Multisite phosphorylation of Pin1-associated mitotic phosphoproteins revealed by monoclonal antibodies MPM-2 and CC-3
Multisite phosphorylation of Pin1-associated mitotic phosphoproteins revealed by monoclonal antibodies MPM-2 and CC-3
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DOI：
10.1186/1471-2121-5-22
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发表时间：
2004-06-01
期刊：
影响因子：
--
通讯作者：
Vincent, M
中科院分区：
文献类型：
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作者：
Albert, AL;Lavoie, SB;Vincent, M
背景：肽基脯氨酰异构酶 Pin1 最近被证明是通过磷酸化调节蛋白质功能的新参与者。 Pin1 使参与多种细胞事件（包括有丝分裂、转录、分化和 DNA 损伤反应）的几种蛋白质中脯氨酸之前的特定磷酸化丝氨酸或苏氨酸残基的肽键异构化。许多 Pin1 底物是磷酸依赖性单克隆抗体 MPM-2 的抗原，MPM-2 与 G2/M 转变时磷酸化的蛋白质子集发生反应。结果：由于 MPM-2 不是有丝分裂磷蛋白的一般标记，并且大多数有丝分裂底物被磷酸化多次，因此我们使用不同的磷酸依赖性抗体 mAb CC-3，通过结合亲和纯化来鉴定其他有丝分裂磷蛋白和最终的 Pin1 底物， MALDI-TOF 质谱和免疫印迹。大多数 CC-3 反应性磷蛋白似乎是已知或新型 MPM-2 抗原，包括 RNA 结合蛋白 p54(nrb)/nmt55、剪接体蛋白 SAP155、Ki-67 抗原、MAP-1B、DNA 拓扑异构酶 II α 和 β、延伸因子 hSpt5 和 RNA 聚合酶 II 的最大亚基。 CC-3 有丝分裂抗原也被证明是 Pin1 靶标。 RNA 聚合酶 II 羧基末端结构域的精细 CC-3 和 MPM-2 表位图谱证实，这些表位是不同的，并且可以在体外由不同的激酶产生。最后，当选择性抑制剂胡桃醌阻断细胞 Pin1 活性时，CC-3 和 MPM-2 抗原的有丝分裂后去磷酸化被阻止。结论：这些观察结果表明与 Pin1 相关的有丝分裂磷蛋白在多个位点被磷酸化，表明底物识别和异构化的组合调节。
Background: The peptidyl-prolyl isomerase Pin1 recently revealed itself as a new player in the regulation of protein function by phosphorylation. Pin1 isomerizes the peptide bond of specific phosphorylated serine or threonine residues preceding proline in several proteins involved in various cellular events including mitosis, transcription, differentiation and DNA damage response. Many Pin1 substrates are antigens of the phosphodependent monoclonal antibody MPM-2, which reacts with a subset of proteins phosphorylated at the G2/M transition.Results: As MPM-2 is not a general marker of mitotic phosphoproteins, and as most mitotic substrates are phosphorylated more than once, we used a different phosphodependent antibody, mAb CC-3, to identify additional mitotic phosphoproteins and eventual Pin1 substrates by combining affinity purification, MALDI-TOF mass spectrometry and immunoblotting. Most CC-3-reactive phosphoproteins appeared to be known or novel MPM-2 antigens and included the RNA-binding protein p54(nrb)/nmt55, the spliceosomal protein SAP155, the Ki-67 antigen, MAP-1B, DNA topoisomerases II alpha and beta, the elongation factor hSpt5 and the largest subunit of RNA polymerase II. The CC-3 mitotic antigens were also shown to be Pin1 targets. The fine CC-3- and MPM-2-epitope mapping of the RNA polymerase II carboxy-terminal domain confirmed that the epitopes were different and could be generated in vitro by distinct kinases. Finally, the post-mitotic dephosphorylation of both CC-3 and MPM-2 antigens was prevented when cellular Pin1 activity was blocked by the selective inhibitor juglone.Conclusion: These observations indicate that the mitotic phosphoproteins associated with Pin1 are phosphorylated on multiple sites, suggesting combinatorial regulation of substrate recognition and isomerization.