NIH Image 1.62 software and a ratio of phospho-Hrs:Hrs was calculated for each time point. Phospho-Hrs:Hrs after 20-min stimulation was expressed as a percentage of phospho-Hrs:Hrs after 8-min stimulation. Each data point represents mean ± SEM of three experiments. *p < 0.05.

Inhibition of the proteasome has no effect on ligand-induced Met internalization but instead promotes recycling to the plasma membrane. (A) Cell-surface receptors were biotinylated on ice with a disulfide-cleavable biotin as described in MATERIALS AND METHODS. Cells were then rapidly rewarmed to 37°C and cultured in medium with or without HGF/SF and/or proteasome inhibitor for the indicated times to allow internalization of surface Met, after which they were rapidly cooled on ice, and remaining cell-surface biotin stripped with MESNA. After lysis, internalized biotinylated molecules were recovered with NeutrAvidin-conjugated beads, resolved by SDS-PAGE, and examined by Western analysis under reducing conditions using anti-Met^Hu intracellular-domain antibody. Similar results were obtained in three experiments. (B) To investigate recycling of Met after a 10-min HGF/SF “pulse,” cells were rewarmed after MESNA stripping, but in the absence of ligand, for 15 or 30 min at 37°C then subjected to a second round of MESNA stripping before lysis and analysis as described in A. Molecular mass markers are shown on the left.

Isolation of biotinylated Met: efficiency of MESNA reduction and optimization of cell-surface Met biotinylation. (A) HeLa cells were incubated on ice for 15 min ± 500 μg/ml disulfide-cleavable biotin, as described in MATERIALS AND METHODS. The cells were then washed of excess biotin by three quenching rinses and either lysed immediately (lane 2), or after three incubations in a solution containing the reducing agent MESNA (lane 3), plus additional washes. Surface biotinylated molecules were recovered with NeutrAvidin-agarose beads, resolved by 8% SDS-PAGE, and examined by Western blot analysis under reducing conditions using anti-Met^Hu intracellular-domain antibody. MESNA efficiently cleaves all biotin-Met moieties. (B) Biotinylations were carried out with varying concentrations of biotin. Excess biotin was removed by three quenching rinses and the cells were then lysed. i) Surface biotinylated molecules were recovered with NeutrAvidin-agarose beads, resolved by SDS-PAGE, and examined by Western blot analysis under reducing conditions using anti-Met^Hu intracellular-domain antibody. ii) The unbound fraction from each precipitation reaction was also probed with the same anti-Met^Hu antibody. Molecular mass markers (in kilodaltons) are shown on the left. Using 250 μg/ml or more of biotin (lanes 5 and 6) results in only a very small (<5%) p140^Met signal in the unbound fraction, implying that a high proportion of total mature Met, under steady-state conditions, resides at the plasma membrane and is accessible to biotinylation at these concentrations.