Regulation of Peroxiredoxin Ⅰ Peroxidase Activity by Cdc2-dependent phosphoarylation

다국어 초록 (Multilingual Abstract)

Recent evidence suggests that H₂O₂ mediates a variety of cellular responses, including proliferation and differentiation, to many important physiological stimuli. Peroxiredoxin(Prx) is a novel family of peroxidase that exists in multiple isoforms in mammalian cells. Prx Ⅰ and Prx Ⅱ, which exist mainly in the cytosol, are also detected in the nuclei fraction prepared from He La and NIH3T3 cells. Prx Ⅰ and Prx Ⅱ contain a consensus site(Thr90-Pro-Lys-Lys93) for phosphorylation by Cdc2, a major regulator of G₂/M transition in the eukaryotic cell cycle. In this study, we investigated the biological function of Prx Ⅰ phosphorylation by Cdc2 during cell cycle progression.
The ability of Cdc2 to phosphorylate Prx Ⅰ was confirmed by in vitro kinase assay with a highly purified Cdc2-cyclin B complex or Cdc2 immunocomplex prepared from HeLa cells. Using the mutant Prx Ⅰ in which Thr-90 residue had been substituted with Ala, we identified Thr-90 as a residue phosphorylated by Cdc2. Mutation at Thr-90 to Glu or Asp to mimic the phosphorylation state resulted in a marked decrease in peroxidase activity toward H₂O₂. To monitor Prx Ⅰ phosphorylation in intact cells, we prepared rabbit antibodies that specifically bind to the Prx Ⅰ phosphorylated at Thr-90. Using these site-specific, threonine phosphorylation-dependent antibodies, we observed that Thr-90 phosphorylation was readily induced upon activation of Cdc2 by treatment of HeLa cells with okadaic acid and its phosphorylation correlated well with the accumulation of cells in G₂/M phases. Furthermore, cell synchronization studies showed that Thr-90 was highly phosphorylated during mitosis. Thr-90 phosphorylation of Prx Ⅰ in mitotic cells was greatly reduced by the treatment of a specific Cdc2 inhibitor, roscovitine. These results suggest that phosphorylation of Prx Ⅰ by Cdc2 and the resulting inhibition of its peroxidase activity are likely to result in increase in the concetration of H₂O₂ in the nucleus, which might be a critical signaling component in the progression of normal cell division.