Hepatocyte Growth Factor Receptor/Tyrosine-Protein Kinase Met (HGFR/c-MET) is a glycosylated receptor tyrosine kinase that plays an important role in the development of cancer in different organs including liver, kidney and brain. Mature HGFR/c-MET is a disulfide-linked dimer composed of a 50 kDa extracellular α-chain and a 145 kDa transmembrane β-chain. Interaction of HGF with HGFR/c-MET triggers its internalization and subsequent proteasome-dependent degradation. It also stimulates phosphorylation of tyrosine residues in the cytoplasmic region, which leads to activation of the kinase domain and exposure of docking sites for multiple SH2-containing molecules. HGFR/c-MET has been implicated in the progression of glioblastoma, which is one of the most aggressive brain tumors. High expression levels of HGFR/c-MET has been shown to be indicative of shorter survival periods. To investigate the spatial distribution of HGFR/c-MET and its phosphorylated forms, we generated antibodies against its extracellular domain, as well as against specific phosphorylation sites in its intracellular kinase domain (Y1234/Y1235) and intracellular multisubstrate docking site (Y1349). Multiplex immunocytochemistry and immunohistochemistry were used to analyze colocalization of these domains in untreated and HGF-treated glioblastoma cell lines and in tissue sections of human glioblastoma. Treatment of U-251 MG, U-87 MG, U-118-MG and A-172 cells with Recombinant Human HGF induced profound phosphorylation of HGFR/c-MET. Phospho-HGFR/c-MET was detected in both the cytoplasm and cell nuclei. We also saw that HGFR/c-MET was colocalized with either phosphorylated form. In addition, the immunoreactive profiles for phospho-HGFR/c-MET (Y1349) and phospho-HGFR/c-MET (Y1234/Y1235) overlapped in certain instances, but not all, indicating the action of HGF on the same cells has different effects on tyrosine phosphorylation in the different domains. In glioblastoma tissue sections, phospho-HGFR/c-MET (Y1234/Y1235) was also detected in cell nuclei. Our data indicate that there is distinct activation of HGFR/c-MET at a single-cell level by phosphorylation that may underly the regulation of tumor progression.
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