Cited by 63Protein Kinase Regulation and GTPase SignalingMelanoma and MAPK PathwaysProtein Tyrosine Phosphatases

Quantitative Analysis of ERK2 Interactions with Substrate Proteins

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Burkhard, Kimberly A., Chen, Fengming, Shapiro, Paul·2011·Journal of Biological Chemistry

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.

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Crossref · 10.1074/jbc.m110.177899

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Authors
Burkhard, K. A., Chen, F., & Shapiro, P.
Affiliations
University of Maryland, Baltimore (US)
Corresponding author
Shapiro, Paul
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2011-01
Volume
286
Issue
4
Pages
2477-2485
ISSN
0021-9258 (ISSN-L)
OpenAlex
W1980331527
Cited by
62
References
57
Citations by year
2026: 3 · 2025: 4 · 2024: 2 · 2023: 4 · 2022: 5 · 2021: 1 · 2020: 4 · 2019: 5 · 2018: 3 · 2017: 2 · 2016: 1 · 2015: 6 · 2014: 6 · 2013: 3 · 2012: 9
FWCI
2.08
Type
journal article
Language
en
Open access
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
National Cancer Institute (R01CA120215, CA120215)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Melanoma and MAPK Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
Keywords
ERK2, protein-protein interaction, docking site, Elk-1, c-fos, stathmin, substrate phosphorylation, MAPK signaling, binding affinity
MeSH terms
Binding Sites, Humans, Mutation, Phosphorylation, Recombinant Proteins, Mitogen-Activated Protein Kinase 1, Surface Plasmon Resonance, Amino Acid Motifs, Ribosomal Protein S6 Kinases, 90-kDa, Stathmin, ets-Domain Protein Elk-1

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