Since Nurr1 can be phosphorylated by serine/threonine kinases (Nordzell et al., 2004), we speculated that signal-dependent phosphorylation might contribute to the Nurr1-CoREST interaction. Nemo-like kinase (NLK) received our attention because NLK is known to be involved in the repression of various transcription factors (Yasuda et al., 2004). NLK cooperates with TGFβ-activating kinase 1 (TAK1) and homeodomain-interacting kinase 2 (HIPK2) in Wnt signaling (Kanei-Ishii et al., 2004). Knockdown of NLK abolished Nurr1 repression of iNOS-promoter activity, whereas HIPK2 knockdown was much less effective and TAK1 had no effect (Fig. 4C and Fig. S9A). Furthermore, overexpression of kinase-dead NLK (NLKK155M, NLK-KD) in RAW264.7 cells inhibited Nurr1-mediated repression of iNOS in a dose-dependent manner (Fig. S9B). Kinase assays showed that Nurr1, but not CoREST, could be phosphorylated by active NLK in vitro (Fig. 4D). Finally, Nurr1-CoREST interaction was significantly reduced by NLK-knockdown in BV2 cells (Fig. 4E).
