Mapping of tyrosine kinase autophosphorylation sites with synthetic peptide substrates.
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.
Turck
C W
CW
Howard Hughes Medical Institute, University of California, San Francisco.
Edenson
S P
SP
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D017421:Sequence Analysis; D015398:Signal Transduction; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*
D009842:Oligopeptides; D010446:Peptide Fragments; D017468:Receptors, Fibroblast Growth Factor; D014443:Tyrosine; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017479:Receptors, Platelet-Derived Growth Factor