PMID:19492062

Citation	Santos, AA, Carvalho, CM, Florentino, LH, Ramos, HJ and Fontes, EP (2009) Conserved threonine residues within the A-loop of the receptor NIK differentially regulate the kinase function required for antiviral signaling. PLoS ONE 4:e5781
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.
Links	PubMed PMC2686266 Online version:10.1371/journal.pone.0005781
Keywords	Alanine/chemistry; Amino Acid Sequence; Antiviral Agents/pharmacology; Arabidopsis/metabolism; Cell Nucleus/metabolism; Geminiviridae/genetics; Molecular Sequence Data; Mutation; Phosphorylation; Point Mutation; Protein Structure, Tertiary; Ribosomal Proteins/chemistry; Sequence Homology, Amino Acid; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Threonine/chemistry; Tobacco/metabolism; Viruses/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ARATH:NIK1	acts_upstream_of_or_within	GO:0051607: defense response to virus	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete

References

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