Abstract
Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events.
Highlights
Which may lead to activation of protein kinase C
The major peak eluting at about 30% acetonitrile showed increased levels of both phosphotyrosine and phosphothreonine
Since epidermal growth factor (EGF) treatment of cells results in tyrosine phosphorylation of PLC-7 [5,6,7] and phosphorylation of threonine 654 inhibits the tyrosine kinase activity of the EGF receptor [23], we examined the effects of EGF and tetradecanoylphorbol 13-acetate (TPA) on tyrosine phosphorylation of PLC--y in cells expressing wild-type or Ala-654 receptors (Fig. 3)
Summary
&Zarer&-[3zP]Orthophosphate and [?S]methionine/cysteine were from ICN Radiochemicals. NIH-3T3 cells were transfected with pCOl2-EGFR containing the various receptor genes and plasmid pSVneo [28] as described previously [26] and colonies resistant to the antibiotic G418 were selected. Muppmg-Cells were labeled with ls’P]orthophosphate and EGF receptor isolated by immune precipitation and polyacrvlamide electrophoresis as described [25]. Phosphoamino acid analysis of acid hydrolysates of eluted peptides were performed as described previously [25]. Cells were washed once with 1 ml of cold phosphate-buffered saline and lysed in 1 ml of 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1%. Zmmuno~Zot AnuZys&-Cells expressing each receptor type were lysed directly in Laemmli sample buffer [31] and 40 +g of cellular protein were run on 7.2% polyacrylamide gels. Measurements of inositol phosphate levels in the presence of LiCl and binding of ‘*‘I-EGF were performed as [29]
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