Effects of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on metabolism of the epidermal growth factor receptor in normal human fibroblasts.

Abstract

The biosynthesis, phosphorylation, and degradation of the epidermal growth factor (EGF) receptor were examined in normal human fibroblasts. The receptor was initially synthesized as an Mr = 160,000 immature form which matured to an Mr = 170,000 form in a monensin-sensitive manner. Tunicamycin treatment led to the accumulation of an Mr = 130,000 protein. The receptor was phosphorylated on serine and threonine residues in normally growing and quiescent cells, and treatment with EGF or the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) resulted in a two-to threefold increase in receptor-bound phosphate. EGF increased the amount of phosphoserine and phosphothreonine and caused the appearance of a minor amount of phosphotyrosine. TPA increased the levels of phosphoserine and phosphothreonine exclusively. Prior treatment with TPA inhibited the EGF-dependent appearance of phosphotyrosine in the receptor. Analysis of tryptic phosphopeptides revealed that six of the seven major peptides were common to the receptor from cells treated with EGF or TPA. EGF strongly stimulated [3H]thymidine incorporation in confluent cells, increased final saturation density three to fourfold, and increased whole-cell levels of phosphotyrosine about threefold. Treatment of cells with TPA before addition of EGF inhibited all three of these EGF-dependent responses. EGF also decreased the receptor half-life from 15 h to 1 h, but this was not inhibited by TPA. TPA alone had no detectable effect on the receptor half-life.Epidermal growth factor (EGF) binds to a specific plasma membrane receptor protein which functions in mediating EGF-induced mitogenesis. Responses to EGF binding to its receptor include activation of a receptor-associated, tyrosine-specific protein kinase (9, 10), increased phosphorylation of the receptor protein (15, 26) and of other unidentified proteins (11, 21), increased nutrient transport (1, 24), transient influx of Ca2+ and Na2+ ions (38, 45), changes in membrane phospholipid metabolism (40, 43), and ultimately cell division. The primary event is probably an activation of the receptor protein kinase, which could induce the other phenomena through phosphorylation of appropriate protein substrates. EGF-dependent stimulation of the EGF receptor-associated protein kinase has been demonstrated in vitro in membrane preparations from many different cell types (4, 13, 18) and in vivo for the A431 epidermoid carcinoma cell line (26) and 3T3 cells (11). This unusual tyrosine-specific protein kinase activity has also been found associated with the receptors for platelet-derived growth factor (PDGF) (17), insulin (30), and somatomedin C (28) and with the transforming gene products of a number of oncogenic viruses (2). In vivo phosphorylation of the EGF receptor from A431 cells is of a complex nature; phosphate has been found on serine, threonine, and tyrosine residues (12, 15}. The insulin receptor has also been shown to contain these three phosphoamino acids (31). The functional significance of receptor phosphorylation is...

Vaccinia virus growth factor stimulates tyrosine protein kinase activity of A431 cell epidermal growth factor receptors.

“…Phosphate labeling of the EGF receptor at serine and threonine increases by about 100%, but phosphate labeling at tyrosine increases by at least 300% (17). Tyrosine phosphorylation of EGF receptors in EGFtreated fibroblasts has also been detected (9). Most of the phosphotyrosine is found at a single position in the EGF receptor, now identified as tyrosine 1173 (10).…”

Protein kinase Cα activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase

“…Several PKC isoforms physically interact with tyrosine kinase receptors, including epidermal growth factor (EGF) (Chen et al, 1996), insulin-like growth factor-I (IGF-I) (Li et al, 1998) and insulin (Formisano et al, 1998;Caruso et al, 2001;Oriente et al, 2001) receptors. In turn, PKC-mediated serine/threonine phosphorylation has been shown to inhibit EGFR (Decker, 1984;Downward et al, 1984;Chen et al, 1996), insulin receptor (Takayama et al, 1984;Caruso et al, 2001), and hepatocyte growth factor receptor (Met) (Gandino et al, 1994) by attenuating their intrinsic tyrosine-kinase enzymatic activity and their autophosphorylation on tyrosine.…”

The calpain cleavage sites in the epidermal growth factor receptor kinase domain

“…Studies of ligand-induced receptor internalisation in normal and cancer cells, and in perfused rat liver, showed that endocytosis is followed by proteolysis of receptor predominantly in the lysosomes, with half-life depending on cell type (Beguinot et al, 1984;Stoscheck and Carpenter, 1984a,b;Dunn et al, 1986;Gamou and Shimizu, 1987;Sunada et al, 1990). Endocytosis and degradation were originally proposed to provide a mechanism for the generation of a non-membrane-bound second messenger, communicating cell membrane events to the nucleus (Das and Fox, 1978;Decker, 1984;Cohen and Fava, 1985;Kay et al, 1986). However, internalisation-defective EGF receptor transfectants have been shown to respond to mitogenic stimulation by EGF, suggesting that receptor degradation is not an obligatory step in signal transduction (Wells et al, 1990).…”