Regulation of NIK1 Activation Figure 2. Autophosphorylation properties of mutant NIK1 proteins. GST-fusion proteins (as indicated) were incubated with [c-32P]ATP, separated by SDS-PAGE and stained with Coomassie-brilliant blue. Phosphorylated proteins were visualized by autoradiography (A). The coomassie- stained gel (B) was used to normalize protein loading with Multi Gauge V3.0 (Fujifilm) software. The radioactivity incorporated into proteins was quantified by phosphoimaging (C). The relative kinase activity was expressed as Vunits/min/mg protein. Values are given as mean6S.D. of three determinations. doi:10.1371/journal.pone.0005781.g002 conserved Gly-473 residue in the T474A mutant to valine (G473V/T474A) totally abolished autophosphorylation activity and indicated that removing a strong structural constraint by substituting the flexible Gly-473 prevented the A-loop from adopting an active conformation. In contrast, mutation at Thr- 469 caused a much lower impact on autophosphorylation activity of the resulting mutant kinase (10.96103 cpm/mg/min compared to 13.76103; Figure 2C, lane 6) and like T474D, may reflect reduction of the incorporated radioactivity without alteration of the autophosphorylation activity of the protein. In fact, using MALDI-TOF mass spectrometric analyses we detected a mono- phosphorylated form (peak at m/z of 1671.74) of the peptide 459LLDHQDSHVTTAVR472 (peak of m/z 1591.75, Figure 4A). MALDI-TOF/TOF analysis of the respective peptides was further used to examine the phosphorylation site on the monopho- sphorylated form. In Figure 4B, the MS/MS spectrum of the non- phosphorylated 459L-R472 peptide is shown. Phosphorylated peptides were confirmed by identification of a strong signal ([M+H] 1573,74) corresponding to the mass difference of 298.0 Da from the neutral loss of phosphoric acid (H3PO4), as indicated in Figure 4C. Phosphorylation at Thr-469 was confirmed by the presence of the ion b11 [M+H] 1328.10 that corresponds to the fragment LLDHQDSVTT-PO3. To confirm the regulatory role of the A-loop phosphorylation, we made double mutations at T474A and the conserved putative phosphorylation sites, Ser-465, Thr-468 and Thr-469, as indicated in Figure 1B. All double mutants exhibited reduced autopho- sphorylation activity which varied from 34% to 40% of the normal NIK1 activity (Figure 2C). Although we did not obtain a Thr-468 single mutation, the substitution of this residue to alanine in the T469A mutant (T468A/T469A) caused a strong reduction in autophosphorylation down to 34% activity (4.76103 cpm/min/ mg, lane 7) as compared to the normal kinase, suggesting that Thr- 468 also plays a relevant role in the kinase activation. NIK1 kinase inhibition by the geminivirus NSP Previous work had indicated that the NSP binding site on NIK overlaps two kinase sub domains VIb and VIII [18]. This observation raised the possibility that NSP binding to NIK might prevent A-loop transphosphorylation and hence kinase activation (Figure 1C). Inclusion of GST-NSP fusion in phosphorylation assays in vitro promoted a 50% reduction on NIK1 autopho- sphorylation (Figure 5). Except for the T474D mutant, NSP effectively caused a further 50–70% reduction in the residual activity of the mutant kinases independently of the mutation in the conserved residues of NIK1. The capacity of NSP to inhibit the mutant proteins indicates that any effect of the single and double amino acid replacements on NIK structure was too small to impair NSP binding (Figure 1C). Thus, the reduction in kinase activity by Thr replacements within the A-loop may be due to the lack of phosphorylation sites rather than a global misfolding of the kinase domain upon introduction of a different amino acid residue. In contrast to the alanine-substituted NIK A-loop mutants, the T474D mutant showed an attenuated effect of NSP on inhibition of autophosphorylation with only a 20% reduction in kinase activity in the presence of NSP. The lesser effect of NSP on inhibition of the activity of the T474D mutant could be due to a conformational change leading to destabilization of the NSP-NIK complex when PLoS ONE | www.plosone.org 4 June 2009 | Volume 4 | Issue 6 | e5781