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


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 Hsp90alpha and exogenous HA-Hsp90beta 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.
Miao
Robert Q
RQ
Department of Pharmacology and Vascular Biology and Therapeutics Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536, USA.
Fontana
Jason
J
Fulton
David
D
Lin
Michelle I
MI
Harrison
Kenneth D
KD
Sessa
William C
WC
D000256:Adenoviridae; D000818:Animals; D002417:Cattle; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology*; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D008168:Lung / Q000166:cytology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology*
D018841:HSP90 Heat-Shock Proteins; D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt