Abstract
Activation of the EGF receptor tyrosine kinase by ligand indirectly activates a series of other cellular enzymes, including protein kinase C. To test the hypothesis that phosphorylation of the EGF receptor by protein kinase C provides an intracellular negative feedback loop to attenuate EGF receptor signaling, we used scanning EM to follow the characteristic EGF-induced retraction of lamellipodia and concomitant cell shape changes. Wild type and mutant EGF receptors were expressed in receptor-deficient NR6 cells. The mutant receptors were prepared by truncation at C' terminal residue 973 (c'973) to provide resistance to ligand-induced down regulation that strongly attenuates receptor signaling and by replacement of threonine 654 (T654) with alanine (A654) to remove the site of phosphorylation by protein kinase C. Cells expressing WT and c'973 EGF receptors demonstrated characteristic lamellipodial retraction after exposure to EGF, with the non-down regulating c'973 EGF receptors responding more rapidly. Exposure of cells to TPA blocked this response. Replacement of T654 by alanine resulted in EGF receptors that were resistant to TPA. Cells expressing the A654 mutation underwent more rapid and more extensive morphologic changes than cells with the corresponding T654 EGF receptor. In cells expressing T654 EGF receptors, down regulation of protein kinase C resulted in more rapid and extensive EGF-induced changes similar to those seen in cells expressing A654 EGF receptors. These data indicate that activation of protein kinase C and subsequent phosphorylation of the EGF receptor at T654 lead to rapid physiological attenuation of EGF receptor signaling.
Full Text
The Full Text of this article is available as a PDF (3.4 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Anklesaria P., Teixidó J., Laiho M., Pierce J. H., Greenberger J. S., Massagué J. Cell-cell adhesion mediated by binding of membrane-anchored transforming growth factor alpha to epidermal growth factor receptors promotes cell proliferation. Proc Natl Acad Sci U S A. 1990 May;87(9):3289–3293. doi: 10.1073/pnas.87.9.3289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ballester R., Rosen O. M. Fate of immunoprecipitable protein kinase C in GH3 cells treated with phorbol 12-myristate 13-acetate. J Biol Chem. 1985 Dec 5;260(28):15194–15199. [PubMed] [Google Scholar]
- Besterman J. M., Duronio V., Cuatrecasas P. Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6785–6789. doi: 10.1073/pnas.83.18.6785. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bird T. A., Saklatvala J. Down-modulation of epidermal growth factor receptor affinity in fibroblasts treated with interleukin 1 or tumor necrosis factor is associated with phosphorylation at a site other than threonine 654. J Biol Chem. 1990 Jan 5;265(1):235–240. [PubMed] [Google Scholar]
- Bishop W. R., Bell R. M. Attenuation of sn-1,2-diacylglycerol second messengers. Metabolism of exogenous diacylglycerols by human platelets. J Biol Chem. 1986 Sep 25;261(27):12513–12519. [PubMed] [Google Scholar]
- Brachmann R., Lindquist P. B., Nagashima M., Kohr W., Lipari T., Napier M., Derynck R. Transmembrane TGF-alpha precursors activate EGF/TGF-alpha receptors. Cell. 1989 Feb 24;56(4):691–700. doi: 10.1016/0092-8674(89)90591-6. [DOI] [PubMed] [Google Scholar]
- Bretscher A. Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J Cell Biol. 1989 Mar;108(3):921–930. doi: 10.1083/jcb.108.3.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Campos-Gonzalez R., Kanemitsu M., Boynton A. L. Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling. Cell Motil Cytoskeleton. 1990;15(1):34–40. doi: 10.1002/cm.970150106. [DOI] [PubMed] [Google Scholar]
- Chen W. S., Lazar C. S., Lund K. A., Welsh J. B., Chang C. P., Walton G. M., Der C. J., Wiley H. S., Gill G. N., Rosenfeld M. G. Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation. Cell. 1989 Oct 6;59(1):33–43. doi: 10.1016/0092-8674(89)90867-2. [DOI] [PubMed] [Google Scholar]
- Chinkers M., McKanna J. A., Cohen S. Rapid rounding of human epidermoid carcinoma cells A-431 induced by epidermal growth factor. J Cell Biol. 1981 Feb;88(2):422–429. doi: 10.1083/jcb.88.2.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cochet C., Gill G. N., Meisenhelder J., Cooper J. A., 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] [Google Scholar]
- Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
- Collins M. K., Rozengurt E. Homologous and heterologous mitogenic desensitization of Swiss 3T3 cells to phorbol esters and vasopressin: role of receptor and postreceptor steps. J Cell Physiol. 1984 Feb;118(2):133–142. doi: 10.1002/jcp.1041180205. [DOI] [PubMed] [Google Scholar]
- Countaway J. L., McQuilkin P., Gironès N., Davis R. J. Multisite phosphorylation of the epidermal growth factor receptor. Use of site-directed mutagenesis to examine the role of serine/threonine phosphorylation. J Biol Chem. 1990 Feb 25;265(6):3407–3416. [PubMed] [Google Scholar]
- Davis R. J., Czech M. P. Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts. J Biol Chem. 1987 May 15;262(14):6832–6841. [PubMed] [Google Scholar]
- Davis R. J. Independent mechanisms account for the regulation by protein kinase C of the epidermal growth factor receptor affinity and tyrosine-protein kinase activity. J Biol Chem. 1988 Jul 5;263(19):9462–9469. [PubMed] [Google Scholar]
- Decker S. J., Ellis C., Pawson T., Velu T. Effects of substitution of threonine 654 of the epidermal growth factor receptor on epidermal growth factor-mediated activation of phospholipase C. J Biol Chem. 1990 Apr 25;265(12):7009–7015. [PubMed] [Google Scholar]
- Downward J., Waterfield M. D., Parker P. J. Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity. J Biol Chem. 1985 Nov 25;260(27):14538–14546. [PubMed] [Google Scholar]
- Friedman B., Frackelton A. R., Jr, Ross A. H., Connors J. M., Fujiki H., Sugimura T., Rosner M. R. 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. doi: 10.1073/pnas.81.10.3034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Glenney J. R., Jr, Chen W. S., Lazar C. S., Walton G. M., Zokas L. M., Rosenfeld M. G., Gill G. N. Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies. Cell. 1988 Mar 11;52(5):675–684. doi: 10.1016/0092-8674(88)90405-9. [DOI] [PubMed] [Google Scholar]
- Haigler H., Ash J. F., Singer S. J., Cohen S. Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3317–3321. doi: 10.1073/pnas.75.7.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heisermann G. J., Wiley H. S., Walsh B. J., Ingraham H. A., Fiol C. J., Gill G. N. Mutational removal of the Thr669 and Ser671 phosphorylation sites alters substrate specificity and ligand-induced internalization of the epidermal growth factor receptor. J Biol Chem. 1990 Aug 5;265(22):12820–12827. [PubMed] [Google Scholar]
- Hepler J. R., Nakahata N., Lovenberg T. W., DiGuiseppi J., Herman B., Earp H. S., Harden T. K. Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-trisphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells. J Biol Chem. 1987 Mar 5;262(7):2951–2956. [PubMed] [Google Scholar]
- Hunter T., Ling N., Cooper J. A. 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. doi: 10.1038/311480a0. [DOI] [PubMed] [Google Scholar]
- Jackowski M. M., Swift L. L., Cohen S., McKanna J. A. Morphologic changes in human carcinoma cells (A-431) stimulated by epidermal growth factor: effect of cholesterol and low-density lipoproteins on the ruffling response. J Cell Physiol. 1990 Mar;142(3):458–468. doi: 10.1002/jcp.1041420304. [DOI] [PubMed] [Google Scholar]
- Kennedy J. R., Williams R. W., Gray J. P. Use of Peldri II (a fluorocarbon solid at room temperature) as an alternative to critical point drying for biological tissues. J Electron Microsc Tech. 1989 Feb;11(2):117–125. doi: 10.1002/jemt.1060110205. [DOI] [PubMed] [Google Scholar]
- King C. S., Cooper J. A. Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation. J Biol Chem. 1986 Aug 5;261(22):10073–10078. [PubMed] [Google Scholar]
- Lin C. R., Chen W. S., Kruiger W., Stolarsky L. S., Weber W., Evans R. M., Verma I. M., Gill G. N., Rosenfeld M. G. Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells. Science. 1984 May 25;224(4651):843–848. doi: 10.1126/science.6326261. [DOI] [PubMed] [Google Scholar]
- Lin C. R., Chen W. S., Lazar C. S., Carpenter C. D., Gill G. N., Evans R. M., Rosenfeld M. G. 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. doi: 10.1016/0092-8674(86)90006-1. [DOI] [PubMed] [Google Scholar]
- Lund K. A., Lazar C. S., Chen W. S., Walsh B. J., Welsh J. B., Herbst J. J., Walton G. M., Rosenfeld M. G., Gill G. N., Wiley H. S. Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation. J Biol Chem. 1990 Nov 25;265(33):20517–20523. [PubMed] [Google Scholar]
- Lund K. A., Opresko L. K., Starbuck C., Walsh B. J., Wiley H. S. Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization. J Biol Chem. 1990 Sep 15;265(26):15713–15723. [PubMed] [Google Scholar]
- Miyata Y., Nishida E., Koyasu S., Yahara I., Sakai H. Protein kinase C-dependent and -independent pathways in the growth factor-induced cytoskeletal reorganization. J Biol Chem. 1989 Sep 15;264(26):15565–15568. [PubMed] [Google Scholar]
- Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
- Olashaw N. E., O'Keefe E. J., Pledger W. J. Platelet-derived growth factor modulates epidermal growth factor receptors by a mechanism distinct from that of phorbol esters. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3834–3838. doi: 10.1073/pnas.83.11.3834. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pruss R. M., Herschman H. R. Variants of 3T3 cells lacking mitogenic response to epidermal growth factor. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3918–3921. doi: 10.1073/pnas.74.9.3918. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schlessinger J., Geiger B. Epidermal growth factor induces redistribution of actin and alpha-actinin in human epidermal carcinoma cells. Exp Cell Res. 1981 Aug;134(2):273–279. doi: 10.1016/0014-4827(81)90426-2. [DOI] [PubMed] [Google Scholar]
- Scott J., Urdea M., Quiroga M., Sanchez-Pescador R., Fong N., Selby M., Rutter W. J., Bell G. I. Structure of a mouse submaxillary messenger RNA encoding epidermal growth factor and seven related proteins. Science. 1983 Jul 15;221(4607):236–240. doi: 10.1126/science.6602382. [DOI] [PubMed] [Google Scholar]
- Shoyab M., De Larco J. E., Todaro G. J. Biologically active phorbol esters specifically alter affinity of epidermal growth factor membrane receptors. Nature. 1979 May 31;279(5712):387–391. doi: 10.1038/279387a0. [DOI] [PubMed] [Google Scholar]
- Slivka S. R., Meier K. E., Insel P. A. Alpha 1-adrenergic receptors promote phosphatidylcholine hydrolysis in MDCK-D1 cells. A mechanism for rapid activation of protein kinase C. J Biol Chem. 1988 Sep 5;263(25):12242–12246. [PubMed] [Google Scholar]
- Stoscheck C. M., Carpenter G. Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts. J Cell Biol. 1984 Mar;98(3):1048–1053. doi: 10.1083/jcb.98.3.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
- Walton G. M., Chen W. S., Rosenfeld M. G., Gill G. N. Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals self-phosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates. J Biol Chem. 1990 Jan 25;265(3):1750–1754. [PubMed] [Google Scholar]
- Wells A., Bishop J. M. Genetic determinants of neoplastic transformation by the retroviral oncogene v-erbB. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7597–7601. doi: 10.1073/pnas.85.20.7597. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wells A., Welsh J. B., Lazar C. S., Wiley H. S., Gill G. N., Rosenfeld M. G. Ligand-induced transformation by a noninternalizing epidermal growth factor receptor. Science. 1990 Feb 23;247(4945):962–964. doi: 10.1126/science.2305263. [DOI] [PubMed] [Google Scholar]
- 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. doi: 10.1083/jcb.103.4.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wiley H. S., Cunningham D. D. The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem. 1982 Apr 25;257(8):4222–4229. [PubMed] [Google Scholar]
- Wong S. T., Winchell L. F., McCune B. K., Earp H. S., Teixidó J., Massagué J., Herman B., Lee D. C. The TGF-alpha precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction. Cell. 1989 Feb 10;56(3):495–506. doi: 10.1016/0092-8674(89)90252-3. [DOI] [PubMed] [Google Scholar]
- Wright T. M., Shin H. S., Raben D. M. Sustained increase in 1,2-diacylglycerol precedes DNA synthesis in epidermal-growth-factor-stimulated fibroblasts. Evidence for stimulated phosphatidylcholine hydrolysis. Biochem J. 1990 Apr 15;267(2):501–507. doi: 10.1042/bj2670501. [DOI] [PMC free article] [PubMed] [Google Scholar]