Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of a M(r) 55,000 substrate with the receptor.
Administration of epidermal growth factor (EGF) to neonatal mice resulted in rapid tyrosine phosphorylation of a number of specific substrates in liver, kidney, lung, bladder, skin, and brain as detected by Western blot analysis of tissue homogenates with anti-phosphotyrosine antibodies. In the liver, three prominent EGF-dependent substrates of M(r) approximately 170,000, 120,000, and 55,000 were detected. A number of less prominent EGF-dependent substrates also were noted. Maximal tyrosine phosphorylation of pp170, pp120, and pp55 occurred within 5 min of subcutaneous injection and the levels of these phosphoproteins remained elevated for at least 45 min. Direct hepatic injection of EGF resulted in the tyrosine phosphorylation of these substrates within 60 sec of treatment. Tyrosine-phosphorylated pp170 was identified as the EGF receptor (EGFR). The tyrosine-phosphorylated pp55 substrate appeared to be associated with EGFR; both pp55 and EGFR were adsorbed to EGF-Affi-Gel, wheat germ lectin-Sepharose, and anti-EGFR antibodies bound to protein A-Sepharose. pp55 was not immunoreactive with anti-EGFR antiserum by Western blot analysis, indicating that it was not a fragment of the receptor. These results were confirmed by repeating the liver experiments using 32P-labeled neonatal mice. Increased amounts of 32P-labeled pp170 and pp55 were detected in anti-EGFR immunoprecipitates from liver extracts of EGF-treated animals as compared with controls. Phospho amino acid analysis of the 32P-labeled phosphoproteins revealed that EGF stimulated both serine and tyrosine phosphorylation in pp55 as well as in EGFR. The neonatal mouse may be a useful model for the study of signal transduction mediated by a variety of growth factors.
Donaldson
R W
RW
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.
Cohen
S
S
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D019000:Phosphotyrosine; D011485:Protein Binding; D015398:Signal Transduction; D014018:Tissue Distribution; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D046911:Macromolecular Substances; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors