Mindbomb 1 , an E 3 ubiquitin ligase , forms a complex with RYK to activate Wnt / - catenin signaling Jason

The Wnt family of secreted glycoproteins plays a critical role in developmental processes including axis patterning, cellular proliferation, planar cell polarity, cell migration, cell fate specification, and neuronal development. In adults, Wnt signaling is involved in tissue homeostasis, regeneration, and stem/progenitor cell function. Moreover, elevated or attenuated Wnt signaling is found in a diversity of diseased tissues (Logan and Nusse, 2004; Moon et al., 2004). Wnt signaling can be divided into CTNNB1-dependent and CTNNB1-independent signaling. The best-characterized family of receptors for Wnts is the Frizzled class of seven-pass transmembrane atypical G protein–coupled receptors. In addition, CTNNB1-dependent signaling requires a coreceptor, the low-density lipoprotein receptor–related proteins 5 and 6 (LRP). More recently, it has become clear that two additional unrelated, single-pass receptor tyrosine kinases, receptor tyrosine kinase-like orphan receptor 2 (ROR2) and receptor-like tyrosine kinase (RYK), can bind to Wnts. In contrast to signaling events downstream of the Frizzled/LRP receptors, the mechanisms of ROR2and RYK-mediated Wnt signaling are poorly understood (Angers and Moon, 2009). RYK has been shown to bind to Frizzleds (Lu et al., 2004; Kim et al., 2008; Li et al., 2009). However, Frizzleds are not required for Wnt/RYK signaling in all contexts (Inoue et al., 2004; Schmitt et al., 2006; Harris and Beckendorf, 2007; Li et al., 2009), which suggests a distinct molecular mechanism of Wnt/RYK signaling. RYK is required for CTNNB1-dependent Wnt signaling, as loss of function of RYK inhibits the ability of Wnt-3A to activate a CTNNB1-dependent transcriptional reporter in human embryonic kidney (HEK293T) cells (Lu et al., 2004). Moreover, RYK is required for Wnt/CTNNB1 signaling in vivo. In the specification of vulva cell fate in Caenorhabditis elegans, LIN-18/RYK shows a genetic interaction with WNT/ MOM-2 and TCF/POP-1 (Inoue et al., 2004; Deshpande et al., 2005). In mice, RYK is required for Wnt-3A–induced neurite outgrowth from explanted dorsal root ganglia (Lu et al., 2004) Receptor-like tyrosine kinase (RYK) functions as a transmembrane receptor for the Wnt family of secreted protein ligands. Although RYK undergoes endocytosis in response to Wnt, the mechanisms that regulate its internalization and concomitant activation of Wnt signaling are unknown. We discovered that RYK both physically and functionally interacts with the E3 ubiquitin ligase Mindbomb 1 (MIB1). Overexpression of MIB1 promotes the ubiquitination of RYK and reduces its steadystate levels at the plasma membrane. Moreover, we show that MIB1 is sufficient to activate Wnt/-catenin (CTNNB1) signaling and that this activity depends on endogenous RYK. Conversely, in loss-of-function studies, both RYK and MIB1 are required for Wnt-3A–mediated activation of CTNNB1. Finally, we identify the Caenorhabditis elegans orthologue of MIB1 and demonstrate a genetic interaction between ceMIB and lin-18/RYK in vulva development. These findings provide insights into the mechanisms of Wnt/RYK signaling and point to novel targets for the modulation of Wnt signaling. Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/-catenin signaling