Identification and biochemical characterization of novel putative substrates for the epidermal growth factor receptor kinase.
To gain insight into the mechanisms which control the mitogenic response to epidermal growth factor (EGF), we have partially purified and characterized several intracellular proteins which are phosphorylated on tyrosine residues following activation of the epidermal growth factor receptor (EGFR). Partial purification was achieved by immunoaffinity chromatography using immobilized anti-phosphotyrosine antibodies. Antisera generated against the partially purified proteins were used to identify at least five novel EGFR putative substrates, designated, on the basis of their apparent molecular weight, p97, p68, p61, p56, and p23. All of these proteins became specifically phosphorylated on tyrosine after EGF treatment of intact cells, as assessed by phosphoamino acid analysis, and none of them represented an EGFR degradation product. The phosphorylation of these proteins appeared to be relatively specific for the EGFR. In particular, an EGFR-related kinase, erbB-2 was much less efficient than EGFR at phosphorylating p97, p56, and p23 and incapable of phosphorylating p68. The identification of these novel EGFR putative substrates should lead to a better understanding of the mechanisms controlling the specificity of EGFR-mediated mitogenic signaling.
Fazioli
F
F
Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Bottaro
D P
DP
Minichiello
L
L
Auricchio
A
A
Wong
W T
WT
Segatto
O
O
Di Fiore
P P
PP
D016475:3T3 Cells; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002678:Chimera; D002846:Chromatography, Affinity; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D051379:Mice; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases; D013379:Substrate Specificity; D014162:Transfection
D000596:Amino Acids; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases