Identification of Tyrosine 489 in the Carboxy Terminus of the Tpr-Met Oncoprotein as a Major Site of Autophosphorylation

The Met receptor tyrosine kinase is the receptor for hepatocyte growth factor/scatter factor. HGF/SF is a multifunctional cytokine that can stimulate proliferation, motility, and morphogenesis in epithelial and endothelial cells. Oncogenic activation of the Met receptor occurs through a genomic rearrangement that generates a hybrid protein in which tpr sequences are directly fused amino terminal to the met receptor kinase domain. The resultant Tpr-Met hybrid protein posseses tyrosine kinase activity, is constitutively phosphorylated on tyrosine residues in vivo, and transforms fibroblasts in culture. We have identified two tyrosine residues within the catalytic domain of the Tpr-Met oncoprotein (Y365, Y366) and Met receptor (Y1234, Y1235) that are phosphorylated and essential for both the catalytic and biological activity of the oncoprotein and receptor. However, a detailed analysis of phosphorylation in these proteins has not been undertaken. In order to determine the sites of tyrosine phosphorylation in the Tpr-Met oncoprotein, in vitro mutagenesis, phosphopeptide mapping, and dephosphorylation protection assays were performed. Here we identify that a single tyrosine (Y489) in the carboxy terminus of the Tpr-Met oncoprotein is highly phosphorylated and is essential for biological activity. In contrast, additional tyrosines (Y482, Y498) located in the carboxy terminus are not phosphorylated at detectable levels and are not essential for the biological activity of the oncoprotein.