Adhesion-dependent tyrosine phosphorylation of β-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 beta-dystrogtycan and utrophin revealed adhesion-and perosyvanadate-dependent mobility shifts which were recognised by anti-phospho-tyrosine antibodies. Using maltose binding protein fusion constructs to the carboxyterminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and beta-dgstroglycan 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.