Regulation of thioredoxin peroxidase I and II by subcellular translocation and C

最近的证据表明，H2O2在响应各种细胞外刺激时产生，可作为细胞内信使。H2O2也是羟基自由基的前体，对细胞成分造成不可逆转的损伤。H2O2的这种二分类函数预示着H2O2的产生时间和局部浓度受到严格的调控。硫氧还蛋白过氧化物酶TPxI和TPx II是过氧化物酶（Prx）家族的成员，通过调节细胞内H2O2浓度参与生长因子和细胞因子信号传导。为了了解TPx酶如何与不同的激动剂信号合作，我们研究了通过差速离心获得的亚细胞组分中TPx蛋白的分布。细胞器、膜和细胞质中检测到TPx I和TPx II。进一步对膜池进行nycodenz -密度梯度分析发现，TPxs I和TPxs II在轻、重膜结构中分布广泛。此外，我们在Triton x -100不溶性膜组分中检测到部分TPx蛋白。免疫荧光染色显示HeLa细胞中TPx II蛋白呈明显的点状，与内体染色相似。这些结果表明，TPxs I和TPxs II可能定位于多种膜结构，包括核内体和脂质筏。有趣的是，用EGF或H2O2处理HeLa细胞诱导了TPxII在核周区域的积累，表明TPxII的刺激依赖性易位。我们还观察到，从HeLa和NIH3T3细胞纯化的细胞核片段中存在TPxs I和II，而不存在其他Prx亚型。核分数中TPxs I和TPxs II的数量在血清刺激下增加，在H2O2和UVB处理下减少。TpxI和TpxII含有一个被细胞周期蛋白依赖性激酶（CDK）磷酸化的位点[TP(K/R)K]。CDK2、CDK4和CDK6免疫沉淀来自于磷酸化野生型TpxI和TpxII的细胞提取物，而不是突变型Tpx蛋白，在假定的磷酸化位点上用Ala代替Thr。苏氨酸磷酸化也被CDKs所证实，CDKs由抗细胞周期蛋白A、D和e的抗体共同免疫沉淀。综上所述，Tpx酶似乎是一种移动蛋白，通过易位响应局部需求。转位到细胞核或它们在细胞核中的催化活性可能受到cdk依赖性磷酸化的调节。

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.