Adhesion-dependent tyrosine phosphorylation of β-dystroglycan regulates its interaction with utrophin

James, Marian; Nuttall, Am M.; Ilsley, Jane L.; Ottersbach, Katrin; Tinsley, Jonathon M.; Sudol, Marius; Winder, Steven J.

doi:10.1242/jcs.113.10.1717

Cited by 120 publications

(33 citation statements)

References 51 publications

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“…Importantly, when tyrosine 892 is mutated to phenylalanine (Y892F), preventing phosphorylation at this site, this immunoreactivity is completely abolished (Figure 1A). In addition, the mobility of phospho-β-dystroglycan was shifted upward, as we and others have previously noted (27,28).…”

Section: Resultssupporting

confidence: 64%

“…Equal protein loading was assessed using a monoclonal antibody against β-dystroglycan (βDG mAb). It is also interesting to note the characteristic upward mobility shift of tyrosine phosphorylated β-dystroglycan, as compared with total β-dystroglycans as we and others have previously described (27,28).…”

Section: Mutation Of Tyrosine 892 To Glutamate (Y892e) Redistributes ...mentioning

confidence: 59%

“…In addition, immunoblot analysis with an anti-β-dystroglycan antibody was also performed. Figure 13A shows the upward mobility shift of tyrosine phosphorylated β-dystroglycan, as compared with total β-dystroglycansas we and others have previously described (27,28).…”

Section: Extracellular Matrix Components (Agrin Laminin and Fibronect...mentioning

confidence: 89%

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Localization of Phospho-β-dystroglycan (pY892) to an Intracellular Vesicular Compartment in Cultured Cells and Skeletal Muscle Fibers in Vivo

et al. 2003

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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.

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Section: Resultssupporting

confidence: 64%

Section: Mutation Of Tyrosine 892 To Glutamate (Y892e) Redistributes ...mentioning

confidence: 59%

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Localization of Phospho-β-dystroglycan (pY892) to an Intracellular Vesicular Compartment in Cultured Cells and Skeletal Muscle Fibers in Vivo

et al. 2003

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“…Alternatively, there may be aspects of these protein–protein interactions, or of protein-antibody interactions, that are not sufficiently appreciated. For example, Winder has described tyrosine phosphorylation of the PPXY motif on beta dystroglycan and shown that this phosphorylation can inhibit utrophin and dystrophin binding. , It may well be that Galgt2 glycosylation inhibits such tyrosine phosphorylation of beta dystroglycan, allowing enriched immunoprecipitation with MANDAG2 and enriched coprecipitation of dystrophin or utrophin and associated proteins.…”

Section: Discussionmentioning

confidence: 99%

Comparative Proteomic Profiling of Dystroglycan-Associated Proteins in Wild Type, mdx, and Galgt2 Transgenic Mouse Skeletal Muscle

Yoon

Johnson

et al. 2012

J. Proteome Res.

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Dystroglycan is a major cell surface glycoprotein receptor for the extracellular matrix in skeletal muscle. Defects in dystroglycan glycosylation cause muscular dystrophy and alterations in dystroglycan glycosylation can impact extracellular matrix binding. Here we describe an immunoprecipitation technique that allows isolation of beta dystroglycan with members of the dystrophin-associated protein complex (DAPC) from detergent solubilized skeletal muscle. Immunoprecipitation, coupled with shotgun proteomics, has allowed us to identify new dystroglycan-associated proteins and define changed associations that occur within the DAPC in dystrophic skeletal muscles. In addition, we describe changes that result from overexpression of Galgt2, a normally synaptic muscle glycosyltransferase that can modify alpha dystroglycan and inhibit the development of muscular dystrophy when it is overexpressed. These studies identify new dystroglycan-associated proteins that may participate in dystroglycan’s roles, both positive and negative, in muscular dystrophy.

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“…Moreover, the disruption of the DG gene results in lethality during mouse early embryogenesis (9). The importance of DG biological function is further stressed by recent studies showing an involvement of DG in signal transduction, as suggested by the presence of potential SH2 and SH3 binding motifs and of a consensus sequence for tyrosine phosphorylation located within the cytodomain of β-DG (10,11).…”

mentioning

confidence: 99%

Structural Characterization by NMR of the Natively Unfolded Extracellular Domain of β-Dystroglycan: Toward the Identification of the Binding Epitope for α-Dystroglycan

et al. 2003

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Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: R-DG, membrane-associated and highly glycosylated, and the transmembrane -DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of R-DG interacts with a recombinant extracellular fragment of -DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of R-DG. In order to elucidate which moieties of -DG are specifically involved in the complex with R-DG, the ectodomain has been recombinantly expressed and purified in a labeled ( 13 C, 15 N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, 1 H-15 N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and 3 J HN,HR coupling constant values confirm that this protein is highly disordered, but 1 H-15 N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of -DG(654-750) to the C-terminal region of the R subunit, R-DG(485-620), has been investigated, showing that the region of -DG(654-750) between residues 691 and 719 is involved in the interaction. † The financial support of CNR, target project "Biotechnology" and

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Adhesion-dependent tyrosine phosphorylation of β-dystroglycan regulates its interaction with utrophin

Cited by 120 publications

References 51 publications

Localization of Phospho-β-dystroglycan (pY892) to an Intracellular Vesicular Compartment in Cultured Cells and Skeletal Muscle Fibers in Vivo

Localization of Phospho-β-dystroglycan (pY892) to an Intracellular Vesicular Compartment in Cultured Cells and Skeletal Muscle Fibers in Vivo

Comparative Proteomic Profiling of Dystroglycan-Associated Proteins in Wild Type, mdx, and Galgt2 Transgenic Mouse Skeletal Muscle

Structural Characterization by NMR of the Natively Unfolded Extracellular Domain of β-Dystroglycan: Toward the Identification of the Binding Epitope for α-Dystroglycan

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