Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release

Jason Fontana, David Fulton, Yan Chen, Todd A. Fairchild, Timothy J. McCabe, Naoya Fujita, Takashi Tsuruo, William C. Sessa

Research output: Contribution to journalArticle

277 Scopus citations

Abstract

Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial, growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation.

Original languageEnglish (US)
Pages (from-to)866-873
Number of pages8
JournalCirculation research
Volume90
Issue number8
DOIs
StatePublished - May 3 2002
Externally publishedYes

Keywords

  • Akt
  • Nitric oxide
  • Scaffold
  • Signaling
  • hsp90

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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