title: The adaptor protein Gab1 couples the stimulation of vascular endothelial growth factor receptor-2 to the activation of phosphoinositide 3-kinase.
abstract: Phosphoinositide 3-kinase (PI3K) mediates essential functions of vascular endothelial growth factor (VEGF), including the stimulation of endothelial cell proliferation and migration. Nevertheless, the mechanisms coupling the receptor VEGFR-2 to PI3K remain obscure. We observed that the Grb2-bound adapter Gab1 is tyrosine-phosphorylated and relocated to membrane fractions upon VEGF stimulation of endothelial cells. We could detect the PI3K regulatory subunit p85 in immunoprecipitates of endogenous Gab1, and vice versa, and measure a Gab1-associated lipid kinase activity upon VEGF stimulation. Furthermore, transfection of the Gab1-YF3 mutant lacking all p85-binding sites strongly repressed PI3K activation measured in vitro. Moreover, Gab1-YF3 severely decreased the cellular amount of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generated in response to VEGF. Furthermore, adenoviral expression of Gab1-YF3 suppressed both Akt phosphorylation and recovery of wounded human umbilical vein endothelial cell monolayers, a VEGF-dependent process involving cell migration and proliferation under PI3K control. Transfection of other Gab1 mutants, lacking Grb2-binding sites or the pleckstrin homology (PH) domain, also prevented Akt activation, further demonstrating Gab1 involvement in PI3K activation. These mutants were also used to show that interactions with both Grb2 and PtdIns(3,4,5)P3 mediate Gab1 recruitment by VEGFR-2. Importantly, Gab1 mobilization was impaired by (i) PI3K inhibitors, (ii) deletion of Gab1 PH domain, (iii) PTEN (phosphatase and tensin homolog deleted on chromosome 10) overexpression to repress PtdIns(3,4,5)P3 production, and (iv) overexpression of a competitor PH domain for PtdIns(3,4,5)P3 binding, which altogether demonstrated that PI3K is also an upstream regulator of Gab1. Gab1 thus appears as a primary actor in coupling VEGFR-2 to PI3K/Akt, recruited through an amplification loop involving PtdIns(3,4,5)P3 and its PH domain.
authors.affiliation: Département Lipoprotéines et Médiateurs Lipidiques, INSERM U563, Bat. C, Hôpital Purpan, B. P. 3028, 31024 Toulouse, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Dance
authors.forename: Alexandra
authors.initials: A
authors.lastname: Montagner
authors.forename: Armelle
authors.initials: A
authors.lastname: Yart
authors.forename: Bernard
authors.initials: B
authors.lastname: Masri
authors.forename: Yves
authors.initials: Y
authors.lastname: Audigier
authors.forename: Bertrand
authors.initials: B
authors.lastname: Perret
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Salles
authors.forename: Patrick
authors.initials: P
authors.lastname: Raynal
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism / Q000502:physiology*; D000256:Adenoviridae / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D017353:Gene Deletion; D006801:Humans; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism / Q000502:physiology*; D014945:Wound Healing
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C098587:GAB1 protein, human; D019869:Phosphatidylinositol 3-Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2