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Epidermal growth factor stimulated phosphorylation of a 120-kilodalton endogenous substrate protein in rat
M Okamoto1, A Karasik, M F White
1Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Abstract:
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.
Insights
Researchers identified a novel protein, pp120, as an endogenous substrate of the epidermal growth factor (EGF) receptor in normal rat hepatocytes. Its phosphorylation is closely linked to EGF-stimulated cell growth.
Area of Science:
- Cellular Biology
- Molecular Signaling
- Biochemistry
Background:
- Endogenous substrates of the epidermal growth factor (EGF) receptor are known in cancer cells, but poorly understood in normal tissues.
- Investigating EGF receptor substrates in normal hepatocytes is crucial for understanding normal cell growth regulation.
Purpose of the Study:
- To characterize EGF receptor phosphorylation in normal rat hepatocytes.
- To identify and characterize endogenous substrates of the EGF receptor in normal rat hepatocytes.
Main Methods:
- Primary rat hepatocytes were labeled with [32P]orthophosphate.
- Proteins were analyzed using anti-phosphotyrosine antibodies and immunoprecipitation.
- Phosphopeptide mapping and phosphoamino acid analysis were performed.
Main Results:
- EGF stimulation induced phosphorylation of 185-kDa (intact EGF receptor), 160-kDa (proteolyzed EGF receptor), and a novel 120-kDa protein (pp120).
- pp120 is distinct from the EGF receptor and other known substrates, phosphorylated on tyrosine and serine.
- pp120 phosphorylation kinetics mirrored EGF receptor autophosphorylation and occupancy, suggesting it's a direct substrate.
Conclusions:
- pp120 is identified as a novel endogenous substrate of the EGF receptor in hepatocytes.
- pp120 phosphorylation is closely associated with EGF receptor signaling and may play a role in EGF-stimulated hepatocyte growth.