Asymmetric receptor contact is required for tyrosine autophosphorylation of fibroblast growth factor receptor in living cells

Abstract

Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

Fig. 1.

The overall structure of asymmetric activated FGFR1 kinase dimer and detailed views of inter receptor contacts. (A) Asymmetric dimer of active phosphorylated FGFR1 is shown in ribbon diagram. Molecules E and S of the asymmetric dimer are colored in cyan and green, respectively. (B) A detailed view of the interface formed between kinases in the asymmetric dimer. ATP analog (AMP-PCP) and interacting residues are shown in stick representation and the magnesium ion is shown as a blue sphere. Residues from molecule S are labeled with primes. The color scheme applied in this figure is used for all figures. Secondary structures are labeled in blue. (C) Surface representation of molecule E is depicted in cyan with interacting residues of the molecule S in stick and ribbon representation. Representative residues from molecule S are labeled. (D) Surface representation of molecule S is shown in green with interacting residues of molecule E (Pale Cyan) in stick and ribbon representation (www.pymol.org).

Fig. 2.

Surface distributions of residues in the asymmetric FGFR1 kinase dimer interface. (A) Overall structures of the asymmetric kinase dimer are shown in ribbon format. (B) Surface presentation of molecule E (the enzyme) is in cyan. The proximal substrate-binding region is shown in red and distal substrate-binding region is shown in yellow. Activation-loop (A-loop) and nucleotide-binding loop (N-loop) are indicated. (C) Surface representation of molecule S (substrate) is in green with the tyrosine autophosphorylation site (Y583) in the kinase insert region of molecule S indicated. Substrate site of molecule S is colored in red and the distal substrate site is in yellow.

Fig. 3.

Autophosphorylation of FGFR1 in vitro and in vivo. Profiles of in vitro phosphorylation reactions of isolated kinase domains of (A) wt-FGFR1 and (B) FGFR1-RE at room temperature as a function of time. (C) Kinase activity of FGFR1-RE in vitro is maintained. Lysates of L6 cells expressing wt-FGFR1 or the FGFR1-RE mutant were subjected to immunoprecipitation with anti-FGFR1 antibodies. The immunoprecipitates were then incubated in the presence or absence of an FGFR1 substrate (PLCγ fragment, described in the results) for 30 min at room temperature followed by SDS-PAGE and immunoblotting with anti-pTyr or anti-FGFR1 antibodies. (D) Autophosphorylation of FGFR1-RE in vivo, is strongly compromised. L6 cells expressing either wt-FGFR1 or its RE mutant were stimulated with increasing concentrations of FGF (as indicated) for 10 min at 37 °C. Lysates of unstimulated or FGF-stimulated cells were subjected to immunoprecipitation using anti-FGFR1 antibodies followed by SDS-PAGE and immunoblotting with antipTyr or anti FGFR1 antibodies. (E) L6 cells expressing wt-FGFR1 or FGFR1-RE were stimulated with 100 ng/ml FGF for different times (as indicated). Lysates of unstimulated or FGF stimulated cells were subjected to SDS-PAGE followed by immunoblotting with anti-pTyr or anti-FGFR1 antibodies.

Fig. 4.

The structures of kinase domains of (A) wt-FGFR1 (PDB ID: 3KY2), (B) FGFR1-RE mutant (PDB ID: 3KXX), and (C) activated FGFR1 (FGFR1-3P) (PDB ID: 3GQI) in a simplified cartoon (Upper) and in a ribbon diagram (Below). The catalytic loop is shown in yellow, and the activation loop in green, helix αC is depicted as a cylinder in the cartoon. Phosphotyrosines are colored in red in the cartoon and in stick representation in the ribbon diagram. (D) Ribbon diagrams of kinase insert loops of FGFR1, FGFR1-RE, and FGFR1-3P are in green, cyan, and blue, respectively. Side chains of R576, R577 and R577E are shown in stick representation. (E) Superposition of kinase insert regions of FGFR1 (Green), FGFR1-RE (Cyan), and FGFR1-3P (Blue) revealing multiple conformations of the kinase insert regions in the three structures.

Fig. 5.

Overall structures of asymmetric FGFR1 and FGFR2 kinase dimers, and distances between sequentially ordered FGFR1 tyrosine autophosphorylation sites. (A, B) Overall structures of asymmetric FGFR1 and FGFR2 kinase dimers are shown in ribbon diagrams (Upper) or as cartoons (Bottom). The proximal and the distal substrate interfaces are marked by a yellow or a red sphere, respectively. The phosphorylated regions and activation loops of both structures are shown. Helix αC is shown as a cylinder. The proximal substrate interface of both structures is marked by a yellow circle, and the distal substrate interface is marked by a red circle. (C) A model of FGFR1 (including residue Y766 not yet observed in an inactive FGFR1 structure) is shown in ribbon diagram and six phosphotyrosine sites in stick representation and colored in red. The sequence of autophosphorylation of the six autophosphorylation sites of FGFR1 is marked with numbers and approximate distances between inter autophosphorylation sites shown. Distances between two phosphotyrosine sites are the average of distance between unphosphorylated and phosphorylated FGFR1 structures, and summarized in the table.