However, our preliminary studies suggest that this apparent discrepancy displays the differences in cell lines used (L cells vs HEK293 cells)

However, our preliminary studies suggest that this apparent discrepancy displays the differences in cell lines used (L cells vs HEK293 cells). pathway Pdgfra choice. Introduction Proteins often undergo post-translational modifications that are critical for their function. The covalent attachment of fatty acids (acylation) is usually one such modification. Proteins can be altered by fatty acids of different chain lengths; palmitoylation is the addition of a 16 carbon fatty acid. Palmitate can be linked to amino groups (N-linked), sulfhydryl groups (S-linked) or alcohol groups (O-linked). S-Palmitoylation of cytosolic proteins and the intracellular domains of transmembrane proteins is known to control membrane association, targeting to lipid rafts and intracellular membranes, intracellular trafficking, protein-protein interactions, biological activity and stability [1], [2], [3], [4], [5]. More recently, the N-,O- and S-palmitoylation of secreted ligands such as Hedgehog, Spitz and Wnt has also been reported to play functions in regulating the activity and distribution of these proteins [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Despite numerous advances, however, the functional role(s) of lipid modifications to secreted proteins remains poorly comprehended. Mass spectrometry studies have definitively exhibited the acylation of Wnt3a with two lipid adducts, fully saturated palmitate (C16:0) on a conserved cysteine (C77; S-palmitoylation) and mono-unsaturated palmitoleic acid (C16:1) on a conserved serine (S209; O-palmiteoylation) [13], [14]. These residues are invariant amongst all of the 19 vertebrate Wnt family members and all, but one, of the known invertebrate Wnt family members [16]. Additional mass spectrometry studies have confirmed the palmitoylation of the cysteine residue in Wnt5a [10]. Thus, it seems likely that this acylation of these residues is usually conserved across family members. As the precise regulation of Wnt signaling is required for proper embryonic development and adult homeostasis [17], [18], [19], [20], [21], [22], it is crucial to fully understand the functional Thiamet G functions of the lipid modifications to the cysteine and serine residues. To achieve this goal, we tested a panel of Wnt1 and Wnt3a constructs encoding differentially acylated Wnt proteins for stability, secretion, and biological activity. We found that mutation of either Thiamet G the cysteine or the serine has comparable effects on stability and secretion, but that this relative importance of each residue for biological activity in ?-catenin dependent and impartial assays differs significantly. Of equivalent importance is the identification of the upstream regulators of these modifications. Porcupine (Porcn) and Wntless (Wls) are upstream regulators of Wnt signaling. While Porcn is usually predicted to play a role in Wnt palmit(e)oylation [13], [23], Thiamet G Wls is usually thought to escort palmit(e)oylated Wnts through the secretory pathway [24], [25], [26], [27]. Though it has not been experimentally exhibited that Porcn functions directly to palmit(e)oylate Wnts, bioinformatic studies have recognized it as a putative O-acyl transferase [23]. Additional studies show that Porcn is required for the palmiteoylation of Wnt3a S209 [13]; however, it is not known if Porcupine participates in the palmitoylation of the cysteine residue [28]. Our studies in L cells show that pharmacological inhibition of Porcn significantly reduces Wnt1 signaling via the ?-catenin dependent pathway, but not a ?-catenin indie pathway (that is yet to be defined). To determine whether Porcn is likely to be involved in the palmit(e)oylation of one or both lipid altered sites, we tested the ability of Porcn to modify GFP-tagged Wnt1 peptides targeted Thiamet G to the secretory pathway. Specifically, we used a hydrophobicity assay along with an assay for co-localization with lipid rafts to identify Wnt1 peptides with sites for Porcn-dependent modifications. Our data from these studies are consistent with a model in which the cysteine residue is usually altered in a Porcn-independent manner while the serine residue is usually altered in a Porcn-dependent manner. In sum, our results strongly suggest that unique enzymes are involved in the lipid.