Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors.
Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors.
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蛋白质组筛查有丝分裂原激活的蛋白激酶对接基序和相互作用子。
DOI：
10.1126/scisignal.abm5518
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发表时间：
2023-01-10
影响因子：
7.3
通讯作者：
中科院分区：
文献类型：
--
作者：
丝裂原活化蛋白激酶（MAPK）的基本功能取决于它们选择性磷酸化有限底物库的能力。MAPK具有位于催化裂缝外部的保守沟，其结合底物和调节剂中发现的短线性序列基序。然而，这些“对接”相互作用的微弱和短暂性质对定义MAPK相互作用组和相关序列基序构成了挑战。在这里，我们描述了一个基于酵母的遗传筛选管道，以评估并行的MAPK对接序列的大集合。使用这个平台，我们分析了基于MAPK激酶MKK 6和MKK 7的对接序列的组合文库，定义了与应激激活的MAPK JNK 1和p38α结合的关键特征。我们筛选了一个由来自人类蛋白质组的约12，000个序列组成的文库，发现了多种MAPK选择性相互作用物，包括许多不符合先前定义的对接基序的相互作用物。p38α/JNK 1交换突变体的分析鉴定了介导选择性结合的特异性对接沟残基。最后，我们验证了在筛选中鉴定的对接序列在体外和培养细胞中的底物募集中起作用。总之，这些研究建立了一种方法来表征MAPK对接序列，并提供了一个资源，为未来的调查信号下游的p38和JNK。
Essential functions of mitogen-activated protein kinases (MAPKs) depend on their capacity to selectively phosphorylate a limited repertoire of substrates. MAPKs harbor a conserved groove located outside of the catalytic cleft that binds to short linear sequence motifs found in substrates and regulators. However, the weak and transient nature of these “docking” interactions poses a challenge to defining MAPK interactomes and associated sequence motifs. Here, we describe a yeast-based genetic screening pipeline to evaluate large collections of MAPK docking sequences in parallel. Using this platform, we analyzed a combinatorial library based on the docking sequences from the MAPK kinases MKK6 and MKK7, defining features critical for binding to the stress-activated MAPKs JNK1 and p38α. Our screen of a library consisting of ~12,000 sequences from the human proteome revealed multiple MAPK-selective interactors, including many that did not conform to previously defined docking motifs. Analysis of p38α/JNK1 exchange mutants identified specific docking groove residues that mediate selective binding. Finally, we verified that docking sequences identified in the screen functioned in substrate recruitment in vitro and in cultured cells. Together, these studies establish an approach to characterize MAPK docking sequences and provide a resource for future investigation of signaling downstream of p38 and JNK.
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影响因子：
14.8
作者：
Gietz, R. Daniel;Schiestl, Robert H.
通讯作者：
Schiestl, Robert H.
影响因子：
4.8
作者：
Ho, DT;Bardwell, AJ;Bardwell, L
通讯作者：
Bardwell, L
DOI：
10.1073/pnas.1312296111
发表时间：
2014-02-18
影响因子：
11.1
作者：
Ivarsson, Ylva;Arnold, Roland;Kim, Philip M.
通讯作者：
Kim, Philip M.
影响因子：
7
作者：
Kinoshita-Kikuta, Emiko;Aoki, Yuri;Koike, Tohru
通讯作者：
Koike, Tohru
影响因子：
16
作者：
Chang, CI;Xu, BE;Goldsmith, EJ
通讯作者：
Goldsmith, EJ