Autophosphorylation modulates the kinase activity and oncogenic potential of the Met receptor tyrosine kinase.
The met oncogene is activated by a genomic rearrangement that generates a hybrid protein containing tpr sequences at its amino terminus fused directly to the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase domain. The resultant p65Tpr-Met hybrid protein possesses tyrosine kinase activity and is constitutively phosphorylated on tyrosine in vivo. A substitution of the conserved lysine in the catalytic domain abolished kinase activity and the transforming potential of Tpr-Met demonstrating the requirement for kinase activity for transformation. To study the role of autophosphorylation at particular tyrosine residues on the transforming activity of Tpr-Met, the major autophosphorylation sites were identified by two dimensional phosphopeptide mapping. Two tyrosine residues in the catalytic domain, tyrosine 365 and tyrosine 366, were found to be the major autophosphorylation sites both in vitro and in vivo. Mutation of these sites singly or together modulates the biochemical and biological properties of Tpr-Met. Compared with the wild-type Tpr-Met, mutants have a reduced in vitro kinase activity when measured by levels of autophosphorylation and by their ability to phosphorylate an exogenous substrate and have little or no transforming activity. These results indicate that autophosphorylation at critical tyrosine residues is required for activation of the kinase activity of Tpr-Met and for cell transformation by this oncogene.
Rodrigues
G A
GA
Royal Victoria Hospital, Department of Medicine, McGill University, Montreal, Canada.
Park
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D017931:DNA Primers; D005786:Gene Expression Regulation; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met; D051381:Rats; D011916:Rats, Inbred F344; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met; D020794:Receptor Protein-Tyrosine Kinases