Abstract
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.
Highlights
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
To determine conditions in which Jun N-terminal kinase (JNK) signaling is regulated by H2O2 in chondrocytes, treatment of chondrocytes with Fibronectin fragments (FN-fs) was compared with addition of exogenous H2O2 or the redox cycling agent menadione
We had previously found that menadione generates high levels of endogenous H2O2 in chondrocytes, resulting in oxidative stress, at least in part through inactivation of Prxs that are critical to cellular ROS detoxification [30]
Summary
H2O2 oxidation of cysteine residues in c-Jun N-terminal kinase 2 (JNK2) contributes to redox regulation in human articular chondrocytes. Pretreatment with dimedone, a reagent that reacts selectively with sulfenylated cysteines [29], blocked FN-f–induced phosphorylation of JNK and its substrate c-Jun, indicating that protein sulfenylation was required for signaling that resulted in JNK and c-Jun phosphorylation Based on these studies, FN-f stimulation of human articular chondrocytes was used as a biologically relevant model system to further study the role of protein thiol oxidation in the regulation of JNK activity. FN-f stimulation was compared with treating cells with various concentrations of exogenous H2O2 as a direct oxidant and with the redox cycling agent menadione, which generates higher levels of intracellular H2O2 We found that both FN-f treatment, which generated low levels of H2O2, and treatment with low micromolar levels of H2O2 stimulated phosphorylation of JNK and its substrate c-Jun. Menadione treatment and treatment with high levels of exogenous H2O2 reversibly inhibited JNK2 kinase activity through oxidation of cysteine residues in JNK2. The mechanism of this inhibition was further explored using mass spectrometry (MS) and sitedirected mutagenesis to identify the sites of oxidation on JNK2␣2
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