Tyrosine phosphorylation of beta-dystroglycan at its WW domain binding motif, PPxY, recruits SH2 domain containing proteins.
beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. However, it remains unknown whether tyrosine-phosphorylated beta-dystroglycan interacts with SH2 domain containing proteins. Here, we show that the tyrosine phosphorylation of beta-dystroglycan is constitutively elevated in v-Src transformed cells. We next reconstituted this phosphorylation ... event in vivo by transiently coexpressing wild-type c-Src with a fusion protein containing full-length beta-dystroglycan. Our results demonstrate that Src-induced tyrosine phosphorylation of beta-dystroglycan is strictly dependent on the presence of a PPxY motif at its extreme C-terminus. In the nonphosphorylated state, this PPxY motif is normally recognized as a ligand by the WW domain; phosphorylation at this site blocks the binding of certain WW domain containing proteins. Using a GST fusion protein carrying the cytoplasmic tail of beta-dystroglycan, we identified five SH2 domain containing proteins that interact with beta-dystroglycan in a phosphorylation-dependent manner, including c-Src, Fyn, Csk, NCK, and SHC. We localized this binding activity to the PPxY motif by employing a panel of beta-dystroglycan-derived phosphopeptides. In addition, tyrosine phosphorylation of beta-dystroglycan in vivo resulted in the coimmunoprecipitation of the same SH2 domain containing proteins, and this binding event required the beta-dystroglycan C-terminal PPxY motif. We discuss the possibility that tyrosine phosphorylation of the PPxY motif within beta-dystroglycan may act as a regulatory switch to inhibit the binding of certain WW domain containing proteins, while recruiting SH2 domain containing proteins.
Mesh Terms:
3T3 Cells, Adaptor Proteins, Signal Transducing, Amino Acid Motifs, Animals, Blotting, Western, Cell Transformation, Neoplastic, Dystrophin, Glutathione Transferase, Intramolecular Transferases, Mice, Oncogene Proteins, Peptide Fragments, Phosphorylation, Precipitin Tests, Protein Binding, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-fyn, Proto-Oncogene Proteins pp60(c-src), Transfection, Tyrosine, src Homology Domains
3T3 Cells, Adaptor Proteins, Signal Transducing, Amino Acid Motifs, Animals, Blotting, Western, Cell Transformation, Neoplastic, Dystrophin, Glutathione Transferase, Intramolecular Transferases, Mice, Oncogene Proteins, Peptide Fragments, Phosphorylation, Precipitin Tests, Protein Binding, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-fyn, Proto-Oncogene Proteins pp60(c-src), Transfection, Tyrosine, src Homology Domains
Biochemistry
Date: Dec. 04, 2001
PubMed ID: 11724572
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