Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry
Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry
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DOI：
10.1016/s1097-2765(04)00161-3
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发表时间：
2004-04-09
期刊：
影响因子：
16
通讯作者：
Ahn, NG
中科院分区：
文献类型：
--
作者：
Lee, T;Hoofnagle, AN;Ahn, NG
MAP激酶与底物、激活剂和支架蛋白之间的蛋白质相互作用受对接位点基序的调节，一个基序含有Leu-X-Leu(DEJL)附近的碱性残基，另一个含有Phe-X-Phe(DEF)。氢交换质谱仪被用来鉴定MAP激酶中通过对接基序相互作用而不受溶剂保护的区域。在p38α和ERK2中跨越7-β8和αD-αE的环上观察到DEJL多肽结合的保护作用。相反，DEF与ERK2的结合保护揭示了在P+1位点、αF螺旋和MAP激酶插入之间形成的Phe-X-Phe结合的明显疏水口袋。在非活性ERK2中，这一口袋被与激活LIP中残基的分子内相互作用所阻断。体外实验证实ELK1和核孔蛋白结合依赖于ERK2的磷酸化，并为活性ERK优先参与底物结合和核孔蛋白相互作用提供了结构基础。
Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.