Constitutive Induction of p-Erk1/2 Accompanied by Reduced Activities of Protein Phosphatases 1 and 2A and MKP3 Due to Reactive Oxygen Species during Cellular Senescence

Abstract

The mechanism of senescence-associated cytoplasmic induction of p-Erk1/2 (SA-p-Erk1/2) proteins in human diploid fibroblasts was investigated. p-Erk1/2 proteins were efficiently dephosphorylated in vitro by protein phosphatases 1 and 2A (PP1/2A) and MAPK phosphatase 3 (MKP3). Specific activity of PP1/2A and MKP3 activity significantly decreased during cellular senescence, whereas their protein expression levels did not. To investigate possible mechanism of phosphatase inactivation, we measured reactive oxygen species (ROS) generation by fluorescence-activated cell sorting analysis and found it was much higher in mid-old cells than the young cells. Treating the young cells once with 1 mm H2O2 remarkably induced p-Erk1/2 expression; however, it was transient unless repeatedly treated until 72 h. Multiple treatment of the cells with 0.2 mm H2O2 significantly duplicated inactivation of PP1/2A; however, thiol-specific reagents could reverse the PP1/2A activities, suggesting the oxidation of cysteine molecule in PP1/2A by the increased ROS. When the cells were pretreated with 10 mm N-acetyl-l-cysteine for 1 h, Erk1/2 activation was completely blocked. To elucidate which cysteine residue and/or metal ion in PP1/2A was modified by H2O2, electrospray ionization-tandem mass spectrometry analyses were performed with purified PP1C-alpha and found Cys62-SO3H and Cys105-SO3H, implicating the tertiary structure perturbation. H2O2 inhibited purified PP1C-alpha activity by both oxidation of Cys residues and metal ion(s), evidenced by dithiothreitol and ascorbate-restoration assay. In summary, SA-p-Erk1/2 was most likely due to the oxidation of PP1/2A, which resulted from the continuous exposure of the cells to vast amounts of ROS generated during cellular senescence by oxidation of Cys62 and Cys105 in PP1C-alpha and metal ion(s).