Lee Thomas, Hoofnagle Andrew N, Kabuyama Yukihito, Stroud James, Min Xiaoshan, Goldsmith Elizabeth J, Chen Lin, Resing Katheryn A, Ahn Natalie G
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA.
Mol Cell. 2004 Apr 9;14(1):43-55. doi: 10.1016/s1097-2765(04)00161-3.
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.
丝裂原活化蛋白激酶（MAP激酶）与底物、激活剂及支架蛋白之间的蛋白质相互作用由对接位点基序调控，一种基序在靠近亮氨酸- X -亮氨酸（DEJL）处含有碱性残基，另一种含有苯丙氨酸- X -苯丙氨酸（DEF）。氢交换质谱法用于鉴定通过对接基序相互作用免受溶剂影响的MAP激酶区域。在p38α和ERK2中跨越β7 - β8和αD - αE的环中观察到DEJL肽结合的保护作用。相比之下，DEF与ERK2结合的保护作用揭示了一个在P + 1位点、αF螺旋和MAP激酶插入片段之间形成的用于苯丙氨酸- X -苯丙氨酸结合的独特疏水口袋。在无活性的ERK2中，该口袋被与激活环中的残基的分子内相互作用所封闭。体外试验证实了Elk1和核孔蛋白结合对ERK2磷酸化的依赖性，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。
