IQGAP1-Dependent Signaling Pathway Regulates Endothelial Cell Proliferation and Angiogenesis

Background Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood. Methodology/Principal Findings In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM). Conclusions/Significance Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2; it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regulate VEGFR-2 function in a combinatorial manner by supporting both diversity of recruitment of angiogenic signaling proteins to VEGFR-2, and its ability to promote angiogenesis.

[1]  D. Stupack,et al.  Role of Raf in Vascular Protection from Distinct Apoptotic Stimuli , 2003, Science.

[2]  Peter Carmeliet,et al.  Angiogenesis in life, disease and medicine , 2005, Nature.

[3]  Susan S. Taylor,et al.  Regulation of protein kinases; controlling activity through activation segment conformation. , 2004, Molecular cell.

[4]  G. Gallick,et al.  Src family kinases in tumor progression and metastasis , 2003, Cancer and Metastasis Reviews.

[5]  Janet Rossant,et al.  Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice , 1995, Nature.

[6]  A J Warner,et al.  The Shc-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells. , 2000, The Biochemical journal.

[7]  N. Rahimi,et al.  Receptor Chimeras Indicate That the Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1) Modulates Mitogenic Activity of VEGFR-2 in Endothelial Cells* , 2000, The Journal of Biological Chemistry.

[8]  Tony Pawson,et al.  SH2 domain specificity and activity modified by a single residue , 1994, Nature.

[9]  N. Rahimi,et al.  VEGFR-1 and VEGFR-2: two non-identical twins with a unique physiognomy. , 2006, Frontiers in bioscience : a journal and virtual library.

[10]  Sakae Tanaka,et al.  c-Cbl is downstream of c-Src in a signalling pathway necessary for bone resorption , 1996, Nature.

[11]  G. Bloom,et al.  IQGAP1, a Novel Vascular Endothelial Growth Factor Receptor Binding Protein, Is Involved in Reactive Oxygen Species–Dependent Endothelial Migration and Proliferation , 2004, Circulation research.

[12]  N. Rahimi,et al.  Substitution of C-terminus of VEGFR-2 with VEGFR-1 promotes VEGFR-1 activation and endothelial cell proliferation , 2004, Oncogene.

[13]  P. Schwartzberg,et al.  Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. , 1999, Molecular cell.

[14]  N. Rahimi,et al.  Identification of Tyrosine Residues in Vascular Endothelial Growth Factor Receptor-2/FLK-1 Involved in Activation of Phosphatidylinositol 3-Kinase and Cell Proliferation* , 2001, The Journal of Biological Chemistry.

[15]  Ha-won Jeong,et al.  IQGAP1 Binds Rap1 and Modulates Its Activity* , 2004, Journal of Biological Chemistry.

[16]  L. Claesson‐Welsh,et al.  VEGF receptor signalling ? in control of vascular function , 2006, Nature Reviews Molecular Cell Biology.

[17]  N. Rahimi,et al.  The Presence of a Single Tyrosine Residue at the Carboxyl Domain of Vascular Endothelial Growth Factor Receptor-2/FLK-1 Regulates Its Autophosphorylation and Activation of Signaling Molecules* , 2002, The Journal of Biological Chemistry.

[18]  D. Mukhopadhyay,et al.  Tyrosine Residues 951 and 1059 of Vascular Endothelial Growth Factor Receptor-2 (KDR) Are Essential for Vascular Permeability Factor/Vascular Endothelial Growth Factor-induced Endothelium Migration and Proliferation, Respectively* , 2001, The Journal of Biological Chemistry.

[19]  N. Rahimi,et al.  A critical role for the E3-ligase activity of c-Cbl in VEGFR-2-mediated PLCγ1 activation and angiogenesis , 2007, Proceedings of the National Academy of Sciences.

[20]  D. Sacks,et al.  IQGAP proteins are integral components of cytoskeletal regulation , 2003, EMBO reports.

[21]  M. Shibuya,et al.  Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[22]  K. Igarashi,et al.  Sck interacts with KDR and Flt-1 via its SH2 domain. , 1998, Biochemical and biophysical research communications.

[23]  N. Rahimi,et al.  The carboxyl terminus of VEGFR-2 is required for PKC-mediated down-regulation. , 2005, Molecular biology of the cell.

[24]  K. Stoletov,et al.  Sck is expressed in endothelial cells and participates in vascular endothelial growth factor-induced signaling , 2002, Oncogene.

[25]  M. Shibuya,et al.  VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells , 1999, Oncogene.

[26]  Jonathan A. Cooper,et al.  Src family kinases are required for integrin but not PDGFR signal transduction , 1999, The EMBO journal.

[27]  David A. Schultz,et al.  A mechanosensory complex that mediates the endothelial cell response to fluid shear stress , 2005, Nature.

[28]  M. Shibuya,et al.  A single autophosphorylation site on KDR/Flk‐1 is essential for VEGF‐A‐dependent activation of PLC‐γ and DNA synthesis in vascular endothelial cells , 2001, The EMBO journal.

[29]  R. Baron,et al.  Identification and functional characterization of an Src homology domain 3 domain‐binding site on Cbl , 2006, The FEBS journal.

[30]  N. Rahimi,et al.  Recruitment and Activation of Phospholipase Cγ1 by Vascular Endothelial Growth Factor Receptor-2 Are Required for Tubulogenesis and Differentiation of Endothelial Cells* , 2003, The Journal of Biological Chemistry.

[31]  Y. Yarden,et al.  Src promotes destruction of c-Cbl: Implications for oncogenic synergy between Src and growth factor receptors , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[32]  D. Sacks,et al.  IQGAP1 modulates activation of B-Raf , 2007, Proceedings of the National Academy of Sciences.

[33]  L. Claesson‐Welsh,et al.  The Adaptor Protein Shb Binds to Tyrosine 1175 in Vascular Endothelial Growth Factor (VEGF) Receptor-2 and Regulates VEGF-dependent Cellular Migration* , 2004, Journal of Biological Chemistry.