Oxido-reductive regulation of vascular remodeling by receptor tyrosine kinase ROS1
Ziad A. Ali, … , Thomas Quertermous, Euan A. Ashley
Ziad A. Ali, … , Thomas Quertermous, Euan A. Ashley
Published November 17, 2014
Citation Information: J Clin Invest. 2014;124(12):5159-5174. https://doi.org/10.1172/JCI77484.
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Research Article

Oxido-reductive regulation of vascular remodeling by receptor tyrosine kinase ROS1

Abstract

Angioplasty and stenting is the primary treatment for flow-limiting atherosclerosis; however, this strategy is limited by pathological vascular remodeling. Using a systems approach, we identified a role for the network hub gene glutathione peroxidase-1 (GPX1) in pathological remodeling following human blood vessel stenting. Constitutive deletion of Gpx1 in atherosclerotic mice recapitulated this phenotype of increased vascular smooth muscle cell (VSMC) proliferation and plaque formation. In an independent patient cohort, gene variant pair analysis identified an interaction of GPX1 with the orphan protooncogene receptor tyrosine kinase ROS1. A meta-analysis of the only genome-wide association studies of human neointima-induced in-stent stenosis confirmed the association of the ROS1 variant with pathological remodeling. Decreased GPX1 expression in atherosclerotic mice led to reductive stress via a time-dependent increase in glutathione, corresponding to phosphorylation of the ROS1 kinase activation site Y2274. Loss of GPX1 function was associated with both oxidative and reductive stress, the latter driving ROS1 activity via s-glutathiolation of critical residues of the ROS1 tyrosine phosphatase SHP-2. ROS1 inhibition with crizotinib and deglutathiolation of SHP-2 abolished GPX1-mediated increases in VSMC proliferation while leaving endothelialization intact. Our results indicate that GPX1-dependent alterations in oxido-reductive stress promote ROS1 activation and mediate vascular remodeling.

Authors

Ziad A. Ali, Vinicio de Jesus Perez, Ke Yuan, Mark Orcholski, Stephen Pan, Wei Qi, Gaurav Chopra, Christopher Adams, Yoko Kojima, Nicholas J. Leeper, Xiumei Qu, Kathia Zaleta-Rivera, Kimihiko Kato, Yoshiji Yamada, Mitsutoshi Oguri, Allan Kuchinsky, Stanley L. Hazen, J. Wouter Jukema, Santhi K. Ganesh, Elizabeth G. Nabel, Keith Channon, Martin B. Leon, Alain Charest, Thomas Quertermous, Euan A. Ashley

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Figure 8

s-Glutathiolation of SHP-2 catalytic and backdoor cysteines inhibits ROS1 deactivation.

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s-Glutathiolation of SHP-2 catalytic and backdoor cysteines inhibits ROS...
(A) HEK293 cells transfected with myc-tagged ROS1 and incubated with biotinylated GSH-ethyl-ester induced cysteine activation and s-glutathiolation of many proteins inhibitable by the reducing agent DTT. (B) s-Glutathiolation of ROS1 was not detected following immunoprecipitation; however, other GSS-protein conjugates were detected. Peptide mass fingerprinting showed that SHP-1 and SHP-2 were coimmunoprecipitated with ROS1, and (C) immunoprecipitation identified s-glutathiolation of SHP-2. LC-ESI-MS/MS identified the sites of glutathiolation. Extracted ion chromatograms of the SHP-2 peptides (D) Q450-R469 and (E) S326-R343 in the native form (top panel, control), s-glutathiolated form (middle panel), and reduced form (bottom panel) identify the catalytic Cys 463 and backdoor Cys 333 as the sites of s-glutathiolation. Insets are the representative MS/MS spectra of the Q450-R469 and S326-R343 peptide, providing site-specific localization of s-glutathiolation in the case of the modified forms. Upper insets represent the specific B and y ions as detected in the MS/MS spectra, showing inter-residue bond breakage generating b13 and y7 ions for Cys 463 and b8 and y11 for Cys 333, providing absolute confirmation. (F) (Upper panel and inset on left) Schematic representation of SHP-2 showing catalytic Cys 463 and backdoor Cys 333 and Cys 367 in the active PTP domain. (Inset on right) Modeling of s-glutathiolation of catalytic Cys 463 depicted by the spheres, demonstrating a physical presence within the active phosphatase domain of the enzyme.