The interaction of dystrophin with beta-dystroglycan is regulated by tyrosine phosphorylation.

Dystrophin and the dystrophin-associated protein complex (DAPC) have recently been implicated in cell signalling events. These proteins are ideally placed to transduce signals from the extracellular matrix (ECM) to the cytoskeleton. Here we show that beta-dystroglycan is tyrosine-phosphorylated in C2/C4 mouse myotubes. Tyrosine phosphorylation was detected by mobility shifts on SDS-polyacrylamide gels (SDS-PAGE) 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 amino acids of beta-dystroglycan disrupts its interaction with dystrophin. The tyrosine residue in beta-dystroglycan's WW-binding motif PPPY appears to be the most crucial in disrupting the beta-dystroglycan-dystrophin interaction. beta-dystroglycan forms the essential link between dystrophin and the rest of the DAPC. This regulation by tyrosine phosphorylation may have implications in the pathogenesis and treatment of Duchenne's muscular dystrophy (DMD).

Adhesion-dependent tyrosine phosphorylation of (beta)-dystroglycan regulates its interaction with utrophin.

Many cell adhesion-dependent processes are regulated by tyrosine phosphorylation. In order to investigate the role of tyrosine phosphorylation of the utrophin-dystroglycan complex we treated suspended or adherent cultures of HeLa cells with peroxyvanadate and immunoprecipitated (beta)-dystroglycan and utrophin from cell extracts. Western blotting of (&bgr;)-dystroglycan and utrophin revealed adhesion- and peroxyvanadate-dependent mobility shifts which were recognised by anti-phospho-tyrosine antibodies. Using maltose binding protein fusion constructs to the carboxy-terminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and (beta)-dystroglycan by co-immunoprecipitation with endogenous (beta)-dystroglycan. In extracts from cells treated with peroxyvanadate, where endogenous (beta)-dystroglycan was tyrosine phosphorylated, (beta)-dystroglycan was no longer co-immunoprecipitated with utrophin fusion constructs. Peptide 'SPOTs' assays confirmed that tyrosine phosphorylation of (beta)-dystroglycan regulated the binding of utrophin. The phosphorylated tyrosine was identified as Y(892) in the (beta)-dystroglycan WW domain binding motif PPxY thus demonstrating the physiological regulation of the (beta)-dystroglycan/utrophin interaction by adhesion-dependent tyrosine phosphorylation.