Abstract 17971: Thioredoxin-Interacting Protein/Thioredoxin1 Translocation to the Plasma Membrane is Required for Vascular Endothelial Growth Factor Receptor-2 Activation by Ros
Background: Signal transduction mediated by reactive oxygen species (ROS) such as H2O2 involves control of local generation by oxidases as well as degradation by reductases and scavenging by antioxidants. Recently peroxiredoxinI (PRXI) was shown to control growth factor stimulated local H2O2 production at the plasma membrane. Because PRXI requires thioredoxin (TRX1) for reduction of oxidized proteins the mechanisms that control TRX1 subcellular location will be important. We hypothesized that the scaffold protein TRX1-interacting protein (TXNIP), which binds to TRX1 in a redox-dependent manner would mediate TRX1 plasma membrane function.
Methods and Results: Immunofluorescence and cell fractionation studies showed that both TRX1 and TXNIP translocated to the plasma membrane in response to a physiologic level of H2O2 (30 μmol/L, peak= 30min). TRX1 translocation required direct interaction since the non-binding TXNIP-C247S mutant did not cause TRX1 plasma membrane association. H2O2 stimulated a 6-fold increase in tyrosine phosphorylation of membrane-associated proteins that was specific for TRX1 since depletion of glutaredoxin had no effect. VEGFR2 tyrosine phosphorylation was increased 8-fold by H2O2 which was dependent upon TXNIP-TRX1 association with the membrane. VEGFR2 was essential for membrane protein tyrosine phosphorylation and downstream signaling (ERK1/2 and Akt), which were inhibited by TRX1 siRNA, TXNIP siRNA and SU1498. When TXNIP was depleted in endothelial cells by transfection with TXNIP siRNA, apoptosis (23.1 ± 0.8%) was significantly increased compared to cells transfected with control siRNA (8.2 ± 0.6%). Rescue experiments showed that TXNIP binding to TRX1 was required because rescue with TXNIP-C247S did not prevent apoptosis (21.4 ± 0.9%), while rescue with TXNIP-wild-type (WT) prevented cell death (7.5 ± 0.8%).
Conclusions: Translocation of TRX1 to the PM by TXNIP is essential for localized VEGFR2 activation and protein phosphorylation.
- © 2010 by American Heart Association, Inc.