Regulation of NIK1 Activation Figure 1. Conservation in the LRRII-RLK family and sequence position of mutant NIKs. (A). Sequence alignment of the 29 amino acid activation segment (boxed) of NIKs and other members of the LRRII-RLK family. The activation segment of NIK1 was compared to its counterparts from tomato (LeNIK) and soybean (GmNIK) and to others members of the Arabidopsis LRRII-RLK family, as indicated. Conserved residues as potential phosphorylation sites are shown in bold letters. The corresponding region of IRAK4 (30 amino acids) is shown below for comparison. (B). Schematic representation of the site-directed mutations within the NIK1 A-loop. The mutant kinases are indicated by their names and their respective mutations are indicated in bold letters. The arrows in the NIK1 sequence show mutated amino acid residues and the numbers refer to their respective positions. Boxed sequence delimits the activation segment. (C). Schematic representation of the kinase domain of two truncated forms of NIK1. The solid gray box represents the serine/threonine kinase domain; NBS represents the nucleotide binding site; AS, the active site; AL, the activation loop; NSP BS, the NSP binding site. The numbers correspond to amino acid positions on NIK1 primary structure and asterisks represent the relative position of serine residues. doi:10.1371/journal.pone.0005781.g001 also analyzed by MALDI-TOF mass spectroscopy. The absence of the ion 2010.9504, corresponding to 1930.9504 Da (473GAV- GHIAPEYLSTGQSSEK491) +80.00 Da (see arrow in Figure 3B), is indicative that the mutated peptide was not phosphorylated, confirming that the Thr-474 residue represents the phosphoryla- tion site on wild type peptide 473G-K491. Taken together, these results indicate that phosphorylation of the Thr-474 residue is crucial for NIK activity, as introduction of a correctly positioned carboxylate from aspartate at this position restored autopho- sphorylation activity. In addition, they are suggestive that regulation of kinase activity depends on a phosphorylation- induced conformational change of the A-loop, because a mutant with glutamate (T474E) had only 9.6% residual activity. We interpret this drop as reflecting antagonism of the activation process by imposition of steric constraints to a required conformational change. In support of this model, changing the PLoS ONE | www.plosone.org 3 June 2009 | Volume 4 | Issue 6 | e5781