Journal of Biological Chemistry
Volume 293, Issue 42, 19 October 2018, Pages 16376-16389
Journal home page for Journal of Biological Chemistry

Signal Transduction
H2O2 oxidation of cysteine residues in c-Jun N-terminal kinase 2 (JNK2) contributes to redox regulation in human articular chondrocytes

https://doi.org/10.1074/jbc.RA118.004613Get rights and content
Under a Creative Commons license
open access

Reactive oxygen species (ROS), in particular H2O2, regulate intracellular signaling through reversible oxidation of reactive protein thiols present in a number of kinases and phosphatases. H2O2 has been shown to regulate mitogen-activated protein kinase (MAPK) signaling depending on the cellular context. We report here that in human articular chondrocytes, the MAPK family member c-Jun N-terminal kinase 2 (JNK2) is activated by fibronectin fragments and low physiological levels of H2O2 and inhibited by oxidation due to elevated levels of H2O2. The kinase activity of affinity-purified, phosphorylated JNK2 from cultured chondrocytes was reversibly inhibited by 5–20 μm H2O2. Using dimedone-based chemical probes that react specifically with sulfenylated cysteines (RSOH), we identified Cys-222 in JNK2, a residue not conserved in JNK1 or JNK3, as a redox-reactive site. MS analysis of human recombinant JNK2 also detected further oxidation at Cys-222 and other cysteines to sulfinic (RSO2H) or sulfonic (RSO3H) acid. H2O2 treatment of JNK2 resulted in detectable levels of peptides containing intramolecular disulfides between Cys-222 and either Cys-213 or Cys-177, without evidence of dimer formation. Substitution of Cys-222 to alanine rendered JNK2 insensitive to H2O2 inhibition, unlike C177A and C213A variants. Two other JNK2 variants, C116A and C163A, were also resistant to oxidative inhibition. Cumulatively, these findings indicate differential regulation of JNK2 signaling dependent on H2O2 levels and point to key cysteine residues regulating JNK2 activity. As levels of intracellular H2O2 rise, a switch occurs from activation to inhibition of JNK2 activity, linking JNK2 regulation to the redox status of the cell.

mitogen-activated protein kinase (MAPK)
redox regulation
redox signaling
chondrocyte
integrin
reactive oxygen species (ROS)
hydrogen peroxide
c-Jun N-terminal kinase (JNK)
disulfide
cellular redox status

Cited by (0)

This work was supported by National Institutes of Health Grants R37 AR049003 and RO1 AG044034 (to R. F. L.), F31 AG032796 (to E. A. E.), R33 CA126659 (to L. B. P.), and R33 CA177461 (to C. M. F.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains Figs. S1–S7 and Table S1.

1

Present address: National Institutes of Health Undiagnosed Diseases Program, National Institutes of Health, Bethesda, MD 20892.

2

Present address: Dept. of Biochemistry and Molecular Genetics, University of Colorado, Anschutz Medical Campus, Aurora, CO 80045.

3

Present address: Dept. of Nano Science and Engineering, South Dakota School of Mines and Technology, Rapid City, SD 57701.