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. 2011 Jan 15;22(2):266–277. doi: 10.1091/mbc.E10-07-0605

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© 2011 Ishitani et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — NLK requires Thr-286 and Cys-425 for kinase activity. (A, B) Both Thr-286 and Cys-425 in NLK are essential for autophosphorylation. (A) Flag-NLK (WT), NLK(K155M) (KM), NLK(T286V) (TV), and NLK(C425Y) (CY) were expressed in HEK293 cells, and the proteins were immunopurified. Aliquots of purified NLK were subjected to nonradioisotope (non-RI) in vitro kinase assay with or without ATP and then immunoblotted (IB) with anti-Flag and anti-pNLK antibodies. The molecular-weight size markers are indicated at the right side of the panel. (B) Flag-NLK proteins subjected to non-RI in vitro kinase assay were treated with or without PPase as indicated and then immunoblotted with anti-Flag antibody. (C and D) Both Thr-286 and Cys-425 in NLK are essential for Tcf4 phosphorylation as a substrate in vitro (C) and in vivo (D). (C) Flag-NLK (WT), NLK(K155M) (KM), NLK(T286V) (TV), and NLK(C425Y) (CY) were expressed in HEK293 cells, and the proteins were immunopurified. Flag-Tcf4 was also expressed in HEK293 cells, and the proteins were purified. Aliquots of purified Tcf4 and NLK proteins were subjected to non-RI in vitro kinase assay and then immunoblotted with anti-Flag antibody (top). Purified NLK proteins were also confirmed by immunoblotting with anti-Flag antibody (bottom). (D) HEK293 cells were transfected with the plasmids encoding Flag-NLK (WT), NLK(K155M) (KM), NLK(T286V) (TV), NLK(C425Y) (CY), and Flag-Tcf4, and then lysates were immunoblotted with anti-Flag antibody. (E and G) Comparison of amino acid sequences around the activation loop (E) and conserved cysteine-rich region (G) of C. elegans NLK (LIT-1), Drosophila NLK (Nemo), mouse NLK, mouse ERK1, mouse JNK1, and mouse p38α. Arrow indicates the conserved threonine residues in the activation loop (E). Arrowheads indicate the conserved glutamic acid residues in NLK family proteins and tyrosine residues in MAPK family proteins (E) and the conserved cysteine residues in NLK family proteins (G). (F) Anti-pNLK antibody recognizes phosphorylated NLK. Flag-NLK was expressed in HEK293 cells, and the proteins were immunopurified. Immunoprecipitates were treated with or without PPase as indicated and then immunoblotted with anti-Flag and anti-pNLK antibodies.