Figure 2.

PP2Cα is susceptible to glutathionylation at non-conserved Cys 314.A, PP2Cα structure. A model shows phosphate (P_i) and Mn²⁺ in the active site and locations of three relatively surface-exposed cysteines (C72, C204, and C314). B, comparisons of pK_a and accessible surface area (ASA) for cysteines in PP2Cα. C, the amino acid sequence alignment around C314 among different species, showing limited conservation of C314. D, a scheme of a clickable glutathione approach for analyzing S-glutathionylation. Cells expressing GS M4 are incubated with azido-Ala, which enables biosynthesis of clickable glutathione (azido-glutathione, N₃-GSH). N₃-GSH forms S-glutathionylation in oxidative stress. Alternatively, purified protein can form glutathionylation in the presence of N₃-GSH, which is analyzed after the click reaction. E, glutathionylation of purified PP2Cα in vitro. Purified PP2Cα was incubated with N₃-GSH and H₂O₂ for 15 min (top) or oxidized azido-glutathione (N₃-GSSG-N₃) for 30 min (bottom). PP2Cα glutathionylated by azido-glutathione was conjugated with rhodamine-alkyne via click chemistry and visualized by Coomassie stain (CM, protein level) and fluorescence (FL, SSG level) (n = 3). F, MALDI-TOF and MS/MS analyses of glutathionylated peptides in PP2Cα. Purified PP2Cα glutathionylated by azido-glutathione was click-conjugated by biotin-DADPS-alkyne, enriched by streptavidin-agarose, and digested by trypsin on beads. Glutathionylated peptides were eluted and analyzed by MALDI-TOF (left) and LC-MS/MS (right), finding a glutathionylated peptide at C314 (YLEC₃₁₄∗R, m/z 1127). In the MS2 spectrum, all y ions were found with additional ions (Y¹_tag and Y_tag) resulting from fragmentation in glutathione modification (n = 3). G, global glutathionylation in MDA-MB-231. MDA-MB-231 cells expressing GS M4 (MDA-MB-231/GS M4) were stimulated in low or high glucose conditions. After the click reaction of lysates with rhodamine-alkyne, proteins were analyzed by fluorescence and Coomassie stain (n = 3). H, PP2Cα glutathionylation in MDA-MB-231. MDA-MB-231/GS M4 cells expressing HA-PP2Cα WT or C/S mutant were incubated in low or high glucose conditions for 24 h or with H₂O₂ for 15 min. After the click reaction of lysates with biotin-alkyne, glutathionylated PP2Cα was probed by Western blot before (protein level) and after (SSG) enrichment by streptavidin-agarose (n = 3). Data are representative of three independent experiments.