title: Phosphorylation of the beta-galactoside-binding protein galectin-3 modulates binding to its ligands.
abstract: The beta-galactoside-binding protein galectin-3 has pleiotropic biological functions and has been implicated in cell growth, differentiation, adhesion, RNA processing, apoptosis, and malignant transformation. Galectin-3 may be phosphorylated at N-terminal Ser(6), but the role of phosphorylation in determining interactions of this endogenous lectin with its ligands remains to be elucidated. We therefore studied the effect of phosphorylation on binding of galectin-3 to two of its reported ligands, laminin and purified colon cancer mucin. Human recombinant galectin-3 was phosphorylated in vitro by casein kinase I, and separated from the native species by isoelectric focusing for use in solid phase binding assays. Non-phosphorylated galectin-3 bound to laminin and asialomucin in a dose-dependent manner with half-maximal binding at 1.5 microg/ml. Phosphorylation reduced saturation binding to each ligand by >85%. Ligand binding could be fully restored by dephosphorylation with protein phosphatase type 1. Mutation of galectin-3 at Ser(6) (Ser to Glu) did not alter galectin ligand binding. Metabolic labeling or separation by isoelectric focusing confirmed the presence of phosphorylated galectin-3 species in vivo in the cytosol of human colon cancer cells from which ligand mucin was purified. Phosphorylation significantly reduces the interaction of galectin-3 with its ligands. The process by which phosphorylation modulates protein-carbohydrate interactions has important implications for understanding the biological functions of this protein, and may serve as an "on/off" switch for its sugar binding capabilities.
authors.affiliation: Gastrointestinal Cancer Research Laboratory, Henry Ford Health Sciences Center, Detroit, Michigan 48202, USA.
authors.forename: N
authors.initials: N
authors.lastname: Mazurek
authors.forename: J
authors.initials: J
authors.lastname: Conklin
authors.forename: J C
authors.initials: JC
authors.lastname: Byrd
authors.forename: A
authors.initials: A
authors.lastname: Raz
authors.forename: R S
authors.initials: RS
authors.lastname: Bresalier
mesh_terms: D000943:Antigens, Differentiation / Q000032:analysis / Q000378:metabolism*; D001212:Asialoglycoproteins / Q000378:metabolism*; D047388:Casein Kinases; D003110:Colonic Neoplasms / Q000737:chemistry / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism*; D037502:Galectin 3; D006801:Humans; D007525:Isoelectric Focusing; D007797:Laminin / Q000378:metabolism*; D008024:Ligands; D009077:Mucins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000943:Antigens, Differentiation; D001212:Asialoglycoproteins; D005697:Galactosides; D037502:Galectin 3; D007797:Laminin; D008024:Ligands; D009077:Mucins; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D011494:Protein Kinases; D047388:Casein Kinases; D010749:Phosphoprotein Phosphatases