Autophosphorylation of soluble insulin receptor protein-tyrosine kinases. 1H NMR spectral changes observed during phosphorylation of mobile tyrosine residues.
Autophosphorylation of a soluble approximately 48-kDa derivative of the insulin receptor protein-tyrosine kinase occurs at multiple tyrosine residues (analogous to tyrosines 1158, 1162, and 1163 in the kinase homology region of the native receptor and tyrosines 1328 and 1334 in the carboxyl-terminal tail) and is accompanied by an increase in the specific activity of the enzyme toward exogenous substrates. A comparison of 1H NMR spectra of approximately 48- and approximately 38-kDa forms of enzyme (the latter generated by tryptic deletion of approximately 10 kDa from the carboxyl terminus of the approximately 48-kDa protein) allows a correlation of observed mobile tyrosine resonances to two of the known sites of autophosphorylation (residues 1328 and 1334). Furthermore, spectra acquired during autophosphorylation of the approximately 48-kDa enzyme reveal a rapid downfield shift in the resonances of these mobile tail tyrosines consistent with their phosphorylation (as confirmed by two-dimensional tryptic phosphopeptide mapping performed under identical conditions). This experimental strategy now provides a means by which to monitor protein-tyrosine kinase autophosphorylation in solution in real time.
Levine
B A
BA
Inorganic Chemistry Laboratory, University of Oxford, United Kingdom.
Tavaré
J M
JM
Alejos
E
E
Clack
B
B
Sayed
N
N
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D006859:Hydrogen; D007313:Insecta; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin; D014357:Trypsin; D014443:Tyrosine
D000596:Amino Acids; D010748:Phosphopeptides; D014443:Tyrosine; D006859:Hydrogen; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D014357:Trypsin