More Favorable Palmitic Acid Over Palmitoleic Acid Modification of Wnt3 Ensures Its Localization and Activity in Plasma Membrane Domains

Abstract

While the lateral organization of plasma membrane components has been shown to control binding of Wnt ligands to their receptors preferentially in the ordered membrane domains, the role of posttranslational lipid modification of Wnt on this selective binding is unknown. Here, we identify that the canonical Wnt is presumably acylated by palmitic acid, a saturated 16-carbon fatty acid, at a conserved serine residue. Acylation of Wnt3 is dispensable for its secretion and binding to Fz8 while it is essential for Wnt3's proper binding and domain-like diffusion in the ordered membrane domains. We further unravel that non-palmitoylated Wnt3 is unable to activate Wnt/β-catenin signaling either in zebrafish embryos or in mammalian cells. Based on these results, we propose that the lipidation of canonical Wnt, presumably by a saturated fatty acid, determines its competence in interacting with the receptors in the appropriate domains of the plasma membrane, ultimately keeping the signaling activity under control.

Wnt3 distribution in the zebrafish brain is determined by expression, diffusion and multiple molecular interactions

“…The diffusion coefficients of proteins range roughly between 0.1 and 2 µm 2 /s in cell membranes and depend on their localization and interactions within the membrane. As shown in our previous studies, Wnt3 is associated with lipid domains on the membrane and is influenced by various interventions that change the lipid content of membranes in cells and in vivo ( Azbazdar et al, 2019 ; Ng et al, 2016 ; Sezgin et al, 2017 ). Therefore, the slow component likely represents the fraction of Wnt3EGFP on the membrane.…”

“…In zebrafish, Wnt3 directs neural stem cell proliferation and differentiation, making it indispensable for brain development ( Clements et al, 2009 ). Our group showed that in zebrafish embryos, Wnt3 associates with domains on the membrane ( Azbazdar et al, 2019 ; Ng et al, 2016 ; Sezgin et al, 2017 ). Blocking the activity of Porc and thus reducing Wnt acylation resulted in reduced domain confinement and defective brain development in zebrafish embryos ( Ng et al, 2016 ; Teh et al, 2015 ).…”

Regulation of Wnt Signaling Pathways at the Plasma Membrane and Their Misregulation in Cancer

“…This heterogeneity leads to formation of ordered membrane domains, also known as lipid rafts or lipid nanodomains, which are assembled from saturated lipids, sphingolipids, sterols, glycolipids, glycoproteins and certain lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and fatty acylated proteins, leaving the relatively disordered domains occupied by unsaturated lipids and a large fraction of membrane proteins ( Sezgin et al, 2015 , 2017b ; Fakhree et al, 2019 ; Kusumi et al, 2020 ; Figure 1 ). The ordered domains of the plasma membrane have been shown to be essential for cell signaling events by controlling membrane-cytoskeleton communication, ligand-receptor interaction and receptor clustering ( Simons and Toomre, 2000 ; Thomas et al, 2004 ; Ozhan et al, 2013 ; Agarwal et al, 2018 ; Azbazdar et al, 2019 ). For example, activation of epidermal growth factor receptor (EGFR) signaling by the epidermal growth factor (EGF) has been proposed to induce coalescence of different lipid rafts and formation of signaling platforms ( Hofman et al, 2009 ; Irwin et al, 2011 ).…”

“…Strikingly, several non-acylated mutants of Wnt1, Wnt3a, and Wnt8 from different species have been identified to vary dramatically in their rates of secretion, interactions with the receptor Fz and abilities to activate signaling in vitro or in vivo ( Speer et al, 2019 ). Studies on the zebrafish canonical Wnt ligands have reported that Wnt palmitoylation is essential for activation of signaling but may be dispensable for secretion ( Luz et al, 2014 ; Azbazdar et al, 2019 ). APT1-mediated depalmitoylation has been shown to be important for asymmetric localization of β-catenin and Wnt signaling activity during development ( Stypulkowski et al, 2018 ).…”

Exploring the mechanistic and temporal regulation of LRP6 endocytosis in canonical WNT signaling

“…This means that our simulation results indicate a raft-and clathrin-independent endocytosis pathway with similar internalization dynamics as raft-dependent caveolin-mediated internalization. However, caveolin is raft associated, which makes it highly unlikely that caveolin also mediates the internalization of non-raft associated LRP6 (Azbazdar et al, 2019;Sonnino and Prinetti, 2009). Recently a flotillin-dependent internalization mechanism of LRP6 has been discussed (Yamamoto et al, 2017).…”