Structural Studies of ERK2 Protein Complexes

  • Johannes F. Weijman
  • Stefan J. Riedl
  • Peter D. Mace
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1487)

Abstract

ERK1 and ERK2 (ERK1/2) are the primary effector kinases of the RAS-RAF-MEK-ERK signaling pathway. A variety of substrates and regulatory partners associate with ERK1/2 through distinct D-peptide- and DEF-docking sites on their kinase domains. While understanding of D-peptides that bind to ERK1/2 has become increasingly clear over the last decade, only more recently have structures of proteins interacting with other binding sites on ERK1/2 become available. PEA-15 is a 130-residue ERK1/2 regulator that engages both the D-peptide- and DEF-docking sites of ERK kinases, and directly sequesters the ERK2 activation loop in various different phosphorylation states. Here we describe the methods used to derive crystallization-grade complexes of ERK2-PEA-15, which may also be adapted for other regulators that associate with the activation loop of ERK1/2.

Key words

ERK2 ERK1 MAPK PEA-15 D-Peptide DEJL DEF-docking Crystallization Activation loop 

Notes

Acknowledgements

This work was supported by NIH grants R01AA017238, 1R01CA160457 (to S.J.R.), and a DOD-BCRP Fellowship BC100466 to P.D.M. P.D.M. and J.F.W. are currently supported by a Rutherford Discovery Fellowship from the New Zealand government administered by the Royal Society of New Zealand (to P.D.M.). The original pET-LIC vector used in this work was kindly gifted by the Netherlands Cancer Institute (NKI) Protein Facility with funding from grant no. 175.010.2007.012.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Johannes F. Weijman
    • 1
  • Stefan J. Riedl
    • 2
  • Peter D. Mace
    • 1
  1. 1.Biochemistry Department, Otago School of Medical SciencesUniversity of OtagoDunedinNew Zealand
  2. 2.Cell Death and Survival Networks ProgramSanford Burnham Prebys Medical Discovery InstituteLa JollaUSA

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