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Spatial and Temporal Analysis of Active ERK in the C. elegans Germline
08:40

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Published on: November 29, 2016

The gatekeeper residue controls autoactivation of ERK2 via a pathway of intramolecular connectivity

Michelle A Emrick1, Thomas Lee, Paul J Starkey

  • 1Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309, USA.

Proceedings of the National Academy of Sciences of the United States of America
|November 23, 2006
PubMed

Insights

Mutations in the gatekeeper residue of MAP kinase ERK2 unexpectedly cause autoactivation. This occurs via an intramolecular mechanism involving an N-terminal hydrophobic cluster that maintains the kinase in an inactive state.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Protein kinases play crucial roles in cellular signaling pathways.
  • The gatekeeper residue is known to confer selectivity for nucleotide and inhibitor binding in kinases.
  • ERK2 is a key mitogen-activated protein kinase involved in cell proliferation and differentiation.

Purpose of the Study:

  • To investigate the role of the gatekeeper residue in the regulation of MAP kinase ERK2 activity.
  • To elucidate the mechanism by which mutations at the gatekeeper site lead to kinase autoactivation.
  • To identify novel structural elements involved in maintaining ERK2 in an inactive state.

Main Methods:

  • Site-directed mutagenesis of the gatekeeper residue and other interacting residues in ERK2.
  • Biochemical assays to measure kinase activity and autophosphorylation.
  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe protein dynamics.
  • Structural modeling to predict communication pathways within the kinase.

Main Results:

  • Mutations at the ERK2 gatekeeper residue unexpectedly resulted in kinase autoactivation.
  • Autoactivation was attributed to enhanced autophosphorylation of regulatory sites on the activation lip via an intramolecular mechanism.
  • Mutations in an N-terminal hydrophobic cluster, including the gatekeeper, also caused autoactivation.
  • HDX-MS studies revealed perturbations in the DFG motif, suggesting a communication route from the hydrophobic cluster to the activation lip.

Conclusions:

  • The gatekeeper residue indirectly constrains the flexibility of the activation lip, preventing premature autophosphorylation.
  • An N-terminal hydrophobic cluster, including the gatekeeper, forms a novel structural unit that maintains ERK2 in an inactive 'off' state.
  • This structural unit is critical for regulating ERK2 activity in response to cellular signals.

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