title: Localization of phospho-beta-dystroglycan (pY892) to an intracellular vesicular compartment in cultured cells and skeletal muscle fibers in vivo.
abstract: beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. Tyrosine 892 is now thought to be the principal site for recognition by the c-Src tyrosine kinase; however, little is known about the regulation of this phosphorylation event in vivo. Here, we generated a novel monoclonal antibody probe that recognizes only tyrosine 892 phosphorylated beta-dystroglycan (pY892). We show that upon tyrosine phosphorylation, beta-dystroglycan undergoes a profound change in its sub-cellular localization (e.g., from the plasma membrane to an internal membrane compartment). One possibility is that the net negative charge at position 892 causes the redistribution of beta-dystroglycan to this intracellular vesicular location. In support of this notion, mutation of tyrosine 892 to glutamate (Y892E) is sufficient to drive this intracellular localization, while other point mutants (Y892F and Y892A) remain at the plasma membrane. Interestingly, our colocalization studies with endosomal markers (EEA1, transferrin, and transferrin receptor) suggest that these phospho-beta-dystroglycan containing internal vesicles represent a subset of recycling endosomes. At the level of these internal vesicular structures, we find that tyrosine phosphorylated beta-dystroglycan is colocalized with c-Src. In addition, we demonstrate that known ligands for alpha-dystroglycan, namely, agrin and laminin, are able to induce the tyrosine phosphorylation of beta-dystroglycan. Finally, we show that tyrosine phosphorylated beta-dystroglycan is also detectable in skeletal muscle tissue lysates and is localized to an internal vesicular membrane compartment in skeletal muscle fibers in vivo. The generation of a phospho-specific beta-dystroglycan (pY892) mAb probe provides a new powerful tool for dissecting the role of dystroglycan phosphorylation in normal cellular functioning and in the pathogenesis of muscular dystrophies.
authors.affiliation: Department of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Federica
authors.initials: F
authors.lastname: Sotgia
authors.forename: Gloria
authors.initials: G
authors.lastname: Bonuccelli
authors.forename: Mark
authors.initials: M
authors.lastname: Bedford
authors.forename: Andrea
authors.initials: A
authors.lastname: Brancaccio
authors.forename: Ulrike
authors.initials: U
authors.lastname: Mayer
authors.forename: Mark T
authors.initials: MT
authors.lastname: Wilson
authors.forename: Roberto
authors.initials: R
authors.lastname: Campos-Gonzalez
authors.forename: James W
authors.initials: JW
authors.lastname: Brooks
authors.forename: Marius
authors.initials: M
authors.lastname: Sudol
authors.forename: Michael P
authors.initials: MP
authors.lastname: Lisanti
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015415:Biomarkers; D019556:COS Cells; D002451:Cell Compartmentation / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D049030:Dystroglycans; D011992:Endosomes / Q000737:chemistry / Q000648:ultrastructure; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D003598:Cytoskeletal Proteins; C483081:DAG1 protein, human; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D049030:Dystroglycans; D014443:Tyrosine; D016392:Proto-Oncogene Proteins pp60(c-src)