Conserved threonine residues within the A-loop of the receptor NIK differentially regulate the kinase function required for antiviral signaling

PLoS One. 2009 Jun 3;4(6):e5781. doi: 10.1371/journal.pone.0005781.

Abstract

NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry
  • Amino Acid Sequence
  • Antiviral Agents / pharmacology*
  • Arabidopsis / metabolism
  • Arabidopsis Proteins
  • Cell Nucleus / metabolism
  • Geminiviridae / genetics
  • Molecular Sequence Data
  • Mutation
  • Phosphorylation
  • Point Mutation
  • Protein Structure, Tertiary
  • Ribosomal Protein L10
  • Ribosomal Proteins / chemistry
  • Sequence Homology, Amino Acid
  • Signal Transduction
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Threonine / chemistry*
  • Tobacco / metabolism
  • Viruses / metabolism

Substances

  • Antiviral Agents
  • Arabidopsis Proteins
  • Ribosomal Proteins
  • ribosomal protein L10, Arabidopsis
  • Threonine
  • Alanine