Proteomic Investigation of Phosphorylation Sites in Poly(ADP-ribose) Polymerase-1 and Poly(ADP-ribose) Glycohydrolase
Proteomic Investigation of Phosphorylation Sites in Poly(ADP-ribose) Polymerase-1 and Poly(ADP-ribose) Glycohydrolase
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DOI：
10.1021/pr800810n
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发表时间：
2009-02-01
影响因子：
4.4
通讯作者：
Poirier, Guy G.
中科院分区：
文献类型：
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作者：
Gagne, Jean-Philippe;Moreel, Xavier;Poirier, Guy G.
磷酸化是一种非常常见的翻译后修饰事件，已知可调节广泛的生物反应。除了蛋白质活性的调节之外，磷酸化与其他翻译后机制的相互关系负责控制各种信号通路。一些观察结果表明，磷酸化的聚（ADP-核糖）聚合酶-1（PARP-1）调节其活性。也有越来越多的证据表明PARP-1相关多蛋白复合物的磷酸化依赖性组装的建立。虽然证明一个确定的靶点的磷酸化是相对简单的，但对许多实验室来说，鉴定所涉及的实际氨基酸仍然是一个技术挑战。结合使用基于生物信息学的通用和激酶特异性磷酸化位点预测工具，体外磷酸化测定和质谱分析，我们研究了PARP-1和聚（ADP-核糖）糖水解酶（PARG）的磷酸化谱，这两种主要酶负责聚（ADP-核糖）营业额。质谱分析揭示了这两种酶的重要调节结构域和基序内几个丝氨酸/苏氨酸残基的磷酸化。使用体内微辐射诱导的DNA损伤，我们表明，改变磷酸化在特定的网站可以修改的动态组装和拆卸的PARP-1的DNA损伤的网站。通过记录和注释一系列已知和新发现的磷酸化位点，这项靶向蛋白质组学研究显著提高了我们对磷酸化在PARP-1和PARG调节中作用的理解。
Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.,