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.
James
M
M
Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK.
Nuttall
A
A
Ilsley
J L
JL
Ottersbach
K
K
Tinsley
J M
JM
Sudol
M
M
Winder
S J
SJ
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000378:metabolism*; D049030:Dystroglycans; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism*; D049411:Utrophin; D014638:Vanadates / Q000494:pharmacology
D003598:Cytoskeletal Proteins; C483081:DAG1 protein, human; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D049411:Utrophin; D049030:Dystroglycans; D014638:Vanadates; D014443:Tyrosine