EGF-receptor specificity for phosphotyrosine-primed substrates provides signal integration with Src

Abstract

Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK pathway and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Using peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, thus promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly than singly phosphorylated peptide to the Ras activator Grb2; this binding is a key step in activating the Ras-MAPK pathway. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras.

Figure 1

Determination of EGFR optimal substrate motif. (a) Schematic representation of the peptide libraries used in the positional scanning peptide library assay (Z, fixed amino acid; X, degenerate mixture of amino acids). (b) Peptide library results for wild-type and L858R EGFR kinase domains. Representative images from 3 independent experiments are shown. Quantification is provided in Supplementary Table 1. (c) Primary and secondary selections determined from the EGFR peptide library results. An “X’ denotes no selectivity.

Figure 2

Shc1 is phosphorylated by EGFR at Tyr239. (a) EGFR kinase assays with synthetic peptides corresponding to the sequence surrounding Tyr239 of Shc1 (PDHQYYNDAKKK = Y-Y; PDHQYpYNDAKKK = Y-pY; PDHQpYYNDAKKK = pY-Y). Error bars, s.d. (n=3 technical replicates) (b) Reversed-phase HPLC separation of Shc1 peptide standards (PDHQpYpYNDAKKK = pY-pY). The retention times of each peptide were established by individual injections (data not shown). (c) HPLC analysis of an EGFR kinase assay with the Shc1 Y-Y peptide at multiple time points. (d) Mass spectrometry based analysis of the 120 min peak in panel C. The LC-MS/MS spectrum for the singly phosphorylated peptide PDHQpYYNDAKKK is shown. (e) Percentage of peptide spectra detected by mass spectrometry phosphorylated at Tyr239 or Tyr240.

Figure 3

Src primes Shc1 for EGFR phosphorylation by phosphorylating Tyr240. (a) Peptide library results for Src. Representative image from 3 independent experiments is shown. Quantification is provided in Supplementary Table 2. (b) Src kinase assays with synthetic peptides corresponding to the sequence surrounding Tyr239 of Shc1 (PDHQYYNDAKKK = Y-Y; PDHQYpYNDAKKK = Y-pY; PDHQpYYNDAKKK = pY-Y). Error bars, s.d. (n=3 technical replicates) (c) HPLC analysis at of a Src kinase assay with the Shc1 Y-Y peptide at multiple time points. (d) Mass spectrometry based analysis of the 120 min peak in panel C. The LC-MS/MS spectrum for the singly phosphorylated peptide PDHQYpYNDAKKK is shown. (e) Percentage of peptide spectra detected by LC-MS/MS phosphorylated at Tyr239 or Tyr240. (f) HPLC analysis of an in vitro kinase assay containing Src, EGFR and the Y-Y Shc1 peptide at multiple time points.

Figure 4

Dual phosphorylation of Tyr239 and Tyr240 is controlled by EGFR and Src in cells and enhances Shc1 binding to Grb2. (a) Immunoblot analysis of cell lysates prepared from MCF10A cells pretreated with dasatinib for 1 hour then stimulated with 1ng/mL EGF for 3 min. (b) Pulldown assay and anti-Grb1 immunoblot. Samples are lysates from A431 cells incubated with the indicated biotinylated phosphopeptides. (Lysate = L; Control Beads = B; DHQYYNDFPGKE = Y-Y; DHQYpYNDFPGKE = Y-pY; DHQpYYNDFPGKE = pY-Y; DHQpYpYNDFPGKE = pY-pY). (c) Structure of a pseudo-peptide based on the sequence of Shc1 phosphorylated at Tyr239 and Tyr240 (mAZ-pY-(αME)pY-N-NH2) bound to the SH2 domain of Grb2 (PDB ID:1JYQ). Hydrogen bonds are indicated with dashed lines. (d) Model for the integration of EGFR and Src signaling by coordinated phosphorylation of Shc1. EGFR phosphorylation of Shc1 at Tyr239 creates a binding site for Grb2, leading to activation of the Ras-MAPK pathway. Active Src enhances activation by phosphorylating Tyr845 of the EGFR as well as Tyr240 of Shc1. Phosphorylation of Tyr240 of Shc1 potentiates phosphorylation at Tyr239 by the EGFR and enhances the binding of Grb2. Full-size images are shown in Supplementary Data Set 1.

Figure 5

Shc1 phosphopeptide binding to the EGFR kinase domain. (a) Overall structure of the EGFR L858R kinase domain bound to a Shc1 peptide phosphorylated at Tyr240 (PDHQYpYNDF). The peptide is shown in stick form. (b) Detailed view of interactions between EGFR and the primed Shc1 substrate peptide. Hydrogen bonds are indicated with dashed lines.