Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation
Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation
复制标题
DOI：
10.1074/mcp.m600453-mcp200
复制
发表时间：
2007-08-01
影响因子：
7
通讯作者：
Hart, Gerald W.
中科院分区：
文献类型：
--
作者：
Wang, Zihao;Pandey, Akhilesh;Hart, Gerald W.
核蛋白和细胞质蛋白丝氨酸和苏氨酸侧链上的o - glcn酰化受到各种环境和生物刺激的动态调节。o - glcn酰化与o -磷酸化非常相似，似乎在细胞调节中与o -磷酸有动态的相互作用。对抑制特定激酶对o - glcn酰化的影响进行系统的糖蛋白组学分析，将有助于揭示这两种丰富的翻译后修饰之间的全局和特异性动态关系。在这里，我们报道了O-GlcNAc扰动对糖原合成酶激酶-3 （GSK-3）抑制的反应，GSK-3是一种参与许多信号通路的关键激酶。通过结合免疫亲和层析和基于SILAC（细胞培养中氨基酸的稳定同位素标记）的定量质谱，我们鉴定了45个潜在的o - glcn酰化蛋白。定量测量表明，至少有10种蛋白质在锂对GSK-3的抑制作用下，o - glcnac酰化明显增加，而令人惊讶的是，其他19种蛋白质表现出降低。o - glcn酰化在蛋白质子集上的变化被后续实验证实。结合新的O-GlcNAc肽富集方法和β -消除法，再加上DTT的Michael加法，我们还绘制了vimentin的O-GlcNAc位点（Ser-55），显示GSK-3抑制后O-GlcNAc酰化明显增加。基于MS数据，我们进一步研究了o- glcnnac对宿主细胞因子-1（转录共激活因子）的潜在作用，发现o- glcn酰化对宿主细胞因子-1的动态调控影响其亚细胞分布。综上所述，这些数据表明磷酸化和o - glcn酰化之间复杂的相互作用发生在信号网络中。
O-GlcNAcylation on serine and threonine side chains of nuclear and cytoplasmic proteins is dynamically regulated in response to various environmental and biological stimuli. O-GlcNAcylation is remarkably similar to O-phosphorylation and appears to have a dynamic interplay with O-phosphate in cellular regulation. A systematic glycoproteomics analysis of the affects of inhibiting specific kinases on O-GlcNAcylation should help reveal both the global and specific dynamic relationships between these two abundant post-translational modifications. Here we report the O-GlcNAc perturbations in response to inhibition of glycogen synthase kinase-3 (GSK-3), a pivotal kinase involved in many signaling pathways. By combining immunoaffinity chromatography and SILAC ( stable isotope labeling with amino acids in cell culture)-based quantitative mass spectrometry, we identified 45 potentially O-GlcNAcylated proteins. Quantitative measurements indicated that at least 10 proteins had an apparent increase of O-GlcNAcylation upon GSK-3 inhibition by lithium, whereas surprisingly 19 other proteins showed decreases. O-GlcNAcylation changes on a subset of the proteins were confirmed by follow-up experiments. By combining a new O-GlcNAc peptide enrichment method and beta-elimination followed by Michael addition with DTT, we also mapped the O-GlcNAc site (Ser-55) of vimentin, which showed an apparent increase of O-GlcNAcylation upon GSK-3 inhibition. Based on the MS data, we further investigated potential roles of O-GlcNAc on host cell factor-1, a transcription co-activator, and showed that dynamic regulation of O-GlcNAcylation on host cell factor-1 influenced its subcellular distribution. Taken together, these data indicated the complex interplay between phosphorylation and O-GlcNAcylation that occurs within signaling networks.