Regulation of PTEN by superoxide and H2O2 through formation of a disulfide bond

肿瘤抑制因子PTEN通过与pi3 -激酶联合调控pi3,4,5 - p3的水平，在细胞凋亡、细胞周期和发育的调控中发挥重要作用。然而，PTEN的活性如何在响应细胞外刺激时被调节尚不清楚。在没有生长因子刺激的情况下，PTEN的过表达可阻止pi3,4,5 - p3效应因子Akt的基础水平激活。然而，生长因子刺激这些细胞可通过pi3激酶依赖途径激活Akt。这表明在受生长因子刺激的细胞中存在PTEN活性被抑制和PIP3积累的机制。越来越多的证据表明，超氧化物和H2O2是在各种细胞外刺激下短暂产生的，其细胞内水平受生成酶如NADPH氧化酶和脂氧化酶以及清除酶如超氧化物歧化酶、过氧化氢酶和过氧化物还毒素（Prxs）的控制。PTEN在活性位点有一个必需的Cys124。与H2O2孵育导致纯化的PTEN以依赖于时间和H2O2浓度的方式失活。对各种Cys突变体的研究和色氨酸的直接质谱分析表明，Cys124在H2O2和超氧阴离子氧化作用下与Cys71形成二硫化物。将h2o2灭活后的PTEN与不同的电子供体[DTT、GSH、硫氧还蛋白（Trx）、谷胱甘肽（Grx）]孵育，并监测其再激活情况。DTT和Trx（5微米）的再激活速度最快，而GSH（5毫米）的再激活效率最低。PTEN对受体产生的H2O2的敏感性在fMLP刺激的人中性粒细胞中进行了测试。PTEN的氧化量随着fMLP孵育时间的延长而增加，在5 min时达到最大值，20 min时恢复到基础值，提示PTEN氧化是可逆的。与硫氧还蛋白还原酶抑制剂1-氯-2,4-二硝基苯孵育细胞可使还原的PTEN转化为氧化形式，而谷胱甘肽生物合成抑制剂丁硫氨酸磺酰亚胺则没有任何影响。这些结果表明PTEN的活性是通过H2O2的可逆氧化来调节的，而还原主要是由硫氧还蛋白完成的。

The tumor suppressor PTEN plays important roles in the control of apoptosis, cell cycles and development by acting in conjuction with PI3-kinase to regulate the levels of PI 3,4,5-P3. However, how the activity of PTEN is regulated in response to extracellular stimuli is not known. Overexpression of PTEN prevents basal level activation of Akt, a PI 3,4,5-P3 effector, in the absence of growth factor stimulation. However, growth factor stimulation of these cells results in activation of Akt via a PI3-Kinase-dependent pathway. This suggest that there are mechanisms by which PTEN activity is suppressed and PIP3 accumulates in cells stimulated with a growth factor. Increasing evidence suggests that superoxide and H2O2 are transiently produced in response to various extracellular stimuli and their intracellular levels are controlled by the generating enzymes like NADPH oxidases and lipooxygenases as well as the scavenging enzymes like superoxide dismutases, catalases, and peroxiredoxins (Prxs).PTEN has an essential Cys124 at the active site. Incubation with H2O2 resulted in the inactivation of purified PTEN in a manner dependent on time and H2O2 concentration. Studies of various Cys mutants and direct mass spectral analysis of tryptic peptides indicated that Cys124 formed a disulfide with Cys71 upon oxidation with H2O2 and superoxide anion. The H2O2-inactivated PTEN was incubated with various electron donors [DTT, GSH, thioredoxin (Trx), glutaredoxin (Grx)] and reactivation was monitored. The most rapid reactivation was achieved by DTT and Trx (5 microM), whereas GSH (5 mM) was the least efficient. The sensitivity of PTEN to receptor-produced H2O2 was tested in human neutrophils stimulated with fMLP. The amount of oxidized PTEN increased with time of incubation with fMLP, reaching a maximum at 5 min, and returned to the basal value by 20 min, suggesting that the PTEN oxidation is reversible. Incubation of cells with 1-chloro-2,4-dinitrobenzene, an inhibitor of thioredoxin reductase caused the conversion of reduced PTEN to oxidized form, whereas buthioninesulfoximine, an inhibitor of glutathione biosynthesis did not have any effect. These results suggest that the activity of PTEN is regulated through reversible oxidation by H2O2 and the reduction is achieved mainly by thioredoxin.