Autophosphorylation of FGFR1 kinase is mediated by a sequential and precisely ordered reaction.
Tyrosine phosphorylation of cellular proteins induced by extracellular cues serves as a critical mediator in the control of a great variety of cellular processes. Here, we describe an integrated experimental approach including rapid quench methodology and ESI-LC-MS/MS as well as time-resolved ESI-MS to demonstrate that tyrosine autophosphorylation of the catalytic tyrosine kinase domain of FGF-receptor-1 (FGFR1) is mediated by a sequential and precisely ordered reaction. We also demonstrate that the rate of catalysis of two FGFR substrates is enhanced by 50- to 100-fold after autophosphorylation of Y653 in the activation loop, whereas autophosphorylation of the second site in the activation loop (Y654) results in 500- to 1,000-fold increase in the rate of substrate phosphorylation. We propose that FGFR1 is activated by a two-step mechanism mediated by strictly ordered and regulated autophosphorylation, suggesting that distinct phosphorylation states may provide both temporal and spatial resolution to receptor signaling.
Furdui
Cristina M
CM
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Lew
Erin D
ED
Schlessinger
Joseph
J
Anderson
Karen S
KS
D002853:Chromatography, Liquid; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D010446:Peptide Fragments; D051496:Receptor, Fibroblast Growth Factor, Type 1