Abstract 18227: IQGAP1 Compartmentalizes ROS-dependent VEGF Receptor 2 Activation and Signaling Linked to Endothelial Proliferation and Angiogenesis
VEGF stimulation increases reactive oxygen species (ROS) that are involved in VEGF receptor2 tyrosine phosphorylation (VEGFR2-pY) linked to angiogenesis. We previously demonstrated that IQGAP1 is a novel VEGFR2 binding scaffold protein involved in ROS-dependent endothelial cell (EC) proliferation; however, the underlying signaling mechanisms are poorly understood. Using proteomics approaches, here we identified protein tyrosine phosphatase (PTP)1B and dynamin-2 (Dyn-2) as new binding partners of IQGAP1 in human ECs. We previously reported that PTP1B, an ER resident, negatively regulates VEGFR2-pY1175 and EC proliferation. VEGFR2-pY occurs initially at the plasma membrane including in caveolae/lipid rafts (CE/LR), and promotes Dyn-2-dependent VEGFR2 internalization for signal propagation and receptor degradation in intracellular compartments. Here we show that VEGF promotes association of IQGAP1 and PTP1B, and that VEGF induces oxidative inactivation of PTP1B. IQGAP1, PTP1B, VEGFR2, Nox2 NAPDH oxidase are localized in CE/LR. Western analysis and surface biotinylation experiments show that knockdown of IQGAP1 with siRNA inhibits VEGF-induced VEGFR2-pY (45%) at 5min, and promotes VEGFR2 protein degradation at 15 min (55%). Mechanistically, IQGAP1 binds to Cbl, an E3 ubiquitin ligase in basal state, but depletion of IQGAP1 enhances VEGF-induced VEGFR2-Cbl association. IQGAP1 also binds to Dyn-2, and overexpression of GTPase-defective mutant Dyn-2 reduces VEGFR2 stabilization and inhibits EC proliferation. Subcellular fractionation and biotinylation experiments reveal that VEGF promotes VEGFR2 internalization to increase VEGFR2-pY1175 and decreases PTP1B activity at 5 and 15 mins (40% and 32% fold, respectively) in ER/endosome fractions, and that overexpression of PTP1B inhibits VEGFR2-pY at both the cell surface and internal compartment along with EC proliferation. Functionally, ischemia-induced angiogenesis is impaired in IQGAP1 knockout mice in vivo. In summary, IQGAP1 association with PTP1B, a target of ROS, and Dyn-2 plays an important role in ROS-dependent VEGFR2 activation and compartmentalization of signaling linked to EC proliferation, which may contribute to angiogenesis in vivo.
- © 2010 by American Heart Association, Inc.