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Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism

Ying-Xin Fan1, Lily Wong1, Jinhui Ding2

  • 1Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda Maryland 20892.

The Journal of Biological Chemistry
|November 28, 2007
PubMed

Insights

ErbB2 receptor-tyrosine kinase activity is controlled by autoinhibition. Mutations in the alphaC-beta4 loop disrupt this inhibition, leading to increased ErbB2 activity and potential oncogenic effects in cancer.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Oncology

Background:

  • Protein kinase activity is crucial for cellular signaling and is tightly regulated by autoinhibition.
  • ErbB2 (also known as HER2) is a receptor-tyrosine kinase implicated in various cancers, but its catalytic regulation remains poorly understood.
  • Unlike other ErbB receptors, ErbB2 does not bind ligand directly, suggesting unique regulatory mechanisms.

Purpose of the Study:

  • To elucidate the molecular mechanism of ErbB2 catalytic regulation.
  • To investigate the role of the loop connecting the alphaC helix and beta4 sheet in ErbB2 autoinhibition.
  • To determine the functional consequences of mutations within this regulatory loop.

Main Methods:

  • Site-directed mutagenesis of specific glycine residues (G776, G778) in the alphaC-beta4 loop.
  • Kinetic analysis of wild-type and mutant ErbB2 enzymes.
  • Cell-based assays measuring ErbB2 autophosphorylation, ErbB3 phosphorylation, and downstream signaling (MAPK pathway).
  • Molecular modeling of the ErbB2 kinase domain.

Main Results:

  • ErbB2 kinase is strongly autoinhibited, with the alphaC-beta4 loop playing a key regulatory role.
  • Mutations in the alphaC-beta4 loop (G776S, G778S) significantly increased ErbB2 catalytic activity.
  • Mutational activation resulted in enhanced ATP binding affinity and turnover rate.
  • Expression of activated mutants in cells led to ligand-independent ErbB2 activation, ErbB3 phosphorylation, and MAPK signaling.
  • Molecular modeling indicated stabilization of the inactive state by hydrophobic interactions involving the alphaC-beta4 loop.

Conclusions:

  • The alphaC-beta4 loop acts as an intramolecular switch controlling ErbB2 kinase activity.
  • Disruption of alphaC-beta4 loop-mediated autoinhibition contributes to oncogenic activation of ErbB2.
  • Many cancer-associated ErbB2 mutations are located in this critical regulatory loop, highlighting its significance in ErbB2-driven oncogenesis.

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