Dominant-negative Hsp90 reduces VEGF-stimulated nitric oxide release and migration in endothelial cells

Robert Q. Miao, Jason Fontana, David Fulton, Michelle I. Lin, Kenneth D. Harrison, William C. Sessa

Research output: Contribution to journalArticle

44 Scopus citations

Abstract

OBJECTIVES - Heat-shock protein 90 (Hsp90) coordinates the regulation of diverse signaling proteins. We try to develop a new tool to explore the regulatory functions of Hsp90 in endothelial cells (ECs) instead of the existing chemical approaches. METHODS AND RESULTS - We designed a dominant-negative Hsp90 construct by site-direct mutagenesis of residue Asp-88 to Asn (D88N-Hsp90) based on the structure of the ATP/ADP-binding site. Recombinant wild-type Hsp90 protein binds ATP-Sepharose beads in manner inhibited by ATP or 17-AAG, a specific inhibitor for Hsp90, however the binding activity of D88N-Hsp90 was markedly reduced and the inhibitory effects of ATP or 17-AAG were negligible. The dimerization between endogenous Hsp90α and exogenous HA-Hsp90β was confirmed by immunoprecipitation, however the association between eNOS and D88N-Hsp90 was less than WT-Hsp90. Furthermore, adenoviral transduction of bovine aortic ECs with D88N-Hsp90 suppressed VEGF-induced phosphorylation of Akt, eNOS, and NO release and the inhibitory effect was blocked by okadaic acid. Moreover, D88N-Hsp90 abolished VEGF-stimulated Rac activation and suppressed VEGF-induced stress fiber formation. Transduction with D88N-Hsp90 decreased growth medium mediated migration of wild-type ECs, but not Akt1(-/-) ECs suggesting that Akt is key target of Hsp90. CONCLUSIONS - Our data demonstrate that dominant-negative Hsp90 modulates endothelial cell mobility mainly through PP2A-mediated dephosphorylation of Akt and Rac activation.

Original languageEnglish (US)
Pages (from-to)105-111
Number of pages7
JournalArteriosclerosis, thrombosis, and vascular biology
Volume28
Issue number1
DOIs
StatePublished - Jan 2008

Keywords

  • ATP-binding
  • Akt
  • Dominant-negative
  • Hsp90
  • Migration

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Fingerprint Dive into the research topics of 'Dominant-negative Hsp90 reduces VEGF-stimulated nitric oxide release and migration in endothelial cells'. Together they form a unique fingerprint.

  • Cite this