Evidence that the tyrosine kinase domain of a small fraction of epidermal growth factor receptor molecules is exposed on the outer surface of A431 cells.
Intact A431 cells were labeled with [gamma-32P]ATP. The major phosphorylation product of the ecto-kinase activity of A431 cells had the molecular mass of 170 kd and was identified as EGF receptor by specific immunoprecipitation. This phosphorylation was not stimulated by EGF added to the reaction buffer, but replacement of MgCl2 by MnCl2 in the buffer remarkably stimulated phosphorylation. An exogenous protein substrate, alpha-casein, was also phosphorylated by intact A431 cells. The analyses for phospho-amino acids of both EGF receptor and alpha-casein revealed that phosphorylation occurred mainly at phosphotyrosine residues. Tryptic phospho-peptides of the EGF receptor of intact A431 cells labeled with [gamma-32P]ATP were fractionated by HPLC. The elution patterns were essentially the same as that of the autophosphorylated EGF receptor, indicating that the phosphorylation sites of EGF receptor labeled in vivo with [gamma-32P]ATP are located in three tyrosine residues in the carboxyl terminus. These results indicate that the carboxyl-terminal tyrosine kinase domain of a small fraction of the EGF receptor molecules of an A431 cell is exposed on the outer surface of the cells.
Department of Molecular Biology I, School of Medicine, Tokai University, Kanagawa, Japan.
S
S
Ihara
F
F
Maeda-Takekoshi
M
M
Takekoshi
M
M
Yokoyama
S
S
Sakuma
Y
Y
Watanabe
D000255:Adenosine Triphosphate / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure*; D002364:Caseins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D066246:ErbB Receptors / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000032:analysis*; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure
D002364:Caseins; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases