Regulation of NIK1 Activation Figure 10. Opposed roles of Thr-469 and Thr-474 phosphorylation sites in NIK-mediated nuclear relocalization of rpL10. Tobacco leaves were co-agroinfiltrated with YFP-L10 and GFP-fusions, as indicated, and the subcellular localization of the fluorescent fusion proteins was monitored by confocal microscopy. The percentage of co-transfected cells containing YFP-L10 fluorescence over the nucleoplasm was registered. Values are the mean6SD of three determinations from independent experiments. In each experiment, a total of 100 to 150 cells were observed. doi:10.1371/journal.pone.0005781.g010 modulates the consequent adaptive response. Here we separated auto- and substrate phosphorylation and identified Thr-474 as targets for intermolecular autophosphorylation and full kinase activity. Conversely, a Thr-469 residue within the A-loop seems to play an inhibitory role in kinase function as its individual replacement with alanine did not impair autophosphorylation but enhanced substrate phosphorylation. The Thr-468 and Thr-474 residues aligned at the same position as Thr-462 and Thr-468 on SERK1 as well as Thr-449 and Thr-455 on SERK3/BAK1, other members of the LRRII-RLK sub-family. Both threonine residues within SERK1 A-loop and BAK1 A-loop have been shown to play relevant roles in kinase function. Thr-468 on SERK1 is absolutely essential for in vitro kinase activity [33] and the equivalent residue on BAK1, Thr-455, has been shown to play a critical role in BAK1 signaling [34]. Our results implicate Thr-468 and Thr-474 on NIK1 as functional analogs of Thr-462 and Thr-468 on SERK1, as well as of Thr-449 and Thr-455 on SERK3/BAK1, respectively. Individual mutations in NIK1 respective residues promote an 80% reduction in kinase autophosphorylation that in turn leads to equivalent decrease in the substrate phosphorylation activity. Phosphorylation of residues within the A-loop constitutes one of the key regulatory mechanisms not only of Ser/Thr kinases but also of Tyr kinases [35,36,37,38,39]. The four lines of evidence presented here demonstrate that the underlying mechanism for Thr-474-dependent kinase activation is due to phosphorylation. Firstly, MALDI-TOF mass spectrometric analyses of tryptic digests of in vitro phosphorylated GST-KDNIK1 and GST-KDT474A revealed that Thr-474 was the phosphory- lated residue on the tryptic phosphopeptide 473GTpVGHIA- PEYLSTGQSSEK491. Secondly, replacing Thr-474 with either alanine or glutamate reduced kinase activity to similar extent, eliminating the possibility that the variation observed might have been due to a possible effect of the newly introduced amino acid residue on structure. Thirdly, the T474A mutant keeps the capacity to be further inhibited by NSP, indicating that replacement of Thr-474 with alanine did not cause a global misfolding of the kinase domain that would impair NSP binding and affect kinase activity. Finally, the introduction of a negatively charged aspartate residue at position 474 did not alter the autophosphorylation activity but enhanced the efficiency of substrate phosphorylation. The findings extend genetic studies linking inactivation of NIK genes to enhanced susceptibility to geminiviruses and protein structural analysis of residues required for kinase activity. Very likely the presence of a correctly positioned carboxylate mimicked the phosphorylation state of Thr-474 resulting in constitutive activation of the receptor kinase. SNF-1 kinases and plant SOS2 (Salt Overlay Sensitive2)-like protein kinases – PSKs have been shown to be highly activated by substitution of phosphoryalated residues with aspartate within the activation loop [40,41,42]. However, the extent by which A-loop phosphorylation-induced kinases are activated by mutations of phosphorylation residues to aspartate or glutamate has been shown to vary considerably among Tyr kinases and Ser/Thr kinases [reviewed in 43]. In the case of NIK1, we further demonstrated that the correct position of the introduced carboxylate within the A-loop was relevant for activity because there was almost no detectable activity with the T474E mutant. The contrasting results of T474D and T47E on NIK1 activity resemble those found for the A-loop mutants of Protein Kinase C, in which the replacement of its critical Thr-500 residue with glutamate increases kinase activity, whereas aspartate in same position inactivates kinase [44]. While the extra methylene in glutamate may be necessary to position the carboxyl group in the correct orientation for electrostatic interactions that could align catalytic residues in the protein kinase C activation loop, it clearly imposes a conformational constraint for NIK1 activation. Similar results have been reported for the SERK1 protein in which the substitution of Thr-468 residue, which is analogous to the NIK1 Thr-474 residue, with glutamate abolishes autophosphorylation activity [33]. Taken together, our results with the Thr-474 mutants of NIK1 are consistent with the current activation model of kinases in which phosphorylation of the A-loop induces conformational changes to position correctly the residues that interact directly with the substrate and the catalytic domain [reviewed in 31]. In addition they implicate the NIK1 residue Thr-474 as the critical phosphorylation site for kinase activation. We also demonstrated that although Thr-469 within the A-loop is not important for autophosphorylation it functionally antago- nizes Thr-474 by playing an inhibitory role in the substrate phosphorylation activity. In fact, replacing Thr-469 with an alanine residue relieves repression and enhances considerably substrate phosphorylation. One possible explanation is that phosphorylation of these residues would antagonize phosphoryla- tion of the relevant Thr-474 residue by blocking the conforma- tional change that permits unrestricted access of ATP and protein substrates to the kinase active site, as that deduced from crystal structures of phosphorylated active kinases [35,36]. The slight decrease in 32P incorporation into T469A upon autophosphoryla- tion may account for loss of a phosphorylation site in the mutant proteins, favoring the argument that Thr-469 is a target of NIK1 PLoS ONE | www.plosone.org 11 June 2009 | Volume 4 | Issue 6 | e5781