Localization of phospho-beta-dystroglycan (pY892) to an intracellular vesicular compartment in cultured cells and skeletal muscle fibers in vivo.
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.
Sotgia
Federica
F
Department of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Bonuccelli
Gloria
G
Bedford
Mark
M
Brancaccio
Andrea
A
Mayer
Ulrike
U
Wilson
Mark T
MT
Campos-Gonzalez
Roberto
R
Brooks
James W
JW
Sudol
Marius
M
Lisanti
Michael P
MP
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
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)