DEVELOPMENT 2061 RESEARCH ARTICLE INTRODUCTION The Drosophila nemo (nmo) gene was originally found to be required for epithelial planar cell polarity during eye development (Choi and Benzer, 1994). Subsequent analyses have implicated nmo in patterning events during embryogenesis and imaginal disc development as well as in controlling apoptosis (Mirkovic et al., 2002; Verheyen et al., 2001). Nemo is the founding member of the evolutionarily conserved Nemo-like kinase (Nlk) family of proline- directed serine/threonine (S/T) kinases closely related to mitogen- activated protein kinases (MAPK) (Choi and Benzer, 1994). Biochemical and genetic studies implicate Nlk in several pathways (reviewed by Behrens, 2000; Martinez Arias et al., 1999). The best-characterized role for Nlk is in Wnt/Wg signaling in numerous species (Golan et al., 2004; Ishitani et al., 2003a; Ishitani et al., 1999; Kanei-Ishii et al., 2004; Meneghini et al., 1999; Rocheleau et al., 1999; Shin et al., 1999; Smit et al., 2004; Thorpe and Moon, 2004; Zeng and Verheyen, 2004). Nlk phosphorylates Tcf/Lef transcription factors and inhibits their activity. Depending on the cellular context, Nlk either inhibits Wnt-dependent gene expression (Ishitani et al., 2003b; Ishitani et al., 1999; Zeng and Verheyen, 2004) or promotes it (Meneghini et al., 1999; Rocheleau et al., 1999; Thorpe and Moon, 2004). There is increasing evidence that Nlk regulates additional HMG-domain-containing proteins, such as Xenopus Sox11 and HMG2L1 (Hyodo-Miura et al., 2002; Yamada et al., 2003), as well as other transcriptional regulators such as CBP/p300, Stat3 and Myb (Kanei-Ishii et al., 2004; Ohkawara et al., 2004; Yasuda et al., 2004). Nlk can be activated by the MAPK kinase kinase Tak1 (TGF- activated kinase 1) in mammals (also known as Map3k7 – Mouse Genome Informatics) and in C. elegans (also known as MOM-4 – Wormbase) in certain contexts (Ishitani et al., 1999; Meneghini et al., 1999). However, in this study we describe an inhibitory relationship between Nemo and Drosophila TGF- signaling. TGF-  signaling is initiated when a secreted ligand of the TGF-, bone morphogenic protein (BMP) or Activin family binds to a type II S/T kinase receptor (reviewed by Attisano and Wrana, 2002; von Bubnoff and Cho, 2001). This receptor then recruits and phosphorylates a type I S/T kinase receptor, which in turn phosphorylates a member of the R-Smad family of proteins on an SSxS motif at its C-terminus. The phosphorylated R-Smad is released from the receptor and binds the Co-Smad. In the nucleus, the Smad complex forms complexes with transcription factors on the promoters of target genes. Nuclear signaling is abrogated when the R-Smad is dephosphorylated at its C-terminus (Chen et al., 2006; Duan et al., 2006; Knockaert et al., 2006). During Drosophila wing patterning, BMP signaling is carried out by two BMPs, Decapentaplegic (Dpp) and Glass bottom boat (Gbb) (Padgett et al., 1987; Wharton et al., 1991). Dpp acts as a morphogen during the patterning of multiple tissues during embryonic and imaginal disc development (reviewed by Raftery and Sutherland, 1999). Dpp activates the Punt receptor, which in turn phosphorylates Thickveins (Tkv), leading to the activation of the Smads. The Smad1 ortholog, Mothers against dpp (Mad), is phosphorylated by activated Tkv and together with the Co-Smad Medea (Med) accumulates in the nucleus and regulates transcription of target genes (reviewed by Moustakas et al., 2001; Shi and Massague, 2003; ten Dijke and Hill, 2004). In the wing imaginal disc, BMP signaling regulates the expression of several genes, including optomoter blind (omb; also known as bifid – Flybase), spalt major (salm) and vestigial quadrant (vg Q ) enhancer (Burke and Basler, 1996; Grimm and Pflugfelder, 1996; Kim et al., 1997; Lecuit et al., 1996; Lecuit and Cohen, 1998; Nellen et al., 1996). The inhibitory Smad homolog Daughters against dpp (Dad) is also a BMP target gene that acts in a negative-feedback loop to inhibit BMP signaling (Tsuneizumi et al., 1997). Dpp plays several distinct roles during larval and pupal wing development (Segal and Gelbart, 1985; Spencer et al., 1982). During larval disc development, Dpp is expressed along the anterior/posterior (A/P) boundary of the disc in response to Drosophila Nemo antagonizes BMP signaling by phosphorylation of Mad and inhibition of its nuclear accumulation Yi Arial Zeng* ,† , Maryam Rahnama*, Simon Wang, Worlanyo Sosu-Sedzorme and Esther M. Verheyen ‡ Drosophila Nemo is the founding member of the Nemo-like kinase (Nlk) family of serine/threonine protein kinases that are involved in several Wnt signal transduction pathways. Here we report a novel function for Nemo in the inhibition of bone morphogenetic protein (BMP) signaling. Genetic interaction studies demonstrate that nemo can antagonize BMP signaling and can inhibit the expression of BMP target genes during wing development. Nemo can bind to and phosphorylate the BMP effector Mad. In cell culture, phosphorylation by Nemo blocks the nuclear accumulation of Mad by promoting export of Mad from the nucleus in a kinase-dependent manner. This is the first example of the inhibition of Drosophila BMP signaling by a MAPK and represents a novel mechanism of Smad inhibition through the phosphorylation of a conserved serine residue within the MH1 domain of Mad. KEY WORDS: Nemo, Nlk, BMP, Dpp, Mad, MH1, Smad, Drosophila Development 134, 2061-2071 (2007) doi:10.1242/dev.02853 Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada. *These authors contributed equally to this work † Present address: Department of Developmental Biology, Stanford University, Stanford, CA 94305-5323, USA ‡ Author for correspondence (e-mail: everheye@sfu.ca) Accepted 19 March 2007