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Functional characterization of cancer-associated Gab1 mutations

Oncogene volume 32, pages 2696–2702 (2013)Cite this article

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Abstract

Grb2-associated binder 1 (Gab1) is a docking protein that transduces signals from a variety of tyrosine kinases, including Met and the epidermal growth factor receptor (EGFR). Although the related protein Gab2 is strongly implicated in human cancer, a role for Gab1 has been less clear. However, a screen for gene mutations in breast cancer identified two somatic mutations in Gab1, Y83C and T387N. In this paper we describe the functional characterization of these Gab1 mutants. MCF-10A immortalized mammary epithelial cells overexpressing Gab1 Y83C and T387N exhibited a more elongated, fibroblastic phenotype compared with wild-type Gab1 controls. Expression of Gab1 or the mutants promoted epidermal growth factor (EGF)-independent proliferation in monolayer culture to a similar degree. However, in Matrigel culture, both mutants enhanced the formation of acini exhibiting an aberrant, branched morphology. In addition, expression of the mutants modestly increased Erk activation. The two mutants also enhanced branching morphogenesis in a different mammary epithelial cell line, HC11. To gain further insights into the mechanism of action of these mutations, we mapped Gab1 phosphorylation sites by mass spectrometry. This detected phosphorylation of T387 but ;not Y83. Cellular stimulation with EGF or hepatocyte growth factor (HGF) led to a transient, or sustained, induction of T387 phosphorylation, respectively. As T387 corresponds in position to Gab2 T391, which suppresses Gab2 signaling in a phosphorylation-dependent manner, these data support a model in which the T387N mutation abrogates negative-feedback regulation of Gab1. Interrogation of publically-available databases revealed additional cancer-associated mutations at, or in close proximity to, identified serine/threonine phosphorylation sites in other docking proteins. These data indicate that aberrant Gab1 signaling can directly contribute to breast cancer progression, and that negative feedback sites in docking proteins can be targeted by oncogenic mutations.

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References

  1. Wohrle FU, Daly RJ, Brummer T . Function, regulation and pathological roles of the Gab/DOS docking proteins. Cell Commun Signal 2009; 7: 22.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Lock LS, Frigault MM, Saucier C, Park M . Grb2-independent recruitment of Gab1 requires the C-terminal lobe and structural integrity of the Met receptor kinase domain. J Biol Chem 2003; 278: 30083–30090.

    Article  CAS  PubMed  Google Scholar 

  3. Lock LS, Royal I, Naujokas MA, Park M . Identification of an atypical Grb2 carboxyl-terminal SH3 domain binding site in Gab docking proteins reveals Grb2-dependent and -independent recruitment of Gab1 to receptor tyrosine kinases. J Biol Chem 2000; 275: 31536–31545.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Maroun CR, Holgado-Madruga M, Royal I, Naujokas MA, Fournier TM, Wong AJ et al. The Gab1 PH domain is required for localization of Gab1 at sites of cell-cell contact and epithelial morphogenesis downstream from the met receptor tyrosine kinase. Mol Cell Biol 1999; 19: 1784–1799.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Abella JV, Vaillancourt R, Frigault MM, Ponzo MG, Zuo D, Sangwan V et al. The Gab1 scaffold regulates RTK-dependent dorsal ruffle formation through the adaptor Nck. J Cell Sci 2010; 123: 1306–1319.

    Article  CAS  PubMed  Google Scholar 

  7. Mood K . Saucier C, Bong YS, Lee HS, Park M, Daar IO. Gab1 is required for cell cycle transition, cell proliferation, and transformation induced by an oncogenic met receptor. Mol Biol Cell 2006; 17: 3717–3728.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Laramee M, Chabot C, Cloutier M, Stenne R, Holgado-Madruga M, Wong AJ et al. The scaffolding adapter Gab1 mediates vascular endothelial growth factor signaling and is required for endothelial cell migration and capillary formation. J Biol Chem 2007; 282: 7758–7769.

    Article  CAS  PubMed  Google Scholar 

  9. Maroun CR, Naujokas MA, Holgado-Madruga M, Wong AJ, Park M . 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 2000; 20: 8513–8525.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Gual P, Giordano S, Anguissola S, Parker PJ, Comoglio PM . Gab1 phosphorylation: a novel mechanism for negative regulation of HGF receptor signaling. Oncogene 2001; 20: 156–166.

    Article  CAS  PubMed  Google Scholar 

  13. Lynch DK, Daly RJ . PKB-mediated negative feedback tightly regulates mitogenic signalling via Gab2. EMBO J 2002; 21: 72–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Brummer T, Larance M, Herrera Abreu MT, Lyons RJ, Timpson P, Emmerich CH et al. Phosphorylation-dependent binding of 14-3-3 terminates signalling by the Gab2 docking protein. EMBO J 2008; 27: 2305–2316.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Arnaud M, Crouin C, Deon C, Loyaux D, Bertoglio J . Phosphorylation of Grb2-associated binder 2 on serine 623 by ERK MAPK regulates its association with the phosphatase SHP-2 and decreases STAT5 activation. J Immunol 2004; 173: 3962–3971.

    Article  CAS  PubMed  Google Scholar 

  16. Bentires-Alj M, Gil SG, Chan R, Wang ZC, Wang Y, Imanaka N et al. A role for the scaffolding adapter GAB2 in breast cancer. Nat Med 2006; 12: 114–121.

    Article  CAS  PubMed  Google Scholar 

  17. Bocanegra M, Bergamaschi A, Kim YH, Miller MA, Rajput AB, Kao J et al. Focal amplification and oncogene dependency of GAB2 in breast cancer. Oncogene 2010; 29: 774–779.

    Article  CAS  PubMed  Google Scholar 

  18. Brown LA, Kalloger SE, Miller MA, Shih Ie M, McKinney SE, Santos JL et al. Amplification of 11q13 in ovarian carcinoma. Genes Chromosomes Cancer 2008; 47: 481–489.

    Article  CAS  PubMed  Google Scholar 

  19. Chernoff KA, Bordone L, Horst B, Simon K, Twadell W, Lee K et al. GAB2 amplifications refine molecular classification of melanoma. Clin Cancer Res 2009; 15: 4288–4291.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Daly RJ, Gu H, Parmar J, Malaney S, Lyons RJ, Kairouz R et al. The docking protein Gab2 is overexpressed and estrogen regulated in human breast cancer. Oncogene 2002; 21: 5175–5181.

    Article  CAS  PubMed  Google Scholar 

  21. Fleuren ED, O'Toole S, Millar EK, McNeil C, Lopez-Knowles E, Boulghourjian A et al. Overexpression of the oncogenic signal transducer Gab2 occurs early in breast cancer development. Int J Cancer 2010; 127: 1486–1492.

    Article  CAS  PubMed  Google Scholar 

  22. Horst B, Gruvberger-Saal SK, Hopkins BD, Bordone L, Yang Y, Chernoff KA et al. Gab2-mediated signaling promotes melanoma metastasis. Am J Pathol 2009; 174: 1524–1533.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lee SH, Jeong EG, Nam SW, Lee JY, Yoo NJ . Increased expression of Gab2, a scaffolding adaptor of the tyrosine kinase signalling, in gastric carcinomas. Pathology 2007; 39: 326–329.

    Article  CAS  PubMed  Google Scholar 

  24. Zatkova A, Schoch C, Speleman F, Poppe B, Mannhalter C, Fonatsch C et al. GAB2 is a novel target of 11q amplification in AML/MDS. Genes Chromosomes Cancer 2006; 45: 798–807.

    Article  CAS  PubMed  Google Scholar 

  25. Juric D, Lacayo NJ, Ramsey MC, Racevskis J, Wiernik PH, Rowe JM et al. Differential gene expression patterns and interaction networks in BCR-ABL-positive and -negative adult acute lymphoblastic leukemias. J Clin Oncol 2007; 25: 1341–1349.

    Article  CAS  PubMed  Google Scholar 

  26. Ellison DW, Dalton J, Kocak M, Nicholson SL, Fraga C, Neale G et al. Medulloblastoma: clinicopathological correlates of SHH, WNT, and non-SHH/WNT molecular subgroups. Acta Neuropathol 2011; 121: 381–396.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD et al. The consensus coding sequences of human breast and colorectal cancers. Science 2006; 314: 268–274.

    Article  PubMed  Google Scholar 

  28. Soule HD, Maloney TM, Wolman SR, Peterson WD, Brenz R, McGrath CM et al. Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res 1990; 50: 6075–6086.

    CAS  PubMed  Google Scholar 

  29. Drasin DJ, Robin TP, Ford HL . Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity. Breast Cancer Res 2011; 13: 226.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Debnath J, Muthuswamy SK, Brugge JS . Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 2003; 30: 256–268.

    Article  CAS  PubMed  Google Scholar 

  31. Wrobel CN, Debnath J, Lin E, Beausoleil S, Roussel MF, Brugge JS . Autocrine CSF-1R activation promotes Src-dependent disruption of mammary epithelial architecture. J Cell Biol 2004; 165: 263–273.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Muthuswamy SK, Li D, Lelievre S, Bissell MJ, Brugge JS . ErbB2 but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Nat Cell Biol 2001; 3: 785–792.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Brummer T, Schramek D, Hayes VM, Bennett HL, Caldon CE, Musgrove EA et al. Increased proliferation and altered growth factor dependence of human mammary epithelial cells overexpressing the Gab2 docking protein. J Biol Chem 2006; 281: 626–637.

    Article  CAS  PubMed  Google Scholar 

  34. Reginato MJ, Mills KR, Becker EB, Lynch DK, Bonni A, Muthuswamy SK et al. Bim regulation of lumen formation in cultured mammary epithelial acini is targeted by oncogenes. Mol Cell Biol 2005; 25: 4591–4601.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Khwaja A, Lehmann K, Marte BM, Downward J . Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway. J Biol Chem 1998; 273: 18793–18801.

    Article  CAS  PubMed  Google Scholar 

  36. Ball RK, Friis RR, Schoenenberger CA, Doppler W, Groner B . Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. EMBO J 1988; 7: 2089–2095.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Xian W, Schwertfeger KL, Vargo-Gogola T, Rosen JM . Pleiotropic effects of FGFR1 on cell proliferation, survival, and migration in a 3D mammary epithelial cell model. J Cell Biol 2005; 171: 663–673.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Carpten JD, Faber AL, Horn C, Donoho GP, Briggs SL, Robbins CM et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature 2007; 448: 439–444.

    Article  CAS  PubMed  Google Scholar 

  39. Landgraf KE, Pilling C, Falke JJ . Molecular mechanism of an oncogenic mutation that alters membrane targeting: Glu17Lys modifies the PIP lipid specificity of the AKT1 PH domain. Biochemistry 2008; 47: 12260–12269.

    Article  CAS  PubMed  Google Scholar 

  40. Ferguson KM, Kavran JM, Sankaran VG, Fournier E, Isakoff SJ, Skolnik EY et al. Structural basis for discrimination of 3-phosphoinositides by pleckstrin homology domains. Mol Cell 2000; 6: 373–384.

    Article  CAS  PubMed  Google Scholar 

  41. Aitken A . 14-3-3 proteins: a historic overview. Semin Cancer Biol 2006; 16: 162–172.

    Article  CAS  PubMed  Google Scholar 

  42. Janes PW, Daly RJ, deFazio A, Sutherland RL . Activation of the Ras signalling pathway in human breast cancer cells overexpressing erbB-2. Oncogene 1994; 9: 3601–3608.

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia. CEC and DRC are recipients of Cancer Institute New South Wales Early Career Fellowships. DG-O is a National Breast Cancer Foundation and Cure Cancer Australia Fellow. CJO is supported by New South Wales Cancer Council and Banque Nationale de Paris-Paribas Australia. TB is supported by the German Research Foundation (DFG) through the Emmy-Noether-Program, CRC850 and EXC294 BIOSS.

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Authors and Affiliations

  1. Cancer Research Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia

    C Ortiz-Padilla, D Gallego-Ortega, B C Browne, F Hochgräfe, C E Caldon, R J Lyons, D R Croucher, D Rickwood, C J Ormandy & R J Daly

  2. Centre for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University of Freiburg, Freiburg, Germany

    T Brummer

  3. Institute for Biology III, Albert-Ludwigs-University of Freiburg, Freiburg, Germany

    T Brummer

  4. Centre for Biological Signalling Studies BIOSS, Albert-Ludwigs-University of Freiburg, Freiburg, Germany

    T Brummer

  5. St Vincent’s Hospital Clinical School, University of New South Wales, Sydney, New South Wales, Australia

    C J Ormandy & R J Daly

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Ortiz-Padilla, C., Gallego-Ortega, D., Browne, B. et al. Functional characterization of cancer-associated Gab1 mutations. Oncogene 32, 2696–2702 (2013). https://doi.org/10.1038/onc.2012.271

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