Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase.
The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235.
Ferracini
R
R
Department of Biomedical Sciences and Oncology, University of Torino Medical School, Italy.
Longati
P
P
Naldini
L
L
Vigna
E
E
Comoglio
P M
PM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019859:Proto-Oncogene Proteins c-met; D014357:Trypsin