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Constitutive activation of extracellular signal-regulated kinase 2 by synergistic point mutations

M A Emrick1, A N Hoofnagle, A S Miller

  • 1Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.

The Journal of Biological Chemistry
|October 10, 2001
PubMed

Insights

Mutant forms of ERK2 (extracellular signal-regulated kinase 2) exhibit significantly enhanced activity through synergistic mutations, primarily via autophosphorylation. These findings reveal novel insights into ERK2 activation mechanisms and cellular signaling pathways.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Enzymology

Background:

  • Constitutively active mutant signaling enzymes are valuable tools for studying activation mechanisms and downstream effects.
  • Understanding the regulation of ERK2 (extracellular signal-regulated kinase 2) is crucial for deciphering cellular signaling pathways.

Purpose of the Study:

  • To investigate the effects of specific point mutations (L73P and S151D) on ERK2 activity and activation mechanisms.
  • To elucidate the role of intramolecular autophosphorylation in ERK2 activation.
  • To assess the in vivo functionality of active ERK2 mutants.

Main Methods:

  • Site-directed mutagenesis to create L73P and S151D ERK2 mutants.
  • Biochemical assays (specific activity measurements, phosphatase sensitivity) and Western blotting to assess ERK2 activity.
  • Mass spectrometry to identify phosphorylation sites.
  • Molecular dynamics simulations to explore structural mechanisms.
  • Cell-based assays to evaluate downstream target phosphorylation and gene promoter activation.

Main Results:

  • Mutations L73P and S151D individually increased ERK2 specific activity by 8-12 fold; combined mutations yielded a 50-fold increase, indicating synergistic interactions.
  • Mutations enhanced ERK2 activity by promoting intramolecular autophosphorylation, primarily at Tyr-185 and secondarily at Thr-183; both sites are required for activation.
  • Molecular dynamics simulations suggested hydrogen bonding interactions in the active mutant that could facilitate autophosphorylation.
  • In cells, ERK2 mutants phosphorylated Elk-1 and RSK1 and activated the c-fos promoter.
  • Inhibition of MKK1/2 with U0126 only partially reduced mutant ERK2 activity, suggesting significant autophosphorylation-driven activation in vivo.

Conclusions:

  • Specific mutations at L73 and S151 synergistically enhance ERK2 activity through increased intramolecular autophosphorylation.
  • ERK2 activation in vivo involves both MKK1/2-dependent phosphorylation and a substantial autophosphorylation mechanism.
  • These findings provide novel insights into the regulation of ERK2 signaling and offer tools for further research.

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