Figure 8. s-Glutathiolation of SHP-2 catalytic and backdoor cysteines inhibits ROS1 deactivation.

(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.