LPS induces pp60c-src-mediated tyrosine phosphorylation of Hsp90 in lung vascular endothelial cells and mouse lung
Nektarios Barabutis1, Vaishali Handa, Christiana Dimitropoulou
1Vascular Biology Center, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA.
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.
Insights
Inflammation triggers tyrosine phosphorylation of Heat Shock Protein 90 (Hsp90), a modification sensitive to Hsp90 inhibitors. This modification, mediated by pp60src, impacts Hsp90 client binding and contributes to inflammatory responses.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Heat Shock Protein 90 (Hsp90) inhibitors, initially developed for cancer, show anti-inflammatory potential.
- Posttranslational modifications of Hsp90 in tumors affect its function; this study explores modifications in inflammation.
Purpose of the Study:
- To investigate the posttranslational modification of Hsp90 in response to inflammatory stimuli.
- To identify the specific sites and mechanisms of Hsp90 modification during inflammation.
Main Methods:
- Used lipopolysaccharide (LPS) to induce inflammation in endothelial cells and mouse lungs.
- Employed mass spectrometry to identify phosphorylation sites on Hsp90.
- Utilized Hsp90 and pp60src inhibitors, as well as adenovirus and siRNA, to probe signaling pathways.
- Generated Hsp90β mutants to assess the functional impact of phosphorylation.
Main Results:
- LPS induced concentration- and time-dependent tyrosine phosphorylation of Hsp90α and Hsp90β at specific sites (Y309/Y300).
- Hsp90 phosphorylation was inhibited by 17-AAG (Hsp90 inhibitor) and PP2 (pp60src inhibitor).
- pp60src activity mediated LPS-induced Hsp90 phosphorylation.
- Hsp90β Y300F mutant showed reduced client protein binding (eNOS, pp60src) and attenuated LPS-induced barrier dysfunction.
Conclusions:
- Inflammatory stimuli induce Hsp90 posttranslational modifications sensitive to Hsp90 inhibitors.
- pp60src-mediated tyrosine phosphorylation of Hsp90 is a key event in inflammatory signaling.
- These modifications influence Hsp90 client interactions and contribute to inflammatory barrier dysfunction.
