Abstract 1538: Dominant-negative Hsp90 Modulates VEGF Mediated Endothelial Cell Functions
Heat-shock protein 90 (Hsp90) coordinates the trafficking and regulation of diverse signaling proteins. Previous studies have shown that stimulation of endothelial cells (EC) with vascular endothelial growth factor (VEGF) recruits the heterocomplex of eNOS, Akt and Hsp90, thereby facilitating eNOS phosphorylation and enzyme activation. To further explore the regulatory functions of Hsp90 in EC, we designed a dominant-negative Hsp90 construct by site-direct mutagenesis of residue Asp-88 to Asn (D88N-Hsp90) based on the crystal structure of the ATP/ADP-binding site in the Hsp90 N-terminal domain. Results of ATP-binding activity using γ-phosphate-linked ATP-Sepharose beads show that WT-Hsp90 binds ATP-Sepharose beads in manner inhibited by ATP or 17-AAG, a specific inhibitor for Hsp90, however, the ATP binding activity of D88N-Hsp90 was markedly reduced and the effects of ATP or 17-AAG were negligible. In bovine aortic EC (BAEC) infected with adenoviruses harboring HA tagged WT-Hsp90 or D88N-Hsp90, the dimerization between endogenous Hsp90α and exogenous HA-Hsp90β was confirmed by immunoprecipitation with HA antibody. However, the association between eNOS and D88N-Hsp90 is markedly reduced compared to WT-Hsp90. Furthermore, adenoviral transduction of BAEC with D88N-Hsp90 significantly suppressed VEGF-induced phosphorylation of Akt and eNOS and the inhibitory effect was blocked by okadaic acid. Tranduction of BAEC with D88N-Hsp90 inhibited VEGF-stimulated NO release, Rac activation, stress fiber formation and VEGF-induced endothelial cell migration. To elucidate if Akt is a specific cellular target of D88N-Hsp90, the directed migration of wild-type or Akt1(−/ −) mouse lung endothelial cells (MLEC) were evaluated after D88N-Hsp90 adenoviral transduction. Transduction with D88N-Hsp90 decreased serum mediated migration of wild-type MLEC, but not Akt1(−/ −) MLEC. These results demonstrate that DN-Hsp90 modulates endothelial cell mobility mainly through PP2A-mediated dephosphorylation of Akt and its downstream substrates.