on November 1, 2007
Published online before print November 1, 2007, doi: 10.1161/ATVBAHA.107.155499
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Submitted on February 7, 2007
Accepted on October 23, 2007
Dominant-Negative Hsp90 Reduces VEGF Stimulated Nitric Oxide Release and Migration in Endothelial Cells
Robert Q. Miao ;
From the Department of Pharmacology and Vascular Biology & Therapeutics Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Conn.
* To whom correspondence should be addressed. E-mail: william.sessa{at}yale.edu.
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 Hsp90
and exogenous HA-Hsp90
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.
Key words: Hsp90 • ATP-binding • Akt • migration • dominant-negative
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