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Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in

Kimberly A Burkhard1, Fengming Chen, Paul Shapiro

  • 1Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, USA.

The Journal of Biological Chemistry
|November 25, 2010
PubMed

Insights

Extracellular signal-regulated kinases (ERK1/2) use docking sites to interact with substrates. This study quantifies how mutations in these docking sites affect ERK2 interactions and phosphorylation of substrates like ELK-1 and c-Fos.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Protein Kinase Research

Background:

  • Extracellular signal-regulated kinases (ERK1/2) are crucial regulators of cellular processes.
  • ERK proteins possess distinct docking sites: the common docking (CD/ED) domain and the F-site recruitment site (FRS).
  • Substrates interact via specific motifs (DEJL/D-domain and FXF/F-site), but the role of each docking site in substrate interaction and phosphorylation is unclear.

Purpose of the Study:

  • To quantitatively analyze the interactions between ERK2 and its protein substrates.
  • To determine the specific contributions of the CD/ED domain and FRS in mediating these interactions.
  • To evaluate the impact of these interactions on efficient phosphate transfer.

Main Methods:

  • Surface plasmon resonance (SPR) was employed for real-time measurement of protein-protein interactions.
  • Site-directed mutagenesis was used to alter specific residues within the CD/ED and FRS docking sites of ERK2.
  • Interaction affinities (K(D) values) and effects of mutations on substrate binding and phosphorylation were assessed.

Main Results:

  • ERK2 exhibited high-affinity interactions with substrates ELK-1, RSK-1, and c-Fos.
  • Mutations in the CD/ED domain significantly impaired interactions with ELK-1 and RSK-1 but not c-Fos.
  • FRS mutations differentially affected ELK-1 and c-Fos binding, with minimal impact on RSK-1; combined mutations abolished ELK-1 interaction.

Conclusions:

  • The CD/ED and FRS docking sites play distinct, crucial roles in mediating specific ERK2-substrate interactions.
  • The phosphorylation status of ERK2 influences interactions with certain substrates, such as ELK-1 and stathmin.
  • This quantitative analysis provides a detailed understanding of ERK2 docking mechanisms and their role in substrate recognition and signaling.

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