Quantitative analysis of ERK2 interactions with substrate proteins

The extracellular signal‐regulated kinase‐1 and 2 (ERK1/2) proteins are serine/threonine kinases that play a significant role in cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. The protein‐protein interactions of ERK with its substrate proteins is governed by at least two docking sites, the common docking (CD/ED) domain and the F‐site recruitment site (FRS) located on ERK. Substrate proteins interact with ERK docking domains, CD/ED and FRS through a DEJL motif (CD/ED) and a DEF motif (F‐site), respectively. Quantitative analysis of the contributions of the ERK docking sites in mediating substrate interactions allowing for efficient phosphate transfer are largely unknown. The current studies provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance (SPR) and correlate these binding interactions with phosphoryl transfer. ERK2 interacted with ELK‐1(DEF and DEJL motif), RSK‐1 (DEJL motif), and c‐Fos (DEF motif) with K D 's 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. Mutations in ERK's FRS inhibited ELK‐1 interactions by 20 fold with little effect on RSK‐1 or c‐Fos. Mutations in both the CD/ED and FRS docking sites completely inhibited ELK‐1 interactions. Interactions with Stathmin, an ERK substrate whose docking site is unknown, were unchanged with mutations in CD/ED and FRS residues. Phosphorylated ERK2 did not affect interactions with RSK‐1 and c‐Fos but did inhibit interactions with ELK‐1 and Stathmin. The current studies provide a quantitative evaluation of interactions between ERK2 and protein substrates by SPR, allowing for the determination of the contributions of ERK's CD/ED domain and FRS in mediating protein interactions and catalysis.