title: LPS induces pp60c-src-mediated tyrosine phosphorylation of Hsp90 in lung vascular endothelial cells and mouse lung.
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 concentration- and time-dependent tyrosine (Y) phosphorylation of Hsp90α and Hsp90β 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.
authors.affiliation: Vascular Biology Center, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Nektarios
authors.initials: N
authors.lastname: Barabutis
authors.forename: Vaishali
authors.initials: V
authors.lastname: Handa
authors.forename: Christiana
authors.initials: C
authors.lastname: Dimitropoulou
authors.forename: Ruslan
authors.initials: R
authors.lastname: Rafikov
authors.forename: Connie
authors.initials: C
authors.lastname: Snead
authors.forename: Sanjiv
authors.initials: S
authors.lastname: Kumar
authors.forename: Atul
authors.initials: A
authors.lastname: Joshi
authors.forename: Gagan
authors.initials: G
authors.lastname: Thangjam
authors.forename: David
authors.initials: D
authors.lastname: Fulton
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
authors.forename: Vijay
authors.initials: V
authors.lastname: Patel
authors.forename: John D
authors.initials: JD
authors.lastname: Catravas
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008168:Lung / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism
chemical_list: C412373:AG 1879; D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; C533585:HSP90AA2P protein, human; C493165:HSP90AB1 protein, human; D047029:Lactams, Macrocyclic; D008070:Lipopolysaccharides; D011743:Pyrimidines; D034741:RNA, Small Interfering; D014443:Tyrosine; C112765:tanespimycin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D016392:Proto-Oncogene Proteins pp60(c-src)
keywords: Hsp90; LPS; endothelial cells; human; posttranslational modifications