Ligand-stimulated VEGFR2 signaling is regulated by co-ordinated trafficking and proteolysis.
Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.
Bruns
Alexander F
AF
Endothelial Cell Biology Unit, Institute of Molecular and Cellular Biology, LIGHT Laboratories, University of Leeds, Leeds LS2 9JT, UK.
Herbert
Shane P
SP
Odell
Adam F
AF
Jopling
Helen M
HM
Hooper
Nigel M
NM
Zachary
Ian C
IC
Walker
John H
JH
Ponnambalam
Sreenivasan
S
D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008024:Ligands; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009389:Neovascularization, Pathologic / Q000201:enzymology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D015398:Signal Transduction / Q000187:drug effects*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D008024:Ligands; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D017868:Cyclic AMP-Dependent Protein Kinases; D046988:Proteasome Endopeptidase Complex