Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin.
In response to epidermal growth factor (EGF) the microvillar core protein ezrin is phosphorylated transiently to a high level on tyrosine residues in human epidermoid carcinoma A431 cells. Here we report the identification of the tyrosine phosphorylation sites in ezrin using bacterially expressed protein as a substrate for in vitro phosphorylation with the EGF receptor. The two major phosphotyrosine-containing peptides observed in vivo were also phosphorylated in vitro. By secondary digestions and site-directed mutagenesis tyrosines 145 and 353 were identified as the sites of phosphorylation. One of the sites, Tyr145, lies in the N-terminal region of homology that is common to the band 4.1-talin-ezrin protein family. This tyrosine residue and its vicinal amino acids are conserved throughout the family members, including radixin, moesin, and the two phosphotyrosine phosphatases, PTP H1 and PTP MEG, but not in band 4.1 or talin. Tyr353 is localized within the alpha-helical domain of ezrin and comparison of the protein sequences reveals that this site is unique to ezrin.
Krieg
J
J
Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92186.
Hunter
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003598:Cytoskeletal Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D004815:Epidermal Growth Factor; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010750:Phosphoproteins; D019000:Phosphotyrosine; D011994:Recombinant Proteins; D014443:Tyrosine; D000099149:Ezrin