Displacement affinity chromatography of protein phosphatase one (PPI) complexes
Displacement affinity chromatography of protein phosphatase one (PPI) complexes
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DOI：
10.1186/1471-2091-9-28
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发表时间：
2008-11-10
期刊：
影响因子：
--
通讯作者：
Lamond, Angus I.
中科院分区：
文献类型：
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作者：
Moorhead, Greg B. G.;Trinkle-Mulcahy, Laura;Lamond, Angus I.
背景：蛋白磷酸酶1(PPI)是一种普遍表达、高度保守的蛋白磷酸酶，可使靶蛋白丝氨酸和苏氨酸残基去磷酸化。PPI通过与靶向或调节蛋白相互作用定位于其作用部位，其中大部分包含一个被称为RVXF/W基序的主要对接位点。结果：我们证明了基于RVXF/W基序的多肽可以有效地取代保留在磷酸酶亲和基质微囊藻毒素-琼脂糖上的PPI结合蛋白。随后的免疫共沉淀实验证实，每个鉴定的结合蛋白要么是直接的PPI相互作用，要么是在含有PPI的复合体中。我们的结果将PPI与许多新的核功能和蛋白质联系在一起，包括Ki-67、Rif-I、拓扑异构酶IIα、几个核解旋酶、NUP153和TRRAP复合体。结论：改进的微囊藻毒素-琼脂糖凝胶技术为纯化新的PPI调节亚基和相关蛋白提供了一种有效的方法，并为揭示PPI与其他细胞过程之间的联系提供了一种简单的方法。
Background: Protein phosphatase one ( PPI) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PPI is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results: We demonstrate that a peptide based on the RVXF/W motif can effectively displace PPI bound proteins from PPI retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PPI interactor or was in a complex that contains PPI. Our results have linked PPI to numerous new nuclear functions and proteins, including Ki-67, Rif-I, topoisomerase II alpha, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion: This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PPI regulatory subunits and associated proteins and provides a simple method to uncover a link between PPI and additional cellular processes.