LPS induces pp60 c-src -mediated tyrosine phosphorylation of Hsp90 in lung vascular endothelial cells and mouse lung

• Published in: American journal of physiology. Lung cellular and molecular physiology 2013-06, Vol.304 (12), p.L883-L893
• Main Authors: Barabutis, Nektarios, Handa, Vaishali, Dimitropoulou, Christiana, Rafikov, Ruslan, Snead, Connie, Kumar, Sanjiv, Joshi, Atul, Thangjam, Gagan, Fulton, David, Black, Stephen M., Patel, Vijay, Catravas, John D.
• Format: Article
• Language: English
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.00419.2012

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 pp60 src activation. LPS-induced Hsp90 phosphorylation was also prevented by the pp60 src inhibitor PP2. Additionally, Hsp90 phosphorylation was induced by infecting cells with a constitutively active pp60 src adenovirus, whereas either a dominant-negative pp60 src adenovirus or reduced expression of pp60 src 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 pp60 src activation. Furthermore, the Hsp90β Y300F mutant showed a reduced ability to bind the Hsp90 client proteins eNOS and pp60 src 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.