Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics
Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics
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DOI：
10.1039/c1mb05185g
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发表时间：
2011-01-01
影响因子：
--
通讯作者：
Blagoev, Blagoy
中科院分区：
文献类型：
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作者：
Akimov, Vyacheslav;Rigbolt, Kristoffer T. G.;Blagoev, Blagoy
蛋白质泛素化是一种动态的可逆的翻译后修饰，在信号转导、内吞、细胞周期调控、DNA修复和基因转录等众多细胞过程中起着重要的调控作用。已知小蛋白泛素或各种类型和长度的泛素分子链与靶蛋白的缀合改变蛋白质的寿命、定位和功能并调节蛋白质相互作用。尽管它在细胞生命和功能的各个方面都具有核心重要性，但在蛋白质组学规模上研究泛素化的报告数量有限，这主要是由于泛素化的遗传复杂性和异质性。我们在这里描述了一个定量蛋白质组学策略的基础上的特异性泛素结合结构域（UBD）和稳定同位素标记的氨基酸在细胞培养（SILAC）的选择性解码泛素化驱动的过程中参与的细胞信号网络的调节。我们应用这种方法来表征伴随表皮生长因子受体（EGFR）信号转导的泛素化事件的时间动态。我们使用了来自内吞衔接蛋白的重组UBD，用于特异性富集EGFR网络中的泛素化复合物，并随后通过高精度质谱进行定量分析。我们表明，该策略适用于分析发生在单个蛋白质上的泛素化动态以及信号通路中的泛素化依赖性事件。除了在30分钟的配体刺激中EGFR泛素化的详细的七个时间点曲线之外，我们的数据确定了赖氨酸-63泛素分支在EGF信号传导中的显著参与。此外，我们发现两个中心体蛋白，PCM 1和Azi 1，形成一个多蛋白复合物与泛素E3连接酶MIB 1和WWP 2下游的EGFR，从而揭示可能的泛素化之间的串扰EGF信号和中心体依赖性的微管重排。这是一个通用的策略，可用于研究其他细胞系统和翻译后修饰的动力学。
Protein ubiquitination is a dynamic reversible post-translational modification that plays a key role in the regulation of numerous cellular processes including signal transduction, endocytosis, cell cycle control, DNA repair and gene transcription. The conjugation of the small protein ubiquitin or chains of ubiquitin molecules of various types and lengths to targeted proteins is known to alter proteins' lifespan, localization and function and to modulate protein interactions. Despite its central importance in various aspects of cellular life and function there are only a limited number of reports investigating ubiquitination on a proteomic scale, mainly due to the inherited complexity and heterogeneity of ubiquitination. We describe here a quantitative proteomics strategy based on the specificity of ubiquitin binding domains (UBDs) and Stable Isotope Labeling by Amino acids in Cell culture (SILAC) for selectively decoding ubiquitination-driven processes involved in the regulation of cellular signaling networks. We applied this approach to characterize the temporal dynamics of ubiquitination events accompanying epidermal growth factor receptor (EGFR) signal transduction. We used recombinant UBDs derived from endocytic adaptor proteins for specific enrichment of ubiquitinated complexes from the EGFR network and subsequent quantitative analyses by high accuracy mass spectrometry. We show that the strategy is suitable for profiling the dynamics of ubiquitination occurring on individual proteins as well as ubiquitination-dependent events in signaling pathways. In addition to a detailed seven time-point profile of EGFR ubiquitination over 30 minutes of ligand stimulation, our data determined prominent involvement of Lysine-63 ubiquitin branching in EGF signaling. Furthermore, we found two centrosomal proteins, PCM1 and Azi1, to form a multi-protein complex with the ubiquitin E3 ligases MIB1 and WWP2 downstream of the EGFR, thereby revealing possible ubiquitination cross-talk between EGF signaling and centrosomal-dependent rearrangements of the microtubules. This is a general strategy that can be utilized to study the dynamics of other cellular systems and post-translational modifications.