Mapping of tyrosine kinase autophosphorylation sites with synthetic peptide substrates.
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Tyrosine phosphorylation is a key step in the regulation of many cellular events including signal transduction of stimulated growth factor receptor tyrosine kinases. Upon ligand activation, these proteins undergo dimerization and subsequent auto- and transphosphorylation events on specific tyrosine residues, which enables them to interact with several cellular signaling proteins. We have used synthetic peptides encompassing all the tyrosine residues of a tyrosine kinase and employed them as substrates in in vitro kinase reactions. Using this assay we have shown that short tyrosine-containing peptides derived from the cytoplasmic domain of the mouse platelet-derived growth factor beta receptor (PDGF-R) can serve as specific phosphorylation targets for the kinase. These peptides include 7 out of 8 tyrosines that are known auto- or transphosphorylation sites in vivo, as previously determined by peptide mapping and mutational analyses. We have also identified 10 additional tyrosine-containing peptides that are phosphorylated and represent possible novel auto- or transphosphorylation sites of PDGF-R. The presented method greatly simplifies the mapping of auto- or transphosphorylation sites in tyrosine kinases and provides a valuable tool in the analysis of signaling mechanisms involving these proteins.