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.
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.
Authors: Vishwaraj Sontake; Yunguan Wang; Rajesh K Kasam; Debora Sinner; Geereddy B Reddy; Anjaparavanda P Naren; Francis X McCormack; Eric S White; Anil G Jegga; Satish K Madala Journal: JCI Insight Date: 2017-02-23
Authors: Erich R Mackow; Nadine A Dalrymple; Velasco Cimica; Valery Matthys; Elena Gorbunova; Irina Gavrilovskaya Journal: Virus Res Date: 2014-01-08 Impact factor: 3.303
Authors: Chunzhang Yang; Shervin Rahimpour; Jie Lu; Karel Pacak; Barbara Ikejiri; Roscoe O Brady; Zhengping Zhuang Journal: Proc Natl Acad Sci U S A Date: 2012-12-31 Impact factor: 11.205