title: Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, USA.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Burkhard
authors.forename: Fengming
authors.initials: F
authors.lastname: Chen
authors.forename: Paul
authors.initials: P
authors.lastname: Shapiro
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050777:Stathmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods; D050807:ets-Domain Protein Elk-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C494112:ELK1 protein, human; D011994:Recombinant Proteins; C493995:STMN1 protein, human; D050777:Stathmin; D050807:ets-Domain Protein Elk-1; C069329:RPS6KA1 protein, human; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1