Abstract
Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.
Highlights
Cell signal transduction is a summation of positive stimuli and homeostatic negative feedback
We have found that p38 is oxidized following exposure of cells to exogenous hydrogen peroxide or prostaglandin J2, and that oxidation results in kinase inactivation despite continued phosphorylation on the activating residues detected by immunoblot
We found that both osmotic shock and hydrogen peroxide, or both, induced phosphorylation of p38 detected with anti-phospho-p38 antibodies (Figure 1A, top)
Summary
Cell signal transduction is a summation of positive stimuli and homeostatic negative feedback. Other than the action of phosphatases that remove the activating phosphorylation, inhibitory or negative feedback mechanisms for damping or silencing protein kinase cascades are less well understood. Mainly due to the utility of anti-phosphoepitope antibodies in identifying phosphate on specific proteins, it has become common in the literature for the presence of an activating phosphorylation on a signaling kinase to be interpreted as an ‘active’ kinase pathway. This unfortunate simplification ignores more subtle means of signal regulation
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