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1: Arterioscler Thromb Vasc Biol. 2008 Jan;28(1):105-11. Epub 2007 Nov 1.
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Dominant-negative Hsp90 reduces VEGF-stimulated nitric oxide release and migration in endothelial cells.

Miao RQ, Fontana J, Fulton D, Lin MI, Harrison KD, Sessa WC.

Department of Pharmacology and Vascular Biology and Therapeutics Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536, USA.

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. 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 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.

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PMID: 17975117 [PubMed - indexed for MEDLINE]