Wnt proteins contain an unusual lipid modification palmitoleic acid. were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation. Wnt Unc5b proteins are a family of secreted signaling glycoproteins that play major roles in coordinating tissue development and cell fate determination during embryogenesis as well as tissue homeostasis and oncogenesis in adults (Clevers 2006 Logan and Nusse 2004 Activation of the canonical Wnt signaling pathway stabilizes the transcriptional co-activator β-catenin which translocates to the nucleus where it binds the T-Cell Factor (TCF) family of transcription factors and activates expression of Wnt target genes. In order to signal correctly Wnt proteins need to be processed modified and secreted. All Wnt ligands contain a signal sequence at the N-terminus several N-glycosylation sites and a cysteine-rich domain. In addition Wnts undergo a unique and essential lipid modification: the cis-Δ9-mono-unsaturated fatty acid palmitoleate (C16:1Δ9) is attached to a highly conserved serine residue corresponding to Ser 209 on Wnt3a (Takada et al. 2006 Lipid modification is required for Wnt secretion as mutants lacking the Ser modification site are retained in the endoplasmic reticulum (ER) (Takada et al. 2006 and are unable to interact with Wntless (Wls) (Coombs et al. 2010 Herr and Basler 2012 a conserved membrane protein dedicated to the secretion of Wnt proteins. In addition palmitoleic Brexpiprazole acid plays a major structural role in mediating the interaction of Wnt with its receptor Frizzled (Janda et al. 2012 Kurayoshi et al. 2007 Thus Wnt fatty acylation is necessary in order to produce Brexpiprazole secreted fully active Wnt protein. Genetic (Kadowaki et al. 1996 Tanaka et al. 2000 van den Heuvel et al. 1993 and biochemical (Chen et al. 2009 Takada et al. 2006 studies have identified Porcupine (Porcn) as the acyltransferase responsible for lipid modification of Wnts. Porcn is a member of the membrane-bound O-acyltransferase (MBOAT) family (Hofmann 2000 and is predicted to modify all Wnt family members containing the conserved Ser 209 equivalent (Takada et Brexpiprazole al. 2006 Wnt signaling is tightly linked to and fine tuned by Porcn expression (Proffitt and Virshup 2012 placing Porcn as an attractive target for the development of drugs that modulate Wnt pathway activity in Wnt-driven diseases (Chen et al. 2009 Dodge et al. 2012 Proffitt et al. 2013 A small-molecule inhibitor of Porcn LGK974 (commercially available as WntC59) has been developed and is currently in early phase clinical trials. Although a role for Porcn as a Wnt acyltransferase has been established it is not known how Porcn recognizes its fatty acid substrate and why a mono-unsaturated fatty acid is attached to Wnt proteins. All studies of Wnt acylation to date have relied on labeling cells with the saturated fatty acid palmitate (Chen et al. 2009 Doubravska et al. 2011 Komekado et al. 2007 Takada et al. 2006 Willert et al. 2003 but mass spectrometric analysis indicates that under these conditions palmitoleate is the major fatty acid attached to Wnt (Takada et al. 2006 Thus a mechanism must exist to convert the saturated fatty acid (SFA) to a monounsaturated fatty acid (MUFA) either prior to or after transfer to Wnt proteins. We hypothesized that Stearoyl-CoA Desaturase (SCD) is responsible for generating the MUFA substrate for Porcn. SCD an ER-resident protein is the rate-limiting enzyme Brexpiprazole in the biosynthesis of MUFAs from saturated fatty acid precursors. It introduces a double bond makes the fatty acyl chain shorter and able to fit into the active site of Porcn. Of note 125 acid (IC15:1) labeling of cells yielded a strikingly strong signal Brexpiprazole (Fig 1e). These data suggest that MUFAs might be better substrates for Porcn than their saturated fatty acid cognates and imply that a cellular fatty acid desaturase is required to generate a suitable fatty acyl CoA substrate for Porcn. SCD inhibition blocks 125I-IC15:0 incorporation into Wnt3a SCD is the major desaturase responsible for generating 16:1 and 18:1 MUFAs.