Epidermal growth factor stimulated phosphorylation of a 120-kilodalton endogenous substrate protein in rat hepatocytes.
Endogenous substrates of the EGF receptor have been described in transformed cells; however, little is known about substrates in normal tissue. To characterize epidermal growth factor (EGF) receptor phosphorylation and search for endogenous substrates in normal rat hepatocytes, cells were labeled with [32P]orthophosphate, and phosphotyrosine-containing proteins were sought by using a high-affinity, specific anti-phosphotyrosine antibody. Exposure of 32P-labeled freshly isolated hepatocytes to 1 microgram/mL EGF caused phosphorylation of several proteins of Mr 185K, 160K, and 120K. The 185- and 160-kDa proteins (pp185 and pp160) were identified as the intact and proteolyzed forms of the EGF receptor by virtue of their immunoprecipitation with anti-EGF receptor antibody. This antibody failed to recognize the 120-kDa phosphoprotein (pp120). The phosphopeptide map derived from pp120 was by trypsinization and HPLC separation different from that of pp185, further indicating that pp120 is distinct from the EGF receptor. This pp120 was also immunologically distinct from the pp120 substrate of the insulin receptor kinase and from ATP-citrate lyase. Phosphoamino acid analysis revealed pp120 to be phosphorylated on both tyrosine and serine residues. Autophosphorylation of EGF receptor and phosphorylation of pp120 were almost maximal within 1 min of EGF stimulation. The dose-response curves for phosphorylation of the EGF receptor and pp120 were identical (ED50 = 30 ng/mL) and were superimposable with the fractional occupancy of the EGF receptor. In A431 cells, a transformed cell line whose growth is inhibited by EGF, EGF produced a decrease in pp120 phosphorylation. These data suggest that pp120 is an endogenous substrate for the EGF receptor in hepatocytes whose phosphorylation may be closely related to EGF stimulation of cell growth.
Okamoto
M
M
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Karasik
A
A
White
M F
MF
Kahn
C R
CR
D000818:Animals; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D011506:Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors