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Temporal regulation of EGF signalling networks by the scaffold protein Shc1

Abstract

Cell-surface receptors frequently use scaffold proteins to recruit cytoplasmic targets, but the rationale for this is uncertain. Activated receptor tyrosine kinases, for example, engage scaffolds such as Shc1 that contain phosphotyrosine (pTyr)-binding (PTB) domains. Using quantitative mass spectrometry, here we show that mammalian Shc1 responds to epidermal growth factor (EGF) stimulation through multiple waves of distinct phosphorylation events and protein interactions. After stimulation, Shc1 rapidly binds a group of proteins that activate pro-mitogenic or survival pathways dependent on recruitment of the Grb2 adaptor to Shc1 pTyr sites. Akt-mediated feedback phosphorylation of Shc1 Ser 29 then recruits the Ptpn12 tyrosine phosphatase. This is followed by a sub-network of proteins involved in cytoskeletal reorganization, trafficking and signal termination that binds Shc1 with delayed kinetics, largely through the SgK269 pseudokinase/adaptor protein. Ptpn12 acts as a switch to convert Shc1 from pTyr/Grb2-based signalling to SgK269-mediated pathways that regulate cell invasion and morphogenesis. The Shc1 scaffold therefore directs the temporal flow of signalling information after EGF stimulation.

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Figure 1: EGF-dependent Shc1 phosphorylation and interactome.
Figure 2: Dynamic phosphorylation of Shc1 and interacting proteins.
Figure 3: Temporal profiles of the Shc1 signalling network.
Figure 4: Grb2-independent, serine/threonine-dependent Shc1 protein interactions.
Figure 5: SGK269 mediates late phase Shc1 protein interactions and regulates acinar morphology of breast epithelial cells in 3D culture.
Figure 6: Model for temporal regulation of Shc1 signalling following Egfr activation.

References

  1. 1

    Good, M. C., Zalatan, J. G. & Lim, W. A. Scaffold proteins: hubs for controlling the flow of cellular information. Science 332, 680–686 (2011)

    CAS  ADS  Article  Google Scholar 

  2. 2

    Lemmon, M. A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010)

    CAS  Article  Google Scholar 

  3. 3

    Uhlik, M. T. et al. Structural and evolutionary division of phosphotyrosine binding (PTB) domains. J. Mol. Biol. 345, 1–20 (2005)

    CAS  Article  Google Scholar 

  4. 4

    Luzi, L., Confalonieri, S., Di Fiore, P. P. & Pelicci, P. G. Evolution of Shc functions from nematode to human. Curr. Opin. Genet. Dev. 10, 668–674 (2000)

    CAS  Article  Google Scholar 

  5. 5

    van der Geer, P., Wiley, S., Gish, G. D. & Pawson, T. The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein-protein interactions. Curr. Biol. 6, 1435–1444 (1996)

    CAS  Article  Google Scholar 

  6. 6

    Pawson, T. Dynamic control of signaling by modular adaptor proteins. Curr. Opin. Cell Biol. 19, 112–116 (2007)

    CAS  Article  Google Scholar 

  7. 7

    Bisson, N. et al. Selected reaction monitoring mass spectrometry reveals the dynamics of signaling through the GRB2 adaptor. Nature Biotechnol. 29, 653–658 (2011)

    CAS  Article  Google Scholar 

  8. 8

    Hardy, W. R. et al. Combinatorial ShcA docking interactions support diversity in tissue morphogenesis. Science 317, 251–256 (2007)

    CAS  ADS  Article  Google Scholar 

  9. 9

    Dankort, D. et al. Grb2 and Shc adapter proteins play distinct roles in Neu (ErbB-2)-induced mammary tumorigenesis: implications for human breast cancer. Mol. Cell. Biol. 21, 1540–1551 (2001)

    CAS  Article  Google Scholar 

  10. 10

    Ursini-Siegel, J. et al. ShcA signalling is essential for tumour progression in mouse models of human breast cancer. EMBO J. 27, 910–920 (2008)

    CAS  Article  Google Scholar 

  11. 11

    Vanderlaan, R. D. et al. The ShcA phosphotyrosine docking protein uses distinct mechanisms to regulate myocyte and global heart function. Circ. Res. 108, 184–193 (2011)

    CAS  Article  Google Scholar 

  12. 12

    Okabayashi, Y. et al. Interaction of Shc with adaptor protein adaptins. J. Biol. Chem. 271, 5265–5269 (1996)

    CAS  Article  Google Scholar 

  13. 13

    Faisal, A., el-Shemerly, M., Hess, D. & Nagamine, Y. Serine/threonine phosphorylation of ShcA. Regulation of protein-tyrosine phosphatase-pest binding and involvement in insulin signaling. J. Biol. Chem. 277, 30144–30152 (2002)

    CAS  Article  Google Scholar 

  14. 14

    Lai, K. M. & Pawson, T. The ShcA phosphotyrosine docking protein sensitizes cardiovascular signaling in the mouse embryo. Genes Dev. 14, 1132–1145 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Min, J. et al. An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-κB. Nature Med. 16, 286–294 (2010)

    CAS  Article  Google Scholar 

  16. 16

    Wang, Y. et al. Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression. Proc. Natl Acad. Sci. USA 107, 10920–10925 (2010)

    CAS  ADS  Article  Google Scholar 

  17. 17

    Müller, T. et al. ASAP1 promotes tumor cell motility and invasiveness, stimulates metastasis formation in vivo, and correlates with poor survival in colorectal cancer patients. Oncogene 29, 2393–2403 (2010)

    Article  Google Scholar 

  18. 18

    Kondo, A. et al. A new paxillin-binding protein, PAG3/Papα/KIAA0400, bearing an ADP-ribosylation factor GTPase-activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration. Mol. Biol. Cell 11, 1315–1327 (2000)

    CAS  Article  Google Scholar 

  19. 19

    Kuroiwa, M., Oneyama, C., Nada, S. & Okada, M. The guanine nucleotide exchange factor Arhgef5 plays crucial roles in Src-induced podosome formation. J. Cell Sci. 124, 1726–1738 (2011)

    CAS  Article  Google Scholar 

  20. 20

    Debily, M. A. et al. Expression and molecular characterization of alternative transcripts of the ARHGEF5/TIM oncogene specific for human breast cancer. Hum. Mol. Genet. 13, 323–334 (2004)

    CAS  Article  Google Scholar 

  21. 21

    Anderson, L. & Hunter, C. L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol. Cell. Proteomics 5, 573–588 (2006)

    CAS  Article  Google Scholar 

  22. 22

    Lange, V. et al. Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring. Mol. Cell. Proteomics 7, 1489–1500 (2008)

    CAS  Article  Google Scholar 

  23. 23

    Picotti, P., Bodenmiller, B., Mueller, L. N., Domon, B. & Aebersold, R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138, 795–806 (2009)

    CAS  Article  Google Scholar 

  24. 24

    Knight, Z. A. et al. A pharmacological map of the PI3-K family defines a role for p110α in insulin signaling. Cell 125, 733–747 (2006)

    CAS  Article  Google Scholar 

  25. 25

    Cantley, L. C. The phosphoinositide 3-kinase pathway. Science 296, 1655–1657 (2002)

    CAS  ADS  Article  Google Scholar 

  26. 26

    Schlessinger, J. Common and distinct elements in cellular signaling via EGF and FGF receptors. Science 306, 1506–1507 (2004)

    CAS  ADS  Article  Google Scholar 

  27. 27

    Tashiro, K. et al. GAREM, a novel adaptor protein for growth factor receptor-bound protein 2, contributes to cellular transformation through the activation of extracellular signal-regulated kinase signaling. J. Biol. Chem. 284, 20206–20214 (2009)

    CAS  Article  Google Scholar 

  28. 28

    Chen, D., Waters, S. B., Holt, K. H. & Pessin, J. E. SOS phosphorylation and disassociation of the Grb2-SOS complex by the ERK and JNK signaling pathways. J. Biol. Chem. 271, 6328–6332 (1996)

    CAS  Article  Google Scholar 

  29. 29

    Tanaka, H., Katoh, H. & Negishi, M. Pragmin, a novel effector of Rnd2 GTPase, stimulates RhoA activity. J. Biol. Chem. 281, 10355–10364 (2006)

    CAS  Article  Google Scholar 

  30. 30

    Ceulemans, H. & Bollen, M. Functional diversity of protein phosphatase-1, a cellular economizer and reset button. Physiol. Rev. 84, 1–39 (2004)

    CAS  Article  Google Scholar 

  31. 31

    Schmandt, R., Liu, S. K. & McGlade, C. J. Cloning and characterization of mPAL, a novel Shc SH2 domain-binding protein expressed in proliferating cells. Oncogene 18, 1867–1879 (1999)

    CAS  Article  Google Scholar 

  32. 32

    Pawson, T. & Nash, P. Assembly of cell regulatory systems through protein interaction domains. Science 300, 445–452 (2003)

    CAS  Article  Google Scholar 

  33. 33

    Charest, A., Wagner, J., Jacob, S., McGlade, C. J. & Tremblay, M. L. Phosphotyrosine-independent binding of SHC to the NPLH sequence of murine protein-tyrosine phosphatase-PEST. Evidence for extended phosphotyrosine binding/phosphotyrosine interaction domain recognition specificity. J. Biol. Chem. 271, 8424–8429 (1996)

    CAS  Article  Google Scholar 

  34. 34

    Sun, T. et al. Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase. Cell 144, 703–718 (2011)

    CAS  Article  Google Scholar 

  35. 35

    Croucher, D. R. et al. Involvement of Lyn and the atypical kinase SgK269/PEAK1 in a basal breast cancer signaling pathway. Cancer Res. 73, 1969–1980 (2013)

    CAS  Article  Google Scholar 

  36. 36

    Rittling, S. R. Clonal nature of spontaneously immortalized 3T3 cells. Exp. Cell Res. 229, 7–13 (1996)

    CAS  Article  Google Scholar 

  37. 37

    Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006)

    CAS  Article  Google Scholar 

  38. 38

    Brummer, T. et al. Increased proliferation and altered growth factor dependence of human mammary epithelial cells overexpressing the Gab2 docking protein. J. Biol. Chem. 281, 626–637 (2006)

    CAS  Article  Google Scholar 

  39. 39

    Unwin, R. D., Griffiths, J. R. & Whetton, A. D. A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS). Nature Protocols 4, 870–877 (2009)

    CAS  Article  Google Scholar 

  40. 40

    R Development Core Team A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. http://www.R-project.org (2010)

  41. 41

    Sarkar, D. Lattice: Multivariate Data Visualization with R (Springer, 2008)

    Book  Google Scholar 

Download references

Acknowledgements

We thank J. Moffat for shRNA lentiviruses and K. Shokat for PI3Kp110 isoform-specific inhibitors. We thank C. Jorgensen, R. Williams, I. Louria-Hayon, R. Tian, and E. Petsalaki for critical input, A. James, V. Nguyen, and B. Larsen for technical assistance and M. M. Stacey, C. Chen and J. Jin for comments on the manuscript. Supported by Genome Canada through the Ontario Genomics Institute, the Ontario Research Fund from the Ontario Ministry of Research and Innovation, a Terry Fox Foundation team grant, the Canadian Institutes of Health Research (MOP-13466-6849), and the Canada Foundation for Innovation. M.A.S. is supported by a Vanier Canada Graduate Studentship and R.B. is supported by a CIHR postdoctoral fellowship. Support for R.J.D. was from the National Health and Medical Research Council of Australia and Cancer Council New South Wales (NSW), and for D.R.C. from Cancer Institute NSW.

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Contributions

Y.Z. conceived and implemented the sMRM approach. C.Z., Y.Z. and L.T. developed and performed sMRM assays. S.A.T. provided technical MS support. Y.Z., M.A.S., N.S.D., D.R.C., R.B. and A.Y.D. performed biochemical and functional experiments. W.R.H. generated Shc1-deficient MEFs and Grb2flox/flox MEFs. Y.Z. and A.P. performed the computational analysis. T.P., R.J.D. and A.-C.G. oversaw the project. Y.Z., M.A.S., K.C. and T.P. wrote the paper with input from J.W.D.

Corresponding author

Correspondence to Tony Pawson.

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Competing interests

S.A.T. is an employee of AB SCIEX. AB SCIEX has provided support for the Ontario Research Fund grant (awarded to T.P.).

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Zheng, Y., Zhang, C., Croucher, D. et al. Temporal regulation of EGF signalling networks by the scaffold protein Shc1. Nature 499, 166–171 (2013). https://doi.org/10.1038/nature12308

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