Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, USA.
J Biol Chem. 2011 Jan 28;286(4):2477-85. doi: 10.1074/jbc.M110.177899. Epub 2010 Nov 22.
Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 μM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.
细胞外信号调节激酶 1 和 2（ERK1/2）蛋白通过与底物蛋白相互作用并使其磷酸化来调节各种细胞功能，包括细胞增殖和分化。ERK 蛋白上已经鉴定出两个对接位点，即常见对接（CD/ED）结构域和 F 位点募集位点（FRS）。ERK 和 JNK 的底物与 ERK 蛋白上的 CD/ED 结构域和 FRS 特异性相互作用，其分别含有 ERK 和 JNK 的对接位点、LXL（DEJL）基序（D 结构域）和 ERK 的对接位点、FXF（DEF）基序（F 结构域）。然而，ERK 对接位点在介导允许有效磷酸转移的底物相互作用中的相对贡献在很大程度上仍是未知的。在这些研究中，我们使用表面等离子体共振来测量实时蛋白质-蛋白质相互作用，对 ERK2 与底物的相互作用进行了定量分析。ERK2 与 ELK-1（DEF 和 DEJL 基序）、RSK-1（DEJL 基序）和 c-Fos（DEF 基序）的相互作用的 K(D)值分别为 0.25、0.15 和 0.97 μM。CD/ED 结构域突变使与 ELK-1 和 RSK-1 的相互作用抑制了 6 倍，但对与 c-Fos 的相互作用没有影响。FRS 残基的选择性突变以不同的方式抑制 ELK-1 或 c-Fos 与 ERK2 的相互作用，但对 RSK-1 相互作用的影响很小。ED 和 FRS 对接位点的突变完全抑制了 ELK-1 的相互作用，但对其未知对接位点的 ERK 底物 stathmin 的相互作用没有影响。ERK2 的磷酸化状态不影响与 RSK-1 或 c-Fos 的相互作用，但确实抑制了与 ELK-1 和 stathmin 的相互作用。这些研究对参与介导 ERK2 与蛋白质底物之间相互作用的特定对接结构域进行了定量评估，并定义了这些相互作用对磷酸转移的贡献。
