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 J Fulton, Stephen Matthew Black, Vijaykumar Surendrakant Patel, John D. Catravas

Research output: Contribution to journalArticle

18 Citations (Scopus)

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)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume304
Issue number12
DOIs
StatePublished - Jun 15 2013

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HSP90 Heat-Shock Proteins
Tyrosine
Endothelial Cells
Phosphorylation
Lung
Post Translational Protein Processing
Adenoviridae

Keywords

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

ASJC Scopus subject areas

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

Cite this

LPS induces pp60c-src-mediated tyrosine phosphorylation of Hsp90 in lung vascular endothelial cells and mouse lung. / Barabutis, Nektarios; Handa, Vaishali; Dimitropoulou, Christiana; Rafikov, Ruslan; Snead, Connie; Kumar, Sanjiv; Joshi, Atul; Thangjam, Gagan; Fulton, David J; Black, Stephen Matthew; Patel, Vijaykumar Surendrakant; Catravas, John D.

In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 304, No. 12, 15.06.2013.

Research output: Contribution to journalArticle

Barabutis, Nektarios ; Handa, Vaishali ; Dimitropoulou, Christiana ; Rafikov, Ruslan ; Snead, Connie ; Kumar, Sanjiv ; Joshi, Atul ; Thangjam, Gagan ; Fulton, David J ; Black, Stephen Matthew ; Patel, Vijaykumar Surendrakant ; Catravas, John D. / LPS induces pp60c-src-mediated tyrosine phosphorylation of Hsp90 in lung vascular endothelial cells and mouse lung. In: American Journal of Physiology - Lung Cellular and Molecular Physiology. 2013 ; Vol. 304, No. 12.
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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.",
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AU - Rafikov, Ruslan

AU - Snead, Connie

AU - Kumar, Sanjiv

AU - Joshi, Atul

AU - Thangjam, Gagan

AU - Fulton, David J

AU - Black, Stephen Matthew

AU - Patel, Vijaykumar Surendrakant

AU - Catravas, John D.

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