Recent evidence suggests that H2O2, produced in response to various extracelluar stimuli, functions as an intracellular messenger. H2O2 is also a precursor of hydroxyl radicals that cause irreversible damage on cellular components. This dichotomous function of H2O2 predicts that the production timing and local concentration of H2O2 be stringently regulated. The thioredoxin peroxidases, TPxI and TPx II, are members of the peroxiredoxin (Prx) family that participates in growth factor and cytokine signaling by modulating intracellular concentration of H2O2. In an effort to understand how TPx enzymes cooperate with different agonist signalings, the distribution of TPx proteins has been studied in subcellular fractions obtained by differential centrifugation. TPx I and II were detected in organelle and membrane fractions, as well as cytosol. When the membrane pool was further analyzed on Nycodenz-density gradient, TPxs I and II showed wide distribution throughout light and heavy membrane structures. Furthermore, we detected a part of TPx proteins in Triton X-100-insoluble membrane fraction. Immunofluorescence staining showed an obvious punctuate pattern of TPx II protein in HeLa cell, similar to endosomal staining. These results indicate that TPxs I and II may localize in various membrane structures, including endosomes and lipid raft. Interestingly, treatment of HeLa cells with EGF or H2O2 induced the accumulation of TPx II in perinuclear region, suggesting a stimulation-dependent translocation of TPxII. We also observed that TPxs I and II, but not other Prx isoforms, are present in nuclei fraction purified from HeLa and NIH3T3 cells. The amount of TPxs I and II in nuclei fraction increased in response to serum stimulation and decreased in response to treatment with H2O2 and UVB. TpxI and TpxII contain a site [TP(K/R)K] of phosphorylation by cyclin-dependent kinase (CDK). CDK2, CDK4, and CDK6 immunoprecipitated from cell extracts phosphorylated wild type TpxI and TpxII, but not mutant Tpx proteins with the Ala for Thr substitution at the putative phosphorylation site. The threonine phosphorylation was also confirmed with CDKs co-immunoprecipitated by antibodies against cyclin A, D, and E. Taken together, Tpx enzymes appear to be mobile protein that respond to the local need by translocation. Either translocation to nucleus or their catalytic activity in nucleus is likely regulated by CDK-dependent phosphorylation.
