interacting kinase 1, NIK1 (At5g16000), NIK2 (At3g25560) and NIK3 (At1g60800) are inserted into the defense group I of the LRRII-RLK sub-family [17] and they have been initially identified as virulence targets of the bipartite geminivirus nuclear shuttle protein, NSP [18]. The NSP-NIK interaction is conserved among geminivirus NSPs and NIK homologs from different hosts. Tomato and soybean NIK homologs also interact stably with NSP from CaLCuV (Cabbage leaf curl virus) and from the tomato-infecting geminiviruses TGMV (Tomato golden mosaic virus) and TCrLYV [Tomato crinkle leaf yellows virus; 18,19,20]. The assignment of NIK as a defense receptor gene was based primarily on the enhanced susceptibility phenotype to geminivirus infection displayed by nik knockout lines [18]. Recent progress towards elucidating the NIK-mediated antiviral signaling includes the identification of the ribosomal protein L10 (rpL10) as the immediate downstream effector in the pathway [21,22]. Phos- phorylation of rpL10 by NIK promotes translocation of the ribosomal protein to the nucleus where it may function to mount a defense response that negatively impacts virus infection. The bipartite geminivirus NSP suppresses NIK activity through specific binding to the kinase domain and hence enhances geminivirus pathogenicity. Thus, upon geminivirus infection, rpL10 is trapped within the cytoplasm to prevent the establishment of a host environment that disfavors virus proliferation and/or spread. Geminiviruses are considered one of the largest and most successful groups of plant viruses that infect a wide range of crops, particularly in tropical and subtropical regions. These plant viruses are characterized by single-stranded circular DNA genomes that are encapsidated in geminate semi-icosahedral particles and replicate via a double-stranded DNA intermediate in the nuclei of infected cells [for review see 23]. Their genome may be organized in either single or double-component configuration. Typically, both genomic components of bipartite geminiviruses (begomoviruses), designated DNA-A and DNA-B, are required for systemic infection. DNA-A encodes proteins involved in DNA replication (Rep and REn), transcriptional activation of viral genes (TrAP), encapsidation of the viral genome (CP) and suppression of RNAi defense functions [AC4 and TrAP; 24,25]. The genes on DNA-B (NSP and MP) provide functions required for systemic movement of the viral genome within infected plants [reviewed in 23,26,27]. The nuclear shuttle protein (NSP) facilitates the intracellular trafficking of viral DNA between the nucleus and the cytoplasm, whereas MP acts as the classic viral movement protein and potentiates the cell-to-cell movement of viral DNA. In addition to interacting with host factors required for basic compatibility functions [28,29,30], NSP from CaLCuV has been demonstrated to act as a virulence factor to suppress the kinase activity of transmembrane receptor NIKs [18]. The NSP binding region of NIK1 was mapped to an 80-amino acid fragment that encompasses the active loop (A-loop) and the active site of the kinase [18]. A-loops of tyrosine and serine/threonine kinases play an essential role in controlling kinase activity through the phosphorylation status of conserved tyrosine or threonine residues within this domain [reviewed in 31]. In addition to being inhibited by the viral NSP, NIKs are presumed to be involved in antiviral defense responses based on our studies showing that loss of NIK function in Arabidopsis is linked to an enhanced susceptibility phenotype to CaLCuV infection [18,21]. Nevertheless, it remains to be determined whether the NIK kinase domain is involved in transducing a defense signal as would be expected from a receptor- like kinase-mediated signaling event. Here we further examined the NIK biochemical properties to elucidate the mechanism of receptor activation as well as its inhibition by viral NSP. We show that NIK requires kinase activation to mediate an antiviral Regulation of NIK1 Activation signaling and thus is an authentic defense receptor. In addition, our data also substantiate the notion that regulated nucleocyto- plasmic trafficking of rpL10 links the antiviral response to receptor activation. Results Mutation at threonine residues within the A-loop of NIK1 impacts autophosphorylation Activation of many kinases requires phosphorylation of the activation segment that is defined by the region delimited by two conserved tripeptide motifs, DFG and APE [31]; Figure 1A. Conserved secondary elements in this segment include the magnesium binding loop, b9, at the N-terminus, the centrally located activation loop (A-loop) and the P+1 loop at the C- terminus. The activation segment is highly conserved among members of the Arabidopsis LRRII-RLK sub-family and NIK counterparts from other plant species, such as tomato (LeNIK) and soybean (GmNIK) (Figure 1A). In the case of SERK1, the conserved Thr-462 and Thr-468 residues within the A-loop have been shown to be intermolecular targets of SERK1 kinase activity in vitro [32]. Likewise, the conserved SERK3/BAK1residues Thr- 446, Thr-449 and Thr-455 have been shown to be phosphorylated in vitro as well as in vivo in response to brassinosteroid signaling [33]. Conservation of more distantly related kinases can also be seen by computer-assisted threading of the amino acid sequence of NIK1 onto the Ser/Thr kinase 2oidB (Interleukin 1 receptor associated kinase 4, IRAK4) as a template [34]. A striking feature is the almost perfect overlay of the activation segments of the two proteins which correspond to regions that have been shown to regulate kinase function. These observations prompted us to investigate the role of putative phosphorylation sites within NIK1 A-loop through site-directed mutagenesis (Figure 1B). The resulting mutant kinase domains were expressed as GST fusions (Figure S1) and examined for autophosphorylation activity (Figure 2). We have previously demonstrated that the kinase domain of NIK1 fused to GST exhibits Mg2+- dependent autophosphorylation activity that occurs intermolecularly [18]. Replacement of Thr-474 with alanine (T474A) strongly inhibited autophosphorylation as the T474A mutant exhibited only 23% residual activity (3.16103 cpm/mg/min) as compared to NIK1 (13.76103 cpm/mg/min; Figure 2). Very likely, this effect was due to the removal of Thr and not due to the nature of the newly introduced amino acid residue because glutamate in the same position (T474E) also promoted a similar inhibition of autophosphorylation from 13.76103 to 1.326103 cpm/mg/min (lane 4). Nevertheless, replacement of Thr-474 with aspartate (T474D) did not promote an accentuated impact on autopho- sphorylation and the T474D mutant retained 72% activity (9.96103 cpm/mg/min; Figure 2C, lane 3). This slight effect on the relative activity of the mutant kinase may be due to the loss of an incorporated radioactive phosphate rather than a decrease in the autophosphorylation activity. We confirmed that the Thr-474 residue can be in vitro phosphorylated by performing matrix- assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analyses of tryptic digests of GST-KDNIK1 and GST-KDT474A mutant (Figure 3). MALDI-TOF analyses of in vitro phosphorylated GST-KDNIK1-derived tryptic fragments revealed a peak at m/z of 1960.854 corresponding to the peptide 473GTVGHIAPEYLSTGQSSEK491 and a peak of m/z 2040.870 representing a shift of 80.02 that corresponds to the addition of a phosphate moiety on residues 473–491. To determine whether the Thr-474 was the site of phosphate incorporation, the tryptic peptides of in vitro phosphorylated GST-KDT474A mutant were PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e5781