Schematic representation of c-Cbl domain architecture and targets of its TKB domain. (A) Structural organization of human c-Cbl. The N-terminal region contains the TKB comprising 4H, EF-hand (EF) and SH-2-like subdomains. The TKB domain is separated from the RING domain by a short linker (L) region. Cbl has a proline-rich stretch following the RING domain and a ubiquitin-associated (UBA) domain at its C terminus. (B) Sequence alignment of previously characterized c-Cbl-TKB binding sites for the PTK, APS and Met subgroups of proteins. Coloured residues refer to the conserved residues in the sequence: pY is coloured red, (pY−2)D/N is green, (pY+1)S/T is dark blue, (pY+4)P is fuschia. (pY−6)R in the APS family is orange, (pY−5)A is brown and the (pY−4)φ represents a hydrophobic residue, coloured violet. pY flanking residues of the Met family (pY−1)D are coloured light blue and (pY+1)R magenta.

The conserved intrapeptidyl hydrogen (H) bond and neighbouring H-bonds for the conserved pTyr of Spry2, EGFR and Met. Close-up view of the tyrosine-binding pocket of the c-Cbl-TKB domain complexed with (A) Spry2^49–61 (cyan), (C) EGFR^1063–1075 (yellow) and (E) Met^997–1009 (pink) shows the conserved intrapeptidyl H-bonds between the pTyr and the (pY−2)Asn of Spry2 or the (pY−1)Arg of Met. Intrapeptidyl H-bonds are shown as dotted red lines and other H-bonds are shown as grey dotted lines. The figure was prepared using Pymol (DeLano, 2002). A schematic linear extended view of the c-Cbl-TKB domain with (B) Spry2, (D) EGFR and (F) Met. The phosphopeptides are coloured red and the Cbl residues are blue. Residues participating in van der Waals contacts are highlighted in gold with the interacting surfaces as gold curved lines. H-bonds are indicated by green dashed lines and the intrapeptidyl H-bond is depicted by a black dashed line between the phosphate group of the conserved tyrosine and the side chain of the Asn53 (Spry2), Arg1068 (EGFR) or the Arg1004 (Met).

Binding affinity measurements with isothermal titration calorimetry. (A) The binding affinities of Spry2^49–61, APS^623–632, Spry4^69–81, EGFR^1063–1075, Src^413–425, Syk^317–329, Met^997–1009, Ron^1011–1023, VEGFR^1327–1338, VEGFR^1327–1338 V1331N and VEGFR^1327–1338 L1332R phosphopeptides to the c-Cbl-TKB domain were measured using ITC. ITC experiments for APS^623–632 and EGFR^1063–1075 were also repeated at pH 8.0 according to Hu and Hubbard (2005). The concentration of the TKB domain was 10.8 μM in all experiments.

Orientation of the intrapeptidyl hydrogen (H) bond within the binding pocket of the Cbl-TKB. A cross-sectional perspective of the binding topograph in Figure 2B and F illustrates the fit of the phosphopeptides on the surface of the Cbl-TKB domain for (A) Spry2^49–61 (cyan) and (B) Met^997–1009 (pink). (C, D) Peptides superimposed from two different angles. (E–G) Schematic diagrams illustrating the importance of the intrapeptidyl H-bond between pTyr and (E) (pY−2)Asn for Spry2 or (F) (pY+1)Arg for Met in the reverse orientation. In the absence of an intrapeptidyl H-bond (G), pTyr is disorientated and unable to dock into the pTyr-binding pocket on Cbl-TKB, as indicated by VEGFR.

Crystal structure and electrostatic surface representations of TKB domain complexed with Spry2, EGFR and Met. Crystal structures of the Cbl-TKB complexed with each of the peptides (A) Spry2^49–61 (cyan), (C) EGFR^1063–1075 (yellow) and (E) Met^997–1009 (pink), and surface representations of the Cbl-TKB complexed with (B) Spry2^49–61, (D) EGFR^1063–1075 and (F) Met^997–1009. The c-Cbl-TKB domain is shown as a ribbon diagram with helices, β-strands and loops coloured light brown. Phosphopeptides are shown as stick models. Intrapeptidyl H-bonds are shown as grey dotted lines. Residues at the extreme N and C termini of the phosphopeptides are disordered and not included. The figure was prepared using Pymol (DeLano, 2002) and the APBS plugin (Baker et al, 2001).

Site-directed mutagenesis to determine the importance of conserved residues. (A) 293T cells were transfected with FGFR1 and FLAG-tagged wild-type Spry2 or Spry2 bearing alanine point mutations as indicated. The cells were lysed 24 h post-transfection and FLAG–Spry2 proteins were immunoprecipitated (IP) using FLAG–M2 beads. IP and total cell lysates (TCL) were immunoblotted with anti-Cbl (C-15), anti-FLAG or anti-FGFR1 (C-15) as labelled. (B) Diagram of c-Cbl with an enlargement of the TKB domain. The point mutations created in the SH2 subdomain of Cbl are described. (C–H) 293T cells were transfected with HA-tagged WT c-Cbl and Cbl bearing various point mutations as described in (B), along with (C) FLAG–Spry2, (D) Spry4, (E) EGFR, (F) Met, (G) myc-APS in combination with insulin receptor-B (IR-B) or (H) alone for endogenous Grb2. Cells were stimulated as described in the Materials and methods and lysed 24 or 48 h post-transfection. In (C) and (D), anti-FLAG and anti-Cbl IPs (C-15) and TCLs were immunoblotted with anti-FLAG and anti-HA (Cbl). In (E), anti-EGFR IPs (528) and TCLs were immunoblotted with anti-HA (Cbl) and anti-EGFR (1005). In (F), anti-HGFR IPs and TCLs were immunoblotted with anti-HA and anti-Met (25H2). In (G), anti-myc IPs (A-14) and TCLs were immunoblotted with anti-HA, anti-c-myc (9E10) and IR-β (C-19). In (H), anti-Grb2 IPs (C-23) and TCLs were immunoblotted with anti-HA and anti-Grb2.

(B–G) Representative ITC profiles for Spry2, EGFR, Met, VEGFR, VEGFR V1331N and VEGFR L1332R are shown respectively. The figures show the injection profile after baseline correction and the bottom panels show the integration (heat release) for each injection (except the first one). The solid lines in the bottom panel show the fit of the data to a function based on a one-site binding model. The binding constants (K_a and K_d), number of binding sites (N), enthalpy (ΔH) and entropy (ΔS) changes of Cbl-N to the various peptides are provided in (A).