Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a Tripartite Protein System
Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a Tripartite Protein System
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DOI：
10.1021/acscentsci.6b00053
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发表时间：
2016-07-27
影响因子：
18.2
通讯作者：
Heck, Albert J. R.
中科院分区：
文献类型：
--
作者：
Loessl, Philip;Brunner, Andrea M.;Heck, Albert J. R.
多位点磷酸化是体内调节蛋白质功能、活性和相互作用模式的常见途径，但常规生化分析往往不足以确定单个磷酸化反应的数量和顺序及其对蛋白质行为的机制影响。在这里，我们整合互补质谱（MS）为基础的方法来表征多位点磷酸化调节的蛋白质系统，包括Polo样激酶1（Plk1）及其共激活剂极光激酶A（Aur-A）和Bora，其中的相互作用是必不可少的DNA损伤诱导的细胞周期停滞后有丝分裂进入。天然MS和交联-MS显示，Aur-A/Bora介导的Plk1激活伴随着Aur-A/Bora和Plk1/Bora异二聚体的形成。我们发现Aur-A/Bora相互作用独立于Bora磷酸化状态，而Plk1/Bora相互作用依赖于广泛的Bora多位点磷酸化。自下而上和自上而下的蛋白质组学分析表明，博拉多位点磷酸化的收益通过一个有序的序列的位点特异性磷酸化反应，从而我们可以揭示多达16个磷酸化博拉残基的参与。离子迁移谱-MS表明，这种多位点磷酸化引发了Bora的大量结构重排，解释了广泛的Bora多位点磷酸化和Plk1/Bora复合物形成之间的相互依赖性。这些结果代表了我们多管齐下的MS策略的第一个基准，突出了其阐明多位点蛋白磷酸化的机制和结构影响的潜力。
Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking-MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry-MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation.