Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Differential phosphorylation of the docking protein Gab1 by c-Src and the hepatocyte growth factor receptor regulates different aspects of cell functions

Oncogene volume 29, pages 698–710 (2010)Cite this article

Abstract

The docking protein Grb2-associated binder1 (Gab1) has a central role in the integration of the growth-factor signaling. In this study, we aimed to examine the significance of Src-mediated Gab1 phosphorylation in the hepatocyte growth factor (HGF) signaling. Using both mutagenesis and mass spectrometry approaches, Y242, Y259, Y317, Y373 and Y627 of Gab1 were identified to be phosphorylated by c-Src. It is interesting to note that the binding of the tyrosine phosphatase SHP2 to the Y627 antagonized the effect of c-Src on the phosphorylation of the other four tyrosine residues. Moreover, the tyrosine residues predominantly phosphorylated by c-Src were different from those predominantly phosphorylated by the HGF receptor. Gab1 overexpression potentiated both mitogenic and motogenic activities of HGF. However, a Gab1 mutant with substitutions of the Src phosphorylation sites (Y242, Y259, Y317 and Y373) failed to promote HGF-induced DNA synthesis, but retained its ability to facilitate HGF-induced chemotaxis. Taken together, our results not only suggest that the phosphorylation of Gab1 by c-Src is important for HGF-induced DNA synthesis, but also provide an example to illustrate how a docking protein (for example, Gab1) is differentially phosphorylated by c-Src and a receptor tyrosine kinase to emanate full spectrum of signals to the downstream.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  • Chan PC, Chen YL, Cheng CH, Yu KC, Cary LA, Shu KH et al. (2003). Src phosphorylates Grb2-associated binder 1 upon hepatocyte growth factor stimulation. J Biol Chem 278: 44075–44082.

    Article  CAS  PubMed  Google Scholar 

  • Chen SY, Chen HC . (2006). Direct interaction of focal adhesion kinase (FAK) with Met is required for FAK to promote hepatocyte growth factor-induced invasion. Mol Cell Biol 26: 5155–5167.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cunnick JM, Mei L, Doupnik CA, Wu J . (2001). Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) conferring binding and activation of SHP2. J Biol Chem 276: 24380–24387.

    Article  CAS  PubMed  Google Scholar 

  • Derman MP, Cunha MJ, Barros EJ, Nigam SK, Cantley LG . (1995). HGF-mediated chemotaxis and tubulogenesis require activation of the phosphatidylinositol 3-kinase. Am J Physiol 268: F1211–F1217.

    CAS  PubMed  Google Scholar 

  • Eulenfeld R, Schaper F . (2009). A new mechanism for the regulation of Gab1 recruitment to the plasma membrane. J Cell Sci 122: 55–64.

    Article  CAS  PubMed  Google Scholar 

  • Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM . (1999). Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20: 601–605.

    Article  CAS  PubMed  Google Scholar 

  • Gu H, Neel BG . (2003). The ‘Gab’ in signal transduction. Trends Cell Biol 13: 122–130.

    Article  CAS  PubMed  Google Scholar 

  • Gual P, Giordano S, Williams TA, Rocchi S, Van Obberghen E, Comoglio PM . (2000). Sustained recruitment of phospholipase Cγ to Gab1 is required for HGF-induced branching tubulogenesis. Oncogene 19: 1509–1518.

    Article  CAS  PubMed  Google Scholar 

  • Holgado-Madruga M, Emlet DR, Moscatello DK, Godwin AK, Wong AJ . (1996). A Grb2-associated docking protein in EGF- and insulin-receptor signaling. Nature 379: 560–564.

    Article  CAS  PubMed  Google Scholar 

  • Itoh M, Yoshida Y, Nishida K, Narimatsu M, Hibi M, Hirano T . (2000). Role of Gab1 in heart, placenta, and skin development and growth factor- and cytokine-induced extracellular signal-regulated kinase mitogen-activated protein kinase activation. Mol Cell Biol 20: 3695–3704.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kallin A, Demoulin JB, Nishida K, Hirano T, Ronnstrand L, Heldin CH . (2004). Gab1 contributes to cytoskeletal reorganization and chemotaxis in response to platelet-derived growth factor. J Biol Chem 279: 17897–17904.

    Article  CAS  PubMed  Google Scholar 

  • Lamorte L, Kamikura DM, Park M . (2000). A switch from p130Cas/Crk to Gab1/Crk signaling correlates with anchorage independent growth and JNK activation in cells transformed by the Met receptor oncoprotein. Oncogene 19: 5973–5981.

    Article  CAS  PubMed  Google Scholar 

  • Lehr S, Kotzka J, Avci H, Sickmann A, Meyer HE, Herkner A et al. (2004). Identification of major ERK-related phosphorylation sites in Gab1. Biochemistry 43: 12133–12140.

    Article  CAS  PubMed  Google Scholar 

  • Lehr S, Kotzka J, Herkner A, Klein E, Siethoff C, Knebel B et al. (1999). Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro. Biochemistry 38: 151–159.

    Article  CAS  PubMed  Google Scholar 

  • Lehr S, Kotzka J, Herkner A, Sikmann A, Meyer HE, Krone W et al. (2000). Identification of major tyrosine phosphorylation sites in the human insulin receptor substrate Gab-1 by insulin receptor kinase in vitro. Biochemistry 39: 10898–10907.

    Article  CAS  PubMed  Google Scholar 

  • Machide M, Kamitori K, Kohsaka S . (2000). Hepatocyte growth factor-induced differential activation of phospholipase Cγ1 and phosphatidylinositol 3-kinase is regulated by tyrosine phosphatase SHP-1 in astrocytes. J Biol Chem 275: 31392–31398.

    Article  CAS  PubMed  Google Scholar 

  • Maroun CR, Naujokas MA, Holgado-Madruga M, Wong AJ, Park M . (2000). The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase. Mol Cell Biol 20: 8513–8525.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Montagner A, Yart A, Dance M, Perret B, Salles JP, Raynal P . (2005). A novel role for Gab1 and SHP2 in epidermal growth factor-induced Ras activation. J Biol Chem 280: 5350–5360.

    Article  CAS  PubMed  Google Scholar 

  • Nishida K, Yoshida Y, Itoh M, Fukada T, Ohtani T, Shirogane T et al. (1999). Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors. Blood 93: 1809–1816.

    CAS  PubMed  Google Scholar 

  • Podar K, Mostoslavsky G, Sattler M, Tai YT, Hayashi T, Catley LP et al. (2004). Critical role for hematopoietic cell kinase (Hck)-mediated phosphorylation of Gab1 and Gab2 docking proteins in interleukin 6-induced proliferation and survival of multiple myeloma cells. J Biol Chem 279: 21658–21665.

    Article  CAS  PubMed  Google Scholar 

  • Riordan SM, Lidder S, Williams R, Skouteris GG . (2000). The beta-subunit of the hepatocyte growth factor/scatter factor (HGF/SF) receptor phosphorylates and associates with CrkII: expression of CrkII enhances HGF/SF-induced mitogenesis. Biochem J 350: 925–932.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rodrigues GA, Falasca M, Zhang Z, Ong SH, Schlessinger J . (2000). A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling. Mol Cell Biol 20: 1448–1459.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sachs M, Brohmann H, Zechner D, Muller T, Hulsken J, Walther I et al. (2000). Essential role of Gab1 for signaling by the c-Met receptor in vivo. J Cell Biol 150: 1375–1384.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schaeper U, Gehring NH, Fuchs KP, Sachs M, Kempkes B, Birchmeier W . (2000). Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses. J Cell Biol 149: 1419–1432.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schaeper U, Vogel R, Chmielowiec J, Huelsken J, Rosario M, Birchmeier W . (2007). Distinct requirements for Gab1 in Met and EGF receptor signaling in vivo. Proc Natl Acad Sci USA 104: 15376–15381.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shi ZQ, Yu DH, Park M, Marshall M, Feng GS . (2000). Molecular mechanism for the Shp-2 tyrosine phosphatase function in promoting growth factor stimulation of Erk activity. Mol Cell Biol 20: 1526–1536.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Takahashi-Tezuka M, Yoshida Y, Fukada T, Ohtani T, Yamanaka Y, Nishida K et al. (1998). Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase. Mol Cell Biol 18: 4109–4117.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Terry DE, Umstot E, Desiderio DM . (2004). Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests. J Am Soc Mass Spectrom 15: 784–794.

    Article  CAS  PubMed  Google Scholar 

  • Weidner KM, Di Cesare S, Sachs M, Brinkmann V, Behrens J, Birchmeier W . (1996). Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature 384: 173–176.

    Article  CAS  PubMed  Google Scholar 

  • Yu CF, Liu ZX, Cantley LG . (2002). ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. J Biol Chem 277: 19382–19388.

    Article  CAS  PubMed  Google Scholar 

  • Yu CF, Roshan B, Liu ZX, Cantley LG . (2001). ERK regulates the hepatocyte growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. J Biol Chem 276: 32552–32558.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are indebted to Dr T Hirano (Osaka University, Osaka, Japan) for Gab1 cDNA and Gab1-null MEFs and Dr DL Wang (Academia Sinica, Taipei, Taiwan) for Flag-tagged SHP2 and its CS mutant. This work is supported by grants NSC97-3112-B-005-001 and NSC97-2628-B-005-001-MY3 from the National Science Council, Taiwan and NHRI-EX97-9730BI from the National Health Research Institutes, Taiwan.

Author information

Author notes

  1. P-C Chan, J N Sudhakar and C-C Lai: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Life Science, National Chung Hsing University, Taichung, Taiwan

    P-C Chan & H-C Chen

  2. Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan

    J N Sudhakar & H-C Chen

  3. Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan

    C-C Lai

Authors
  1. P-C Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J N Sudhakar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C-C Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H-C Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H-C Chen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chan, PC., Sudhakar, J., Lai, CC. et al. Differential phosphorylation of the docking protein Gab1 by c-Src and the hepatocyte growth factor receptor regulates different aspects of cell functions. Oncogene 29, 698–710 (2010). https://doi.org/10.1038/onc.2009.363

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2009.363

Keywords

This article is cited by

Search

Advanced search

Quick links