Regulation of NIK1 Activation Figure 4. MS/MS analyses of GST-KDNIK1 reveal phosphorylation at Thr-467. (A). MALDI-TOF MS spectrum of GST-KDNIK1-derived tryptic fragments showing the ion ([M+H] 1671.74) and a mass shift of 80 Da as an indicative of phosphorylation on peptide 459LLDHQDSHVTTAVR472 ([M+H] 1591.75). (B) and (C). MS/MS spectra of the ions ([M+H] 1591.701) and ([M+H] 1671.70) that correspond to the peptides LLDHQDSVTTAVR and LLDHQDSVTTpAVR, respectively. doi:10.1371/journal.pone.0005781.g004 Figure 5. Inhibition of NIK1 autophosphorylation activity by NSP. GST-fusions (as indicated) were incubated with [c-32P]ATP in the presence and absence of GST-NSP. (A) SDS-PAGE of proteins from the reaction of autophosphorylation. Proteins added to the autopho- sphorylation reactions were analyzed by coomassie-stained SDS-PAGE and further quantified by densitometric scanning to normalize protein levels. (B) Fold variation on autophosphorylation activity in the presence of mutated NIKs. Phosphorylated proteins in paired reactions +/2 GST-NSP were quantified by scintillation counting of excised protein bands. NSP inhibition is expressed as the fold decrease of autophosphorylation activity in the presence of the viral protein. doi:10.1371/journal.pone.0005781.g005 (Figure 1C). Each deletion decreased autophosphorylation activity to the same extent and totally abolished substrate phosphorylation (Figure 7, lanes 6 and 7). These results indicate that the C-terminal fragment, delimited by positions 609 to 638, may exhibit a regulatory role. Inspection of this region revealed the presence of three serine residues at positions 613, 615 and 619 with the potential to be substrates for serine/threonine kinases. Like other receptor-like serine/threonine kinases, NIK1 may possess several other autophosphorylation sites that represent docking sites for substrate recruitment or play distinct regulatory function. Although the biological relevance of this region on kinase function remains to be determined as it extends beyond the scope of the present investigation, these preliminary experiments suggest that NIK1 shares with receptor-like serine/threonine kinases the same complex mechanism of autophosphorylation-dependent activation of kinase function. Thr-474 and Thr-469 exhibit antagonistic roles in regulation of NIK-mediated antiviral signaling Assignment of NIK as a transmembrane signaling receptor that mediates an antiviral defense response has been based on the biochemical properties of the kinase in vitro, its inhibition by the geminivirus NSP and the enhanced susceptibility phenotype to geminivirus infection of knockout lines [18]. To determine whether the NIK-mediated antiviral signaling is indeed transduced through the intracellular kinase domain in vivo, functional complementation assays were performed by expressing the NIK1 wild type protein or a mutant NIK1 harboring an inactive kinase domain (G473A/T474A) in nik1 alleles. We also included the mutants T474A and T469A in the complementation assays to dissect the functional significance of these mutations in vivo. The transformed plants were confirmed by PCR (data not shown) and the transgene expression was monitored by real time RT-PCR for a transgenic line ectopically expressing the wild type protein (NIK*), two independently transformed KO lines Figure 6. NSP interacts with T474D in vitro. Bacterially produced His-NSP (lane 4) was allowed to interact with GST-KDNIK1 (lane 1), GST-T474D (lane 2) or GST (lane 3) previously linked to glutathione-Sepharose beads. The retained proteins were analyzed by Coomassie-stained SDS-PAGE. Lane 6 is the result of a GST-T474D-driven pull down of NSP. GST-KDNIK1 (lane 5) and the control GST (lane 7) were also used to drive NSP pull down on glutathione-Sepharose beads protein. doi:10.1371/journal.pone.0005781.g006 PLoS ONE | www.plosone.org 7 June 2009 | Volume 4 | Issue 6 | e5781