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

• Published in: PloS one Vol. 4; no. 6; p. e5781
• Main Authors: Santos, Anésia A, Carvalho, Claudine M, Florentino, Lilian H, Ramos, Humberto J O, Fontes, Elizabeth P B
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 03.06.2009; Public Library of Science (PLoS)
• Subjects: Abiotic stress; Activation; Alanine - chemistry; Amino Acid Sequence; Amino acids; Antiviral agents; Antiviral Agents - pharmacology; Apoptosis; Arabidopsis; Arabidopsis - metabolism; Arabidopsis Proteins; Arabidopsis thaliana; Begomovirus; Biomedical materials; Cell Nucleus - metabolism; Deoxyribonucleic acid; Disease susceptibility; DNA; Downstream effects; Ectopic expression; Endoplasmic reticulum; Flowers & plants; Geminiviridae - genetics; Genomes; Glycine max; Health aspects; Infection; Infections; Kinases; Molecular Sequence Data; Mutation; Nicotiana - metabolism; Phosphorylation; Plant Biology; Plant Biology/Plant-Biotic Interactions; Plant viruses; Point Mutation; Polymerase chain reaction; Protein Structure, Tertiary; Proteins; Relocation; Residues; Ribosomal Protein L10; Ribosomal Proteins - chemistry; Sequence Homology, Amino Acid; Signal Transduction; Soybeans; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sucrose; Surgical implants; Target recognition; Threonine; Threonine - chemistry; Transgenes; Trends; Virology/Host Antiviral Responses; Virulence; Viruses; Viruses - metabolism; Brazil
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0005781

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.