LPS induces pp60c-src-mediated tyrosine phosphorylation of Hsp90 in lung vascular endothelial cells and mouse lung

Nektarios Barabutis, Vaishali Handa, Christiana Dimitropoulou, Ruslan Rafikov, Connie Snead, Sanjiv Kumar, Atul Joshi, Gagan Thangjam, David Fulton, Stephen M. Black, Vijay Patel, John D. Catravas

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Heat shock protein 90 (Hsp90) inhibitors were initially developed as anticancer agents; however, it is becoming increasing clear that they also possess potent anti-inflammatory properties. Posttranslational modifications of Hsp90 have been reported in tumors and have been hypothesized to affect client protein- and inhibitor-binding activities. In the present study we investigated the posttranslational modification of Hsp90 in inflammation. LPS, a prototypical inflammatory agent, induced concentrationand time-dependent tyrosine (Y) phosphorylation of Hsp90α and Hsp90α in bovine pulmonary arterial and human lung microvascular endothelial cells (HLMVEC). Mass spectrometry identified Y309 as a major site of Y phosphorylation on Hsp90α (Y300 of Hsp90β). LPS-induced Hsp90 phosphorylation was prevented by the Hsp90 inhibitor 17-allyl-amino-demethoxy-geldanamycin (17-AAG) in vitro as well as in lungs from LPS-treated mice, in vivo. Furthermore, 17-AAG prevented LPS-induced pp60src activation. LPS-induced Hsp90 phosphorylation was also prevented by the pp60src inhibitor PP2. Additionally, Hsp90 phosphorylation was induced by infecting cells with a constitutively active pp60src adenovirus, whereas either a dominant-negative pp60src adenovirus or reduced expression of pp60src by a specific siRNA prevented the LPS-induced Y phosphorylation of Hsp90. Transfection of HLMVEC with the nonphosphorylatable Hsp90β Y300F mutant prevented LPS-induced Hsp90β tyrosine phosphorylation but not pp60src activation. Furthermore, the Hsp90β Y300F mutant showed a reduced ability to bind the Hsp90 client proteins eNOS and pp60src and HLMVEC transfected with the mutant exhibited reduced LPS-induced barrier dysfunction. We conclude that inflammatory stimuli cause posttranslational modifications of Hsp90 that are Hsp90-inhibitor sensitive and may be important to the proinflammatory actions of Hsp90.

Original languageEnglish (US)
Pages (from-to)L883-l893
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume304
Issue number12
DOIs
StatePublished - Jun 15 2013

Keywords

  • Endothelial cells
  • Hsp90
  • Human
  • LPS
  • Posttranslational modifications

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

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