Scaffolding Protein Grb2-associated Binder 1 Sustains Epidermal Growth Factor-induced Mitogenic and Survival Signaling by Multiple Positive Feedback Loops*

Grb2-associated binder 1 (GAB1) is a scaffold protein involved in numerous interactions that propagate signaling by growth factor and cytokine receptors. Here we explore in silico and validate in vivo the role of GAB1 in the control of mitogenic (Ras/MAPK) and survival (phosphatidylinositol 3-kinase (PI3K)/Akt) signaling stimulated by epidermal growth factor (EGF). We built a comprehensive mechanistic model that allows for reliable predictions of temporal patterns of cellular responses to EGF under diverse perturbations, including different EGF doses, GAB1 suppression, expression of mutant proteins, and pharmacological inhibitors. We show that the temporal dynamics of GAB1 tyrosine phosphorylation is significantly controlled by positive GAB1-PI3K feedback and negative MAPK-GAB1 feedback. Our experimental and computational results demonstrate that the essential function of GAB1 is to enhance PI3K/Akt activation and extend the duration of Ras/MAPK signaling. By amplifying positive interactions between survival and mitogenic pathways, GAB1 plays the critical role in cell proliferation and tumorigenesis.

[1]  D T Denhardt,et al.  Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. , 1996, The Biochemical journal.

[2]  M. Gold,et al.  The Gab1 Docking Protein Links the B Cell Antigen Receptor to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway and to the SHP2 Tyrosine Phosphatase* , 2001, The Journal of Biological Chemistry.

[3]  L. Cantley,et al.  Activated ERK2 Interacts with and Phosphorylates the Docking Protein GAB1* , 1999, The Journal of Biological Chemistry.

[4]  W. Birchmeier,et al.  Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis , 1996, Nature.

[5]  James E. Ferrell,et al.  Mechanistic Studies of the Dual Phosphorylation of Mitogen-activated Protein Kinase* , 1997, The Journal of Biological Chemistry.

[6]  M. Karin,et al.  Mammalian MAP kinase signalling cascades , 2001, Nature.

[7]  R. Jensen,et al.  Activation of Gab1 in pancreatic acinar cells: effects of gastrointestinal growth factors/hormones on stimulation, phosphospecific phosphorylation, translocation and interaction with downstream signaling molecules. , 2006, Cellular signalling.

[8]  L. Mei,et al.  A role for Grb2-associated binder-1 in growth hormone signaling. , 2002, Endocrinology.

[9]  B. Neel,et al.  The "Gab" in signal transduction. , 2003, Trends in cell biology.

[10]  H. Wiley,et al.  An integrated model of epidermal growth factor receptor trafficking and signal transduction. , 2003, Biophysical journal.

[11]  Joseph Schlessinger,et al.  Common and Distinct Elements in Cellular Signaling via EGF and FGF Receptors , 2004, Science.

[12]  Tony Pawson,et al.  Gene dosage-dependent functions for phosphotyrosine-Grb2 signaling during mammalian tissue morphogenesis , 2001, Current Biology.

[13]  W. S. Hlavacek,et al.  A network model of early events in epidermal growth factor receptor signaling that accounts for combinatorial complexity. , 2006, Bio Systems.

[14]  W. Kolch,et al.  Conferring specificity on the ubiquitous Raf/MEK signalling pathway , 2004, British Journal of Cancer.

[15]  Jie Wu,et al.  Phosphotyrosines 627 and 659 of Gab1 Constitute a Bisphosphoryl Tyrosine-based Activation Motif (BTAM) Conferring Binding and Activation of SHP2* , 2001, The Journal of Biological Chemistry.

[16]  N. Tonks,et al.  A Critical Role for Phosphoinositide 3-Kinase Upstream of Gab1 and SHP2 in the Activation of Ras and Mitogen-activated Protein Kinases by Epidermal Growth Factor* , 2001, The Journal of Biological Chemistry.

[17]  E. Hafen,et al.  Biochemical and genetic analysis of the Drk SH2/SH3 adaptor protein of Drosophila. , 1995, The EMBO journal.

[18]  W. Birchmeier,et al.  Coupling of Gab1 to C-Met, Grb2, and Shp2 Mediates Biological Responses , 2000, The Journal of cell biology.

[19]  A. Godwin,et al.  A Grb2-associated docking protein in EGF- and insulin-receptor signalling , 1996, Nature.

[20]  Roger Brent,et al.  Automatic generation of cellular reaction networks with Moleculizer 1.0 , 2005, Nature Biotechnology.

[21]  Jin Yang,et al.  'On-the-fly' or 'generate-first' modeling? , 2005, Nature Biotechnology.

[22]  T. Hirano,et al.  Gab1 and SHP-2 promote Ras/MAPK regulation of epidermal growth and differentiation , 2002, The Journal of cell biology.

[23]  T Pawson,et al.  Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav , 1994, Molecular and cellular biology.

[24]  A. Ashworth,et al.  Identification of the sites in MAP kinase kinase‐1 phosphorylated by p74raf‐1. , 1994, The EMBO journal.

[25]  W. Kolch.  Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. , 2000, The Biochemical journal.

[26]  James R Faeder,et al.  The complexity of complexes in signal transduction , 2003, Biotechnology and bioengineering.

[27]  E. Gilles,et al.  Computational modeling of the dynamics of the MAP kinase cascade activated by surface and internalized EGF receptors , 2002, Nature Biotechnology.

[28]  R. Dixon,et al.  Lysophosphatidic acid-induced proliferation in opossum kidney proximal tubular cells: role of PI 3-kinase and ERK. , 1999, Kidney international.

[29]  T. Hirano,et al.  Role of Gab1 in Heart, Placenta, and Skin Development and Growth Factor- and Cytokine-Induced Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Activation , 2000, Molecular and Cellular Biology.

[30]  C. Der,et al.  Increasing Complexity of the Ras Signaling Pathway* , 1998, The Journal of Biological Chemistry.

[31]  Tony Pawson,et al.  Mammalian Grb2 Regulates Multiple Steps in Embryonic Development and Malignant Transformation , 1998, Cell.

[32]  S. Kimura,et al.  A computational model on the modulation of mitogen-activated protein kinase (MAPK) and Akt pathways in heregulin-induced ErbB signalling. , 2003, The Biochemical journal.

[33]  B. Kholodenko,et al.  Quantification of Short Term Signaling by the Epidermal Growth Factor Receptor* , 1999, The Journal of Biological Chemistry.

[34]  I. Lax,et al.  The docking protein Gab1 is the primary mediator of EGF-stimulated activation of the PI-3K/Akt cell survival pathway , 2004, BMC Biology.

[35]  M. Hayman,et al.  Molecular Mechanism for a Role of SHP2 in Epidermal Growth Factor Receptor Signaling , 2003, Molecular and Cellular Biology.

[36]  J. Olefsky,et al.  Negative Feedback Regulation and Desensitization of Insulin- and Epidermal Growth Factor-stimulated p21ras Activation (*) , 1995, The Journal of Biological Chemistry.

[37]  Uta Berger,et al.  Pattern-Oriented Modeling of Agent-Based Complex Systems: Lessons from Ecology , 2005, Science.

[38]  M. Gold,et al.  The Gab1 Protein Is a Docking Site for Multiple Proteins Involved in Signaling by the B Cell Antigen Receptor* , 1998, The Journal of Biological Chemistry.

[39]  L. Cantley,et al.  ERK Negatively Regulates the Epidermal Growth Factor-mediated Interaction of Gab1 and the Phosphatidylinositol 3-Kinase* , 2002, The Journal of Biological Chemistry.

[40]  K. Moelling,et al.  Phosphorylation and regulation of Raf by Akt (protein kinase B). , 1999, Science.

[41]  R. Jove,et al.  Roles of Gab1 and SHP2 in Paxillin Tyrosine Dephosphorylation and Src Activation in Response to Epidermal Growth Factor* , 2004, Journal of Biological Chemistry.

[42]  D. Bray,et al.  Predicting temporal fluctuations in an intracellular signalling pathway. , 1998, Journal of theoretical biology.

[43]  T. Hunter,et al.  Signaling—2000 and Beyond , 2000, Cell.

[44]  A. Ullrich,et al.  PTP‐SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal‐regulated kinases ERK1 and ERK2 by association through a kinase interaction motif , 1998, The EMBO journal.

[45]  B. Vanhaesebroeck,et al.  The PI3K-PDK1 connection: more than just a road to PKB. , 2000, The Biochemical journal.

[46]  Yu Zhao,et al.  The Mechanism of Dephosphorylation of Extracellular Signal-regulated Kinase 2 by Mitogen-activated Protein Kinase Phosphatase 3* , 2001, The Journal of Biological Chemistry.

[47]  Richard Marais,et al.  The RAF proteins take centre stage , 2004, Nature Reviews Molecular Cell Biology.

[48]  B N Kholodenko,et al.  Signal processing at the Ras circuit: what shapes Ras activation patterns? , 2004, Systems biology.

[49]  B. Kholodenko,et al.  Tyr-317 Phosphorylation Increases Shc Structural Rigidity and Reduces Coupling of Domain Motions Remote from the Phosphorylation Site as Revealed by Molecular Dynamics Simulations* , 2004, Journal of Biological Chemistry.

[50]  M. Naujokas,et al.  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* , 2000, The Journal of Biological Chemistry.

[51]  Walter Birchmeier,et al.  The Docking Protein Gab1 Is an Essential Component of an Indirect Mechanism for Fibroblast Growth Factor Stimulation of the Phosphatidylinositol 3-Kinase/Akt Antiapoptotic Pathway , 2004, Molecular and Cellular Biology.

[52]  P. Cullen.  Ras effectors: Buying shares in Ras plc , 2001, Current Biology.

[53]  Sos1 rapidly associates with Grb2 and is hypophosphorylated when complexed with the EGF receptor after EGF stimulation. , 1996, Oncogene.

[54]  A. King,et al.  Phosphorylation site specificity of the Pak‐mediated regulation of Raf‐1 and cooperativity with Src , 2001, FEBS letters.

[55]  A. Cherniack,et al.  Disassembly of Son-of-sevenless Proteins from Grb2 during p21 Desensitization by Insulin (*) , 1995, The Journal of Biological Chemistry.

[56]  William S. Hlavacek,et al.  BioNetGen: software for rule-based modeling of signal transduction based on the interactions of molecular domains , 2004, Bioinform..

[57]  B. Kholodenko,et al.  Temperature dependence of the epidermal growth factor receptor signaling network can be accounted for by a kinetic model. , 2002, Biochemistry.

[58]  Morag Park,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 , 1999, Molecular and Cellular Biology.

[59]  T. Hirano,et al.  Gab1 is required for EGF receptor signaling and the transformation by activated ErbB2 , 2003, Oncogene.

[60]  R. Pestell,et al.  Regulation of Cyclin D1 Expression and DNA Synthesis by Phosphatidylinositol 3-Kinase in Airway Smooth Muscle Cells , 2000 .

[61]  H V Westerhoff,et al.  Why cytoplasmic signalling proteins should be recruited to cell membranes. , 2000, Trends in cell biology.

[62]  Shinya Kuroda,et al.  Prediction and validation of the distinct dynamics of transient and sustained ERK activation , 2005, Nature Cell Biology.

[63]  J. Frost,et al.  Phosphorylation of Raf-1 by p21-activated Kinase 1 and Src Regulates Raf-1 Autoinhibition* , 2003, The Journal of Biological Chemistry.

[64]  Yan Liu,et al.  Gab3, a New DOS/Gab Family Member, Facilitates Macrophage Differentiation , 2002, Molecular and Cellular Biology.

[65]  L. Mei,et al.  Requirement of SHP2 Binding to Grb2-associated Binder-1 for Mitogen-activated Protein Kinase Activation in Response to Lysophosphatidic Acid and Epidermal Growth Factor* , 2000, The Journal of Biological Chemistry.

[66]  William S. Hlavacek,et al.  Modeling the early signaling events mediated by FcepsilonRI. , 2002, Molecular immunology.

[67]  Songshu Meng,et al.  Participation of both Gab1 and Gab2 in the activation of the ERK/MAPK pathway by epidermal growth factor. , 2005, The Biochemical journal.

[68]  J. Bertoglio,et al.  Phosphorylation of Grb2-Associated Binder 2 on Serine 623 by ERK MAPK Regulates Its Association with the Phosphatase SHP-2 and Decreases STAT5 Activation1 , 2004, The Journal of Immunology.

[69]  K. Siddle,et al.  Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. , 1998, The Biochemical journal.

[70]  B. Kholodenko,et al.  Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades , 2004, The Journal of cell biology.

[71]  Chen Yan,et al.  ERK1/2 Associates with the c-Met-binding Domain of Growth Factor Receptor-bound Protein 2 (Grb2)-associated Binder-1 (Gab1) , 2004, Journal of Biological Chemistry.

[72]  L. Cantley,et al.  Conditional Inhibition of the Mitogen-activated Protein Kinase Cascade by Wortmannin , 1997, The Journal of Biological Chemistry.

[73]  Joseph Schlessinger,et al.  A Novel Positive Feedback Loop Mediated by the Docking Protein Gab1 and Phosphatidylinositol 3-Kinase in Epidermal Growth Factor Receptor Signaling , 2000, Molecular and Cellular Biology.

[74]  Chi-Ying F. Huang,et al.  Ultrasensitivity in the mitogen-activated protein kinase cascade. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[75]  B. Kholodenko,et al.  Signaling through receptors and scaffolds: independent interactions reduce combinatorial complexity. , 2005, Biophysical journal.

[76]  J. Salles,et al.  A Novel Role for Gab1 and SHP2 in Epidermal Growth Factor-induced Ras Activation* , 2005, Journal of Biological Chemistry.

[77]  J. Downward,et al.  Role of Phosphoinositide 3-Kinase in Activation of Ras and Mitogen-Activated Protein Kinase by Epidermal Growth Factor , 1999, Molecular and Cellular Biology.

[78]  W. R. Burack,et al.  The activating dual phosphorylation of MAPK by MEK is nonprocessive. , 1997, Biochemistry.

[79]  B. Kholodenko,et al.  Negative feedback and ultrasensitivity can bring about oscillations in the mitogen-activated protein kinase cascades. , 2000, European journal of biochemistry.

[80]  A. Sickmann,et al.  Identification of major ERK-related phosphorylation sites in Gab1. , 2004, Biochemistry.

[81]  P. Cohen,et al.  Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines , 1995, Current Biology.

[82]  R. Weinberg,et al.  Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation , 1993, Nature.

[83]  D A Lauffenburger,et al.  Physical modulation of intracellular signaling processes by locational regulation. , 1997, Biophysical journal.

[84]  Boris N Kholodenko,et al.  Trading the micro-world of combinatorial complexity for the macro-world of protein interaction domains. , 2006, Bio Systems.