title: Phosphoproteomic analysis implicates the mTORC2-FoxO1 axis in VEGF signaling and feedback activation of receptor tyrosine kinases.
abstract: The vascular endothelial growth factor (VEGF) signaling pathway plays a pivotal role in normal development and also represents a major therapeutic target for tumors and intraocular neovascular disorders. The VEGF receptor tyrosine kinases promote angiogenesis by phosphorylating downstream proteins in endothelial cells. We applied a large-scale proteomic approach to define the VEGF-regulated phosphoproteome and its temporal dynamics in human umbilical vein endothelial cells and then used siRNA (small interfering RNA) screens to investigate the function of a subset of these phosphorylated proteins in VEGF responses. The PI3K (phosphatidylinositol 3-kinase)-mTORC2 (mammalian target of rapamycin complex 2) axis emerged as central in activating VEGF-regulated phosphorylation and increasing endothelial cell viability by suppressing the activity of the transcription factor FoxO1 (forkhead box protein O1), an effect that limited cellular apoptosis and feedback activation of receptor tyrosine kinases. This FoxO1-mediated feedback loop not only reduced the effectiveness of mTOR inhibitors at decreasing protein phosphorylation and cell survival but also rendered cells more susceptible to PI3K inhibition. Collectively, our study provides a global and dynamic view of VEGF-regulated phosphorylation events and implicates the mTORC2-FoxO1 axis in VEGF receptor signaling and reprogramming of receptor tyrosine kinases in human endothelial cells.
authors.affiliation: Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Guanglei
authors.initials: G
authors.lastname: Zhuang
authors.forename: Kebing
authors.initials: K
authors.lastname: Yu
authors.forename: Zhaoshi
authors.initials: Z
authors.lastname: Jiang
authors.forename: Alicia
authors.initials: A
authors.lastname: Chung
authors.forename: Jenny
authors.initials: J
authors.lastname: Yao
authors.forename: Connie
authors.initials: C
authors.lastname: Ha
authors.forename: Karen
authors.initials: K
authors.lastname: Toy
authors.forename: Robert
authors.initials: R
authors.lastname: Soriano
authors.forename: Benjamin
authors.initials: B
authors.lastname: Haley
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Blackwood
authors.forename: Deepak
authors.initials: D
authors.lastname: Sampath
authors.forename: Carlos
authors.initials: C
authors.lastname: Bais
authors.forename: Jennie R
authors.initials: JR
authors.lastname: Lill
authors.forename: Napoleone
authors.initials: N
authors.lastname: Ferrara
mesh_terms: D017209:Apoptosis / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology*; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D000076225:Mechanistic Target of Rapamycin Complex 2; D046912:Multiprotein Complexes / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D046912:Multiprotein Complexes; D010748:Phosphopeptides; D042461:Vascular Endothelial Growth Factor A; D020794:Receptor Protein-Tyrosine Kinases; D000076225:Mechanistic Target of Rapamycin Complex 2; D058570:TOR Serine-Threonine Kinases