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.

[1]  M. Mann,et al.  Signaling Initiated by Overexpression of the Fibroblast Growth Factor Receptor-1 Investigated by Mass Spectrometry* , 2003, Molecular & Cellular Proteomics.

[2]  S. Hubbard,et al.  Crystal structure of the MuSK tyrosine kinase: insights into receptor autoregulation. , 2002, Structure.

[3]  C. Kahn,et al.  A cascade of tyrosine autophosphorylation in the beta-subunit activates the phosphotransferase of the insulin receptor. , 1988, The Journal of biological chemistry.

[4]  V. P. Eswarakumar,et al.  Cellular signaling by fibroblast growth factor receptors. , 2005, Cytokine & growth factor reviews.

[5]  Joseph Schlessinger,et al.  Structure of the FGF Receptor Tyrosine Kinase Domain Reveals a Novel Autoinhibitory Mechanism , 1996, Cell.

[6]  M. Jaye,et al.  A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1 , 1991, Molecular and cellular biology.

[7]  K. Anderson,et al.  A snapshot of enzyme catalysis using electrospray ionization mass spectrometry. , 2003, Journal of the American Chemical Society.

[8]  S. Hubbard,et al.  Expression, Characterization, and Crystallization of the Catalytic Core of the Human Insulin Receptor Protein-tyrosine Kinase Domain (*) , 1995, The Journal of Biological Chemistry.

[9]  K. Anderson,et al.  Probing the role of tightly bound phosphoenolpyruvate in Escherichia coli 3-deoxy-d-manno-octulosonate 8-phosphate synthase catalysis using quantitative time-resolved electrospray ionization mass spectrometry in the millisecond time range. , 2005, Analytical biochemistry.

[10]  M. Jaye,et al.  Ligand‐induced transphosphorylation between different FGF receptors. , 1991, The EMBO journal.

[11]  B. Mroczkowski,et al.  Characterization and kinetic mechanism of catalytic domain of human vascular endothelial growth factor receptor-2 tyrosine kinase (VEGFR2 TK), a key enzyme in angiogenesis. , 1998, Biochemistry.

[12]  D. Lauffenburger,et al.  Time-resolved Mass Spectrometry of Tyrosine Phosphorylation Sites in the Epidermal Growth Factor Receptor Signaling Network Reveals Dynamic Modules*S , 2005, Molecular & Cellular Proteomics.

[13]  I. Lax,et al.  Critical role for the docking-protein FRS2α in FGF receptor-mediated signal transduction pathways , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Y. Yarden,et al.  Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation. , 1987, Biochemistry.

[15]  T. Hunter,et al.  Oncogenic kinase signalling , 2001, Nature.

[16]  C. Dickson,et al.  Fibroblast growth factor signaling in tumorigenesis. , 2005, Cytokine & growth factor reviews.

[17]  A. Ullrich,et al.  Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Sam A. Johnson,et al.  Phosphoproteomics finds its timing , 2004, Nature Biotechnology.

[19]  Y. Yarden,et al.  Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. , 1987, Biochemistry.

[20]  Stevan R. Hubbard,et al.  Structure and autoregulation of the insulin-like growth factor 1 receptor kinase , 2001, Nature Structural Biology.

[21]  Nanxin Li,et al.  Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis , 1992, Nature.

[22]  M. Mann,et al.  Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics , 2004, Nature Biotechnology.

[23]  M. Jaye,et al.  Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction , 1996, Molecular and cellular biology.

[24]  T. Pawson,et al.  Assembly of Cell Regulatory Systems Through Protein Interaction Domains , 2003, Science.

[25]  M. McTigue,et al.  Mechanistic effects of autophosphorylation on receptor tyrosine kinase catalysis: enzymatic characterization of Tie2 and phospho-Tie2. , 2001, Biochemistry.

[26]  E. Goldsmith,et al.  Autophosphorylation activates the soluble cytoplasmic domain of the insulin receptor in an intermolecular reaction. , 1989, The Journal of biological chemistry.

[27]  Hanno Steen,et al.  Tyrosine Phosphorylation Mapping of the Epidermal Growth Factor Receptor Signaling Pathway* , 2002, The Journal of Biological Chemistry.