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Title: Interactions in the dystrophin-associated protein complex (DAPC) regulated by phosphorylation
Author: Ilsley, Jane
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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Duchenne muscular dystrophy (DMD) is caused by mutations at the DMD locus leading to a lack of the dystrophin protein in muscle cells. Dystrophin associates with a group of membrane-associated proteins called the dystrophin-associated protein complex (DAPC) via an interaction with the membrane spanning glycoprotein 13-dystroglycan. The exact functions of dystrophin and the DAPC have not been elucidated and little is known about the regulation of protein:protein interactions within this complex. However, the proteins are ideally placed to transduce signals from the extracellular matrix to the cytoskeleton and have been implicated in a wide variety of cell signalling events. The aim of this study was to identify novel phosphorylation events within the DAPC and determine their effects on the regulation of protein:protein interactions in the complex. Upon treatment of C2/C4 cells (a mouse muscle cell line) with peroxyvanadate, a potent tyrosine phosphatase inhibitor, 3-dystroglycan is tyrosine-phosphorylated. This phosphorylation event was initially detected by mobility shifts on SDS-polyacrylamide gels, and confirmed by immunoprecipitation and two-dimensional gel electrophoresis. The potential functional significance of this tyrosine phosphorylation was investigated using peptide 'SPOTs' assays. Phosphorylation of tyrosine in the 15 most C-terminal residues of -dystroglycan disrupts its interaction with dystrophin. The tyrosine residue in 13- dystroglycan's WW domain-binding motif PPPY appears to be the most crucial in disrupting the interaction. Therefore the j3-dystroglycan/dystrophin interaction appears to be regulated by tyrosine phosphorylation. Various methods were used to investigate the protein:protein interactions of 0- dystroglycan. In a yeast two-hybrid assay f3-dystroglycan was found to interact with actin. This novel interaction was further characterised by F-actin sedimentation assays and it was demonstrated f3-dystroglycan facilitated the sedimentation of F-actin during low speed centrifugation. Furthermore, electron microscopy studies revealed that 13- dystroglycan bundles actin filaments. These studies highlight the importance of 13-dystroglycan, not only in the regulation of the interaction between dystrophin and the rest of the DAPC but also its possible role in the regulation of the actin cytoskeleton. Understanding more about functions of dystrophin and the DAPC allows a greater insight into the pathogenesis of Duchenne muscular dystrophy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available