Tumor promoters block tyrosine-specific phosphorylation of the epidermal growth factor receptor.

Abstract

Tyrosine-specific phosphorylation of the epidermal growth factor (EGF) receptor in hormonally stimulated A431 cells is blocked by three chemically distinct classes of tumor promoters. Tumor-promoting esters of the diterpene phorbol (phorbol 12-myristate 13-acetate, P-phorbol 12,13-dibutyrate, and (3-phorbol 12,13-didecanoate), indole alkaloids (teleocidin and lyngbyatoxin A), and polyacetates (aplysiatoxin and debromoaplysiatoxin) all inhibited EGF-stimulated phosphorylation of the receptor. Non-tumor-promoting analogs (,Bphorbol, a-phorbol 12,13-didecanoate, and hydrolyzed teleocidin) had no effect on the levels of receptor phosphorylation.The ED5o values of the inhibitory effect (0.1-3 ng/ml) reflected the relative tumor-promoting abilities of these compounds in vivo. None of the tumor promoters tested significantly de-creased the overall specific binding of 125I-labeled EGF to A431 cells. Scatchard analysis, however, revealed two apparent EGF receptors in this cell type. The dose-responses for tumor-promoter inhibition of EGF receptor tyrosine phosphorylation and high-affinity EGF binding were similar, suggesting that the same initial event is responsible for both effects.This demonstrates a correlation between modulation of EGF receptor binding and phosphorylation of tyrosine by tumor promoters. The data suggest a possible role for protein kinase C, the putative cellular receptor for these tumor promoters, in the mechanism of action.Tumor promoters can modulate the action of epidermal growth factor (EGF) by reducing EGF receptor binding (1-4) and internalization (5, 6) and by potentiating the mitogenic activity of EGF in quiescent cells (7,8). Upon binding to cells, EGF stimulates the tyrosine phosphorylation of its receptors (9, 10) via a receptor-associated kinase activity. The potential regulatory role of tyrosine kinase activity is suggested by its association with the action of other growth factors (insulin and platelet-derived growth factor) (11-13) and its apparent requirement for oncogenic transformation by a number of retroviruses (14,15). This led us to examine whether the effects of tumor promoters on the action of EGF involved changes in receptor phosphorylation.We have found that three chemically distinct classes of tumor promoters [esters of the diterpene phorbol, indole alkaloids (16), and polyacetates (17) 32Pi Labeling and Detergent Extraction of Cells. Low-density cultures (105 cells per 35-mm well) of A431 cells were preincubated with 32p; at 0.5 mCi/ml in phosphate-free DME medium plus dialyzed heat-inactivated fetal calf serum for 3-4 hr at 37°C. Dimethyl sulfoxide (Me2SO; final concentration 1%), tumor promoters, or nonpromoting analogs (dissolved in Me2SO) were added for 10 min. EGF was then added for an additional 40 min. The cultures were placed on ice, the binding media were removed, and the cells were scraped and extracted with Tris-buffered Triton (10 mM Tris'HCl/1% Triton X-100/10 mM NaCl/5 mM EDTA/1 mM phenylmethylsulfonyl fluoride/0.1% bovine serum albumin, pH 8).Phosph...

Protein phosphorylation in a tetradecanoyl phorbol acetate-nonproliferative variant of 3T3 cells.

“…The pleiotypic responses include increased transport of ions and hexoses (18,19,29,39,41), increased activity of rate-limiting enzymes in anabolic pathways (e.g., ornithine decarboxylase) (33,34), and increased phosphorylation of a number of cellular proteins (28,36,39). Of particular relevance to the work reported in this communication are the findings that both EGF and TPA cause increased serine/threonine phosphorylation of the EGF receptor (11,17,20,23), that TPA stimulates phosphorylation of an 80-kDa soluble protein (36), and that all the mitogens (including TPA) cause tyrosyl phosphorylation of a 42-kDa cellular protein (4,16,21,31).…”

“…Active protein kinase-C can phosphorylate the EGF receptor both in vivo and in vitro (11,17,20,23). This phosphorylation apparently results in a decreased affinity of the EGF receptor for its ligand.…”

“…For two-dimensional electrophoresis of acidic, 32P-labeled soluble proteins, confluent fibroblasts were exposed to serum-free Dulbecco modified Eagle medium plus 1% bovine serum albumin (crystallized and lyophilized; Sigma Chemical Co.) for 16 (11,17,20,23 (Fig. 1).…”

Protein kinase C-mediated negative-feedback inhibition of unstimulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells

“…Furthermore, the phosphorylation of a protein of Mr 80000, which is a well-characterized protein kinase C substrate (Blackshear et al, 1986;Isacke et al, 1986), is stimulated by TPA in control cultures, but not in the down-modulated cells (Blackshear et al, 1985). We have found (results not shown) that the down-modulated cells fail to exhibit TPA-mediated inhibition of 125I-EGF binding, an effect strongly correlated with activation of protein kinase C (Brown et al, 1979Friedman et al, 1984;Hunter et al, 1984;Davis & Czech, 1985Downward et al, 1985;Lin et al, 1986). Although the molecular mechanism causing the down-modulation of protein kinase C is not fully understood, it appears to involve the proteolytic processing of the activated, translocated, enzyme (Ballester & Rosen, 1985).…”

Control of ion fluxes by mitogenic polypeptides.

“…22 It is known that protein kinase C can specifically phosphorylate the EGF receptor and, as a result, block its ability to act as a tyrosine-specific protein kinase. 23 " 25 It is also known that addition of mitogens to cells often results in increased turnover of phosphatidylinositol, which could yield increased intracellular concentrations of calcium as well as diacylglycerol, both activators of protein kinase C. 21 It seems likely therefore that protein kinase C may act as a negative modulator of the mitogen receptors.…”