A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells
A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells
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DOI：
10.1073/pnas.0804336105
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发表时间：
2008-07-22
影响因子：
11.1
通讯作者：
Tonks, Nicholas K.
中科院分区：
文献类型：
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作者：
Boivin, Benoit;Zhangt, Sheng;Tonks, Nicholas K.
活性氧（ROS）的产生通过瞬时抑制特定蛋白酪氨酸磷酸酶（PTP）的催化活性，对酪氨酸磷酸化依赖性信号转导施加额外的控制。因此，在体内检测PTP可逆氧化的能力对于理解ROS在细胞信号传导控制中的复杂生物学作用至关重要。在这里，我们描述了一种用于识别那些在体内可逆氧化的PTP的测定，其利用在温和的酸性条件下用生物素化的小分子标记活性位点的不变催化Cys残基的独特化学。我们应用半胱氨酰标记法研究血管平滑肌脂肪瘤细胞模型中血小板衍生生长因子（PDGF）受体信号传导。这样做使我们能够检测几种蛋白质对持续PDGF刺激的可逆氧化反应。在其他细胞系统中，我们已经观察到的可逆氧化的经典PTP SHP2和肿瘤抑制磷酸酶PTEN的PDGF刺激的反应。此外，我们还检测到PTPs的另外两个亚类成员的可逆氧化，即受体PTP LAR和双特异性磷酸酶MKP 1。这些数据证明了广泛的选择性的测定，使我们能够检测PTP超家族的所有主要亚组的代表。我们预计这种半胱氨酰标记的富集策略可以广泛应用于研究可逆氧化，作为在多种信号通路中利用PTP催化活性的机制。
The production of reactive oxygen species (ROS) exerts an additional tier of control over tyrosine phosphorylation-dependent signal transduction by transiently inhibiting the catalytic activity of specific protein tyrosine phosphatases (PTPs). Hence, the ability to detect reversible oxidation of PTPs in vivo is critical to understanding the complex biological role of ROS in the control of cellular signaling. Here, we describe an assay for identifying those PTPs that are reversibly oxidized in vivo, which utilizes the unique chemistry of the invariant catalytic Cys residue in labeling the active site with biotinylated small molecules under mildly acidic conditions. We have applied this cysteinyl-labeling assay to the study of platelet-derived growth factor (PDGF) receptor signaling in an angiomyolipoma cell model. Doing so has allowed us to detect reversible oxidation of several proteins in response to sustained PDGF stimulation. As in other cell systems, we have observed the reversible oxidation of the classical PTP SHP2 and the tumor suppressor phosphatase PTEN in response to PDGF stimulation. Furthermore, we detected reversible oxidation of members of two other subclasses of PTPs, the receptor PTP LAR and the dual-specificity phosphatase MKP1. These data demonstrate the broad selectivity of the assay, allowing us to detect representatives of all of the major subgroups of the PTP superfamily. We anticipate that this cysteinyl-labeling enrichment strategy can be applied broadly to study reversible oxidation as a mechanism of harnessing PTP catalytic activity in a variety of signaling pathways.