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

Nature Structural & Molecular Biology volume 22, pages 983–990 (2015)Cite this article

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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.

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Figure 1: Determination of the EGFR optimal substrate motif.
Figure 2: Shc1 is phosphorylated by EGFR at Tyr239.
Figure 3: Src primes Shc1 for EGFR phosphorylation by phosphorylating Tyr240.
Figure 4: Dual phosphorylation of Tyr239 and Tyr240 is controlled by EGFR and Src in cells and enhances Shc1 binding to Grb2.
Figure 5: Shc1 phosphopeptide binding to the EGFR kinase domain.

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Acknowledgements

We thank S. Breitkopf and M. Yuan for help with MS experiments and Y. Zheng for help with HPLC experiments. We thank B. Murray for critical reading of the manuscript. This work was partially supported by US National Institutes of Health grants 2P01CA120964 (J.M.A.) and S10OD010612 (J.M.A.).

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Author notes

  1. Christina A Gewinner

    Present address: Present address: Translational Innovation Group, University College London, London, UK.,

Authors and Affiliations

  1. Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA

    Michael J Begley, Christina A Gewinner, John M Asara & Lewis C Cantley

  2. Centers for Therapeutic Innovation, Pfizer, Boston, Massachusetts, USA

    Michael J Begley, Anthony J Coyle & Irina Apostolou

  3. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Cai-hong Yun & Michael J Eck

  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Cai-hong Yun & Michael J Eck

  5. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    John M Asara

  6. Department of Medicine, Weill Cornell Medical College, New York, New York, USA

    Jared L Johnson & Lewis C Cantley

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Contributions

M.J.B. designed and conducted all biochemical, enzymatic and cell-based experiments. C.Y. determined all crystal structures. C.A.G. and J.L.J., together with M.J.B., performed peptide library experiments. J.M.A. executed mass spectrometry–based experiments. M.J.E. conceived and supervised structural experiments. I.A. and A.J.C. conceived and supervised cell-based experiments. L.C.C., together with M.J.B., conceived the study and wrote the manuscript.

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Correspondence to Lewis C Cantley.

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Integrated supplementary information

Supplementary Figure 1 EGFR and Src peptide library assays using specific inhibitors.

(a) Phosphorylation of the pY+1 peptide library by EGFR WT or EGFR L858R in the presence of increasing concentrations of gefitinib. Activity is graphed as percent of control (no gefitinib). (b) Phosphorylation of the I-1, G+1, pY+1, and Y+3 peptide libraries by Src in the presence of increasing concentrations of dasatinib. Activity is graphed as percent of control (no dasatinib).

Supplementary Figure 2 EGFR activity toward peptides based on the sequence surrounding MET Tyr1234.

Peptides were phosphorylated in vitro with recombinant EGFR (MYDKEYYSVHNKK = Y-Y; MYDKEYpYSVHNKK = Y-pY). Error bars, s.d. (n=3 technical replicates).

Supplementary Figure 3 Specificity of phospho-Shc pY239/240–specific antibody.

Serial dilutions of peptides corresponding to the sequence surrounding Tyr239 of Shc1 were spotted on nitrocellulose and probed with a phospho-Shc pY239/240 antibody (Cell Signaling Technology Cat #2434; PDHQYYNDAKKK = Y-Y; PDHQYpYNDAKKK = Y-pY; PDHQpYYNDAKKK = pY-Y; PDHQpYpYNDAKKK = pY-pY). 40nmol corresponds to 2μL of a 20mM stock solution of each peptide (20mM is the solubility limit of the peptides in Tris buffer).

Supplementary Figure 4 Crystal structures of EGFR in complex with peptides.

(a) (b) Electron density of peptides in complex with EGFR L858R kinase domain. Fo-Fc difference omit maps (in which the peptides were omitted) for the optimal peptide (a) or the primed Shc1 peptide (b) contoured at 3σ. (c) Interactions between the EGFR L858R kinase domain and the optimal substrate peptide (DEEDYpYEIP). Hydrogen bonds are indicated with dashed lines.

Supplementary Figure 5 Peptide library results for ERBB2 and ERBB4.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1 and 2 (PDF 1675 kb)

Supplementary Data Set 1

Original western blot images (PDF 948 kb)

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Begley, M., Yun, Ch., Gewinner, C. et al. EGF-receptor specificity for phosphotyrosine-primed substrates provides signal integration with Src. Nat Struct Mol Biol 22, 983–990 (2015). https://doi.org/10.1038/nsmb.3117

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