Glavy Joseph S, Krutchinsky Andrew N, Cristea Ileana M, Berke Ian C, Boehmer Thomas, Blobel Günter, Chait Brian T
Laboratories of Mass Spectrometry and Gaseous Ion Chemistry, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3811-6. doi: 10.1073/pnas.0700058104. Epub 2007 Feb 28.
The nuclear pore complex (NPC) mediates macromolecular transport between the nucleus and the cytoplasm. Many NPC proteins (nucleoporins, Nups) are modified by phosphorylation. It is believed that phosphorylation regulates the breakdown of the nuclear envelope at mitosis and the disassembly of the NPC into different subcomplexes. In this study, we examined the cell-cycle-dependent phosphorylation of the Nup107-160 subcomplex, a core building block of the NPC. Using in vivo (32)P labeling in HeLa cells, we found that Nup107, Nup96, and Nup133 are phosphorylated during mitosis. To precisely map the phosphorylation sites within the complex, we used a comprehensive multiple-stage MS approach (MS, MS(2), and MS(3)), establishing that Nup160, Nup133, Nup96, and Nup107 are all targets of phosphorylation. We determined that the phosphorylation sites are clustered mainly at the N-terminal regions of these proteins, which are predicted to be natively disordered. In addition, we determined the cell-cycle dependence of the phosphorylation of these sites by using stable isotope labeling and MS(2) analysis. Measurement of the site-specific phosphorylation ratios between mitotic and G(1) cells led us to conclude that several phosphorylation events of the subcomplex are mainly mitotic. Based on these results and our finding that the entire Nup107-160 subcomplex is stable throughout the cell cycle, we propose that phosphorylation does not affect interactions within the Nup107-160 subcomplex, but regulates the association of the subcomplex with the NPC and other proteins.
核孔复合体（NPC）介导细胞核与细胞质之间的大分子运输。许多NPC蛋白（核孔蛋白，Nups）会发生磷酸化修饰。据信，磷酸化作用调节有丝分裂时核膜的解体以及NPC分解为不同的亚复合体。在本研究中，我们检测了NPC的核心构建模块Nup107 - 160亚复合体的细胞周期依赖性磷酸化。通过在HeLa细胞中进行体内³²P标记，我们发现Nup107、Nup96和Nup133在有丝分裂期间发生磷酸化。为了精确绘制该复合体内的磷酸化位点，我们采用了一种全面的多阶段质谱方法（MS、MS²和MS³），确定Nup160、Nup133、Nup96和Nup107均为磷酸化靶点。我们确定磷酸化位点主要聚集在这些蛋白质的N端区域，预计这些区域天然无序。此外，我们通过使用稳定同位素标记和MS²分析确定了这些位点磷酸化的细胞周期依赖性。测量有丝分裂细胞和G1期细胞之间的位点特异性磷酸化比率，使我们得出结论，该亚复合体的几个磷酸化事件主要发生在有丝分裂期。基于这些结果以及我们发现整个Nup107 - 160亚复合体在整个细胞周期中都是稳定的，我们提出磷酸化并不影响Nup107 - 160亚复合体内的相互作用，而是调节该亚复合体与NPC及其他蛋白质的关联。
