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Mol Cell Biol. 1988 June; 8(6): 2302–2308.
PMCID: PMC363427

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.


The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

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Selected References

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  • Adelman JP, Hayflick JS, Vasser M, Seeburg PH. In vitro deletional mutagenesis for bacterial production of the 20,000-dalton form of human pituitary growth hormone. DNA. 1983;2(3):183–193. [PubMed]
  • Beguinot L, Hanover JA, Ito S, Richert ND, Willingham MC, Pastan I. Phorbol esters induce transient internalization without degradation of unoccupied epidermal growth factor receptors. Proc Natl Acad Sci U S A. 1985 May;82(9):2774–2778. [PubMed]
  • Benton WD, Davis RW. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. [PubMed]
  • Brown KD, Dicker P, Rozengurt E. Inhibition of epidermal growth factor binding to surface receptors by tumor promotors. Biochem Biophys Res Commun. 1979 Feb 28;86(4):1037–1043. [PubMed]
  • Carpenter G, Cohen S. Epidermal growth factor. Annu Rev Biochem. 1979;48:193–216. [PubMed]
  • Cochet C, Gill GN, Meisenhelder J, Cooper JA, Hunter T. C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Feb 25;259(4):2553–2558. [PubMed]
  • Davis RJ, Czech MP. Tumor-promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal human fibroblasts at threonine-654. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1974–1978. [PubMed]
  • Friedman B, Frackelton AR, Jr, Ross AH, Connors JM, Fujiki H, Sugimura T, Rosner MR. Tumor promoters block tyrosine-specific phosphorylation of the epidermal growth factor receptor. Proc Natl Acad Sci U S A. 1984 May;81(10):3034–3038. [PubMed]
  • Gillam S, Jahnke P, Astell C, Phillips S, Hutchison CA, 3rd, Smith M. Defined transversion mutations at a specific position in DNA using synthetic oligodeoxyribonucleotides as mutagens. Nucleic Acids Res. 1979 Jul 11;6(9):2973–2985. [PMC free article] [PubMed]
  • Gillam S, Smith M. Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum ologodeoxyribonucleotide length. Gene. 1979 Dec;8(1):81–97. [PubMed]
  • Gillam S, Smith M. Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: II. In vitro selection of mutant DNA. Gene. 1979 Dec;8(1):99–106. [PubMed]
  • Hunter T, Ling N, Cooper JA. Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature. 1984 Oct 4;311(5985):480–483. [PubMed]
  • Hunter T, Sefton BM. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. [PubMed]
  • Iwashita S, Fox CF. Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells. J Biol Chem. 1984 Feb 25;259(4):2559–2567. [PubMed]
  • King AC, Cuatrecasas P. Resolution of high and low affinity epidermal growth factor receptors. Inhibition of high affinity component by low temperature, cycloheximide, and phorbol esters. J Biol Chem. 1982 Mar 25;257(6):3053–3060. [PubMed]
  • Kris RM, Lax I, Gullick W, Waterfield MD, Ullrich A, Fridkin M, Schlessinger J. Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbB protein. Cell. 1985 Mar;40(3):619–625. [PubMed]
  • Lee LS, Weinstein IB. Tumor-promoting phorbol esters inhibit binding of epidermal growth factor to cellular receptors. Science. 1978 Oct 20;202(4365):313–315. [PubMed]
  • Lin CR, Chen WS, Lazar CS, Carpenter CD, Gill GN, Evans RM, Rosenfeld MG. Protein kinase C phosphorylation at Thr 654 of the unoccupied EGF receptor and EGF binding regulate functional receptor loss by independent mechanisms. Cell. 1986 Mar 28;44(6):839–848. [PubMed]
  • Livneh E, Prywes R, Kashles O, Reiss N, Sasson I, Mory Y, Ullrich A, Schlessinger J. Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells. J Biol Chem. 1986 Sep 25;261(27):12490–12497. [PubMed]
  • Livneh E, Reiss N, Berent E, Ullrich A, Schlessinger J. An insertional mutant of epidermal growth factor receptor allows dissection of diverse receptor functions. EMBO J. 1987 Sep;6(9):2669–2676. [PubMed]
  • Messing J, Crea R, Seeburg PH. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. [PMC free article] [PubMed]
  • Prywes R, Livneh E, Ullrich A, Schlessinger J. Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization. EMBO J. 1986 Sep;5(9):2179–2190. [PubMed]
  • Riedel H, Dull TJ, Schlessinger J, Ullrich A. A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor. Nature. 1986 Nov 6;324(6092):68–70. [PubMed]
  • Schlessinger J. Allosteric regulation of the epidermal growth factor receptor kinase. J Cell Biol. 1986 Dec;103(6 Pt 1):2067–2072. [PMC free article] [PubMed]
  • Shoyab M, De Larco JE, Todaro GJ. Biologically active phorbol esters specifically alter affinity of epidermal growth factor membrane receptors. Nature. 1979 May 31;279(5712):387–391. [PubMed]
  • Whiteley B, Glaser L. Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor. J Cell Biol. 1986 Oct;103(4):1355–1362. [PMC free article] [PubMed]

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