Regulation of NIK1 Activation Figure 3. MALDI-TOF analysis of the phosphorylation state of Thr-474 in GST-KDNIK1. Purified GST-KDNIK1 (A) or GST-KDT474A (B) was autophosphorylated in vitro, separated by SDS-PAGE and visualized by Coomassie Blue staining. The stained bands were excised from the gel and subjected to trypsin digestion, and the resulting fragments were analyzed by MALDI-TOF mass spectroscopy. In A, among the detected molecules, peaks with m/z values of 1960.854 and 2040.870 were respectively attributed to the non- and mono-phosphorylated forms of the peptide 473GTVGHIAPEYLSTGQSSEK491. In B, the peak with m/z value of 1930.9504 corresponds to the mutated peptide 473GAVGHIAPEYLSTGQSSEK491 and the arrow indicates the absence of the predicted mono-phosphorylated form (m/z = 2010.9504) of the mutated peptide. Peptide mass was calculated in silico from mutated and wild-type GST-KDNIK1 sequences, using PeptideMass software (www.expasy.ch/). MALDI-TOF spectrum was analyzed using FlexAnalisys package (Bruker Daltonics). Only the regions of the spectra containing the peptides of interest are shown. doi:10.1371/journal.pone.0005781.g003 Thr-474 was replaced with aspartate. To address this issue we produced NSP and T474D in E. coli with different tagging systems and monitored the interaction between the recombinant proteins was monitored by pull down assays (Figure 6). Bacterially expressed His- tagged NSP (lane 4) was incubated with GST-tagged NIK (lane 1), GST-T474D (lane 2) or GST alone (lane 3) and the resulting complexes were isolated on glutathione Sepharose beads. His-tagged NSP bound to GST-NIK (lane 5) and GST-T474D (lane 6) but not to GST alone (lane 7), confirming that NSP binds T474D in vitro. More likely, replacing Thr-474 with aspartate bypassed the inhibitory effect of NSP on Thr phosphorylation at this position. This hypothesis is consistent with an inhibitory mechanism in which the NSP-NIK complex formation modulates the phosphorylation state of the A-loop and thus kinase activation. Conserved threonine residues within the A-loop play distinct roles in kinase function The ribosomal protein L10 has been shown to be a substrate of NIK1 that functions as the immediate downstream component of the NIK-mediated antiviral signaling [21,22]. Inclusion of the recombi- nant GST-L10 fusion in the kinase assay demonstrated that the ribosomal protein was efficiently phosphorylated by NIK1 (Figure 7). There was no measurable 32P incorporation into GST when GST alone was incubated with KDNIK1 and [c32P]ATP (data not shown; Fontes et al., 2004). We also monitored the effect of the point mutations on the substrate phosphorylation activity of NIK1 (Figure 7). In general, mutations that negatively affect the autopho- sphorylation activity of the kinase protein, such as T474A, T474E, G473V/T474A, T474A/T468A, also reduced the capacity of the kinase to phosphorylate the substrate rpL10 (Figures 7B and 7C). These results are consistent with the current model for Ser/Thr kinase regulation in which autophosphorylation promotes kinase activation and precedes substrate phosphorylation [35,36]. In contrast, replacing the essential Thr-474 residue with aspartate caused a 28% reduction in autophosphorylation activity (Figures 2 and 7B, lane 3), but promoted a 50% increase (up to 150% activity) in the substrate phosphorylation activity [Figure 7C, compare lane 1 (0.66104 cpm/mg ptn/min/mg substrate) with lane 3 (0.96104 cpm/ mg ptn/min/mg substrate)]. This observation suggests that introduc- tion of a correctly positioned carboxylate at position 474 causes constitutive activation of the protein by mimicking the induced phosphorylation of Thr-474. In addition, it indicates that phosphor- ylation of Thr-474 is a determinant of kinase activation. Likewise, the T469A mutant caused about 40% decrease in autophosphorylation, but exhibited a 3-fold higher substrate phosphorylation (1.836104 cpm/mg ptn/min/mg substrate) than that of the wild type kinase (Figure 7C, compare lanes 8 and 1). Very likely phosphor- ylation at Thr-469 inhibits substrate phosphorylation, as mutation of this residue to alanine relieves repression and enhances the capacity of the enzyme to phosphorylate rpL10. This inhibitory effect of Thr-469 was further evidenced in T468A/T469A (lane 9) and T474A/T469A (lane 11) double mutants, which exhibited enhanced substrate phosphorylation in comparison with NIK1, in spite of harboring mutations in the relevant Thr-474 or Thr-468 residues for autopho- sphorylation (Figures 7B and 7C). Similarly, phosphorylation at Ser- 465 may inhibit substrate phosphorylation, because mutation at this residue together with mutation at the essential kinase activation residue Thr-474 resulted in a double mutant with reduced autopho- sphorylation but enhanced substrate phosphorylation activity (Figure 7, lane 12). In addition to analyzing the A-loop, we analyzed the C-terminal region by introducing a stop codon at position 609 or 588 PLoS ONE | www.plosone.org 5 June 2009 | Volume 4 | Issue 6 | e5781