Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. Nov 1, 1994; 127(3): 847–857.
PMCID: PMC2120228

Epidermal growth factor receptor-mediated cell motility: phospholipase C activity is required, but mitogen-activated protein kinase activity is not sufficient for induced cell movement

Abstract

We recently have demonstrated that EGF receptor (EGFR)-induced cell motility requires receptor kinase activity and autophosphorylation (P. Chen, K. Gupta, and A. Wells. 1994. J. Cell Biol. 124:547-555). This suggests that the immediate downstream effector molecule contains a src homology-2 domain. Phospholipase C gamma (PLC gamma) is among the candidate transducers of this signal because of its potential roles in modulating cytoskeletal dynamics. We utilized signaling-restricted EGFR mutants expressed in receptor devoid NR6 cells to determine if PLC activation is necessary for EGFR-mediated cell movement. Exposure to EGF (25 nM) augmented PLC activity in all five EGFR mutant cell lines which also responded by increased cell movement. Basal phosphoinositide turnover was not affected by EGF in the lines which do not present the enhanced motility response. The correlation between EGFR-mediated cell motility and PLC activity suggested, but did not prove, a causal link. A specific inhibitor of PLC, U73122 (1 microM) diminished both the EGF- induced motility and PLC responses, while its inactive analogue U73343 had no effect on these responses. Both the PLC and motility responses were decreased by expression of a dominant-negative PLC gamma-1 fragment in EGF-responsive infectant lines. Lastly, anti-sense oligonucleotides (20 microM) to PLC gamma-1 reduced both responses in NR6 cells expressing wild-type EGFR. These findings strongly support PLC gamma as the immediate post receptor effector in this motogenic pathway. We have demonstrated previously that EGFR-mediated cell motility and mitogenic signaling pathways are separable. The point of divergence is undefined. All kinase-active EGFR mutants induced the mitogenic response while only those which are autophosphorylated induced PLC activity. U73122 did not affect EGF-induced thymidine incorporation in these motility-responsive infectant cell lines. In addition, the dominant-negative PLC gamma-1 fragment did not diminish EGF-induced thymidine incorporation. All kinase active EGFR stimulated mitogen-activated protein (MAP) kinase activity, regardless of whether the receptors induced cell movement; this EGF-induced MAP kinase activity was not affected by U73122 at concentrations that depressed the motility response. Thus, the signaling pathways which lead to motility and cell proliferation diverge at the immediate post-receptor stage, and we suggest that this is accomplished by differential activation of effector molecules.

Full Text

The Full Text of this article is available as a PDF (1.5M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ahn NG, Weiel JE, Chan CP, Krebs EG. Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells. J Biol Chem. 1990 Jul 15;265(20):11487–11494. [PubMed]
  • Banno Y, Nakashima T, Kumada T, Ebisawa K, Nonomura Y, Nozawa Y. Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes. J Biol Chem. 1992 Apr 5;267(10):6488–6494. [PubMed]
  • Bar-Sagi D, Rotin D, Batzer A, Mandiyan V, Schlessinger J. SH3 domains direct cellular localization of signaling molecules. Cell. 1993 Jul 16;74(1):83–91. [PubMed]
  • Barrandon Y, Green H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-alpha and epidermal growth factor. Cell. 1987 Sep 25;50(7):1131–1137. [PubMed]
  • Berridge MJ. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. [PubMed]
  • Blay J, Brown KD. Epidermal growth factor promotes the chemotactic migration of cultured rat intestinal epithelial cells. J Cell Physiol. 1985 Jul;124(1):107–112. [PubMed]
  • Bleasdale JE, Thakur NR, Gremban RS, Bundy GL, Fitzpatrick FA, Smith RJ, Bunting S. Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther. 1990 Nov;255(2):756–768. [PubMed]
  • Bornfeldt KE, Raines EW, Nakano T, Graves LM, Krebs EG, Ross R. Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J Clin Invest. 1994 Mar;93(3):1266–1274. [PMC free article] [PubMed]
  • Carpenter G. Receptor tyrosine kinase substrates: src homology domains and signal transduction. FASEB J. 1992 Nov;6(14):3283–3289. [PubMed]
  • Chang CP, Kao JP, Lazar CS, Walsh BJ, Wells A, Wiley HS, Gill GN, Rosenfeld MG. Ligand-induced internalization and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor. J Biol Chem. 1991 Dec 5;266(34):23467–23470. [PubMed]
  • Chen P, Gupta K, Wells A. Cell movement elicited by epidermal growth factor receptor requires kinase and autophosphorylation but is separable from mitogenesis. J Cell Biol. 1994 Feb;124(4):547–555. [PMC free article] [PubMed]
  • Chen WS, Lazar CS, Lund KA, Welsh JB, Chang CP, Walton GM, Der CJ, Wiley HS, Gill GN, Rosenfeld MG. 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. [PubMed]
  • Chen WS, Lazar CS, Poenie M, Tsien RY, Gill GN, Rosenfeld MG. Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. Nature. 328(6133):820–823. [PubMed]
  • Cunningham CC, Stossel TP, Kwiatkowski DJ. Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science. 1991 Mar 8;251(4998):1233–1236. [PubMed]
  • Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed]
  • Decker SJ. Transmembrane signaling by epidermal growth factor receptors lacking autophosphorylation sites. J Biol Chem. 1993 May 5;268(13):9176–9179. [PubMed]
  • Downward J, Parker P, Waterfield MD. Autophosphorylation sites on the epidermal growth factor receptor. Nature. 1984 Oct 4;311(5985):483–485. [PubMed]
  • Fantl WJ, Escobedo JA, Martin GA, Turck CW, del Rosario M, McCormick F, Williams LT. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell. 1992 May 1;69(3):413–423. [PubMed]
  • Goldschmidt-Clermont PJ, Kim JW, Machesky LM, Rhee SG, Pollard TD. Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation. Science. 1991 Mar 8;251(4998):1231–1233. [PubMed]
  • Hack N, Sue-A-Quan A, Mills GB, Skorecki KL. Expression of human tyrosine kinase-negative epidermal growth factor receptor amplifies signaling through endogenous murine epidermal growth factor receptor. J Biol Chem. 1993 Dec 15;268(35):26441–26446. [PubMed]
  • Hepler JR, Nakahata N, Lovenberg TW, DiGuiseppi J, Herman B, Earp HS, Harden TK. 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]
  • Hernández-Sotomayor SM, Carpenter G. Non-catalytic activation of phospholipase C-gamma 1 in vitro by epidermal growth factor receptor. Biochem J. 1993 Jul 15;293(Pt 2):507–511. [PMC free article] [PubMed]
  • Homma Y, Takenawa T. Inhibitory effect of src homology (SH) 2/SH3 fragments of phospholipase C-gamma on the catalytic activity of phospholipase C isoforms. Identification of a novel phospholipase C inhibitor region. J Biol Chem. 1992 Oct 25;267(30):21844–21849. [PubMed]
  • Kim HK, Kim JW, Zilberstein A, Margolis B, Kim JG, Schlessinger J, Rhee SG. PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254. Cell. 1991 May 3;65(3):435–441. [PubMed]
  • Kundra V, Escobedo JA, Kazlauskas A, Kim HK, Rhee SG, Williams LT, Zetter BR. Regulation of chemotaxis by the platelet-derived growth factor receptor-beta. Nature. 1994 Feb 3;367(6462):474–476. [PubMed]
  • Marengere LE, Songyang Z, Gish GD, Schaller MD, Parsons JT, Stern MJ, Cantley LC, Pawson T. SH2 domain specificity and activity modified by a single residue. Nature. 1994 Jun 9;369(6480):502–505. [PubMed]
  • Meisenhelder J, Suh PG, Rhee SG, Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell. 1989 Jun 30;57(7):1109–1122. [PubMed]
  • Nishida E, Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 1993 Apr;18(4):128–131. [PubMed]
  • Nishimura R, Li W, Kashishian A, Mondino A, Zhou M, Cooper J, Schlessinger J. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor. Mol Cell Biol. 1993 Nov;13(11):6889–6896. [PMC free article] [PubMed]
  • Pawson T, Gish GD. SH2 and SH3 domains: from structure to function. Cell. 1992 Oct 30;71(3):359–362. [PubMed]
  • Pelech SL, Sanghera JS. MAP kinases: charting the regulatory pathways. Science. 1992 Sep 4;257(5075):1355–1356. [PubMed]
  • Powis G, Seewald MJ, Gratas C, Melder D, Riebow J, Modest EJ. Selective inhibition of phosphatidylinositol phospholipase C by cytotoxic ether lipid analogues. Cancer Res. 1992 May 15;52(10):2835–2840. [PubMed]
  • Pruss RM, Herschman HR. Variants of 3T3 cells lacking mitogenic response to epidermal growth factor. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3918–3921. [PMC free article] [PubMed]
  • Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. [PubMed]
  • Rotin D, Honegger AM, Margolis BL, Ullrich A, Schlessinger J. Presence of SH2 domains of phospholipase C gamma 1 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor. J Biol Chem. 1992 May 15;267(14):9678–9683. [PubMed]
  • Rotin D, Margolis B, Mohammadi M, Daly RJ, Daum G, Li N, Fischer EH, Burgess WH, Ullrich A, Schlessinger J. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma. EMBO J. 1992 Feb;11(2):559–567. [PMC free article] [PubMed]
  • Rozakis-Adcock M, Fernley R, Wade J, Pawson T, Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 1993 May 6;363(6424):83–85. [PubMed]
  • Sekar MC, Dixon JF, Hokin LE. The formation of inositol 1,2-cyclic 4,5-trisphosphate and inositol 1,2-cyclic 4-bisphosphate on stimulation of mouse pancreatic minilobules with carbamylcholine. J Biol Chem. 1987 Jan 5;262(1):340–344. [PubMed]
  • Sekar MC, Sambandam V, McDonald JM. Bombesin and muscarinic receptor activation in rat pancreas generate cyclic inositol monophosphate: possible involvement of different phospholipase C isoenzymes. Biochem Biophys Res Commun. 1993 May 14;192(3):1079–1085. [PubMed]
  • Smith RJ, Sam LM, Justen JM, Bundy GL, Bala GA, Bleasdale JE. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Exp Ther. 1990 May;253(2):688–697. [PubMed]
  • Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, et al. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. [PubMed]
  • Sorkin A, Helin K, Waters CM, Carpenter G, Beguinot L. Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors. J Biol Chem. 1992 Apr 25;267(12):8672–8678. [PubMed]
  • Stossel TP. On the crawling of animal cells. Science. 1993 May 21;260(5111):1086–1094. [PubMed]
  • Suen KL, Bustelo XR, Pawson T, Barbacid M. Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors. Mol Cell Biol. 1993 Sep;13(9):5500–5512. [PMC free article] [PubMed]
  • Valius M, Kazlauskas A. Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell. 1993 Apr 23;73(2):321–334. [PubMed]
  • Vega QC, Cochet C, Filhol O, Chang CP, Rhee SG, Gill GN. A site of tyrosine phosphorylation in the C terminus of the epidermal growth factor receptor is required to activate phospholipase C. Mol Cell Biol. 1992 Jan;12(1):128–135. [PMC free article] [PubMed]
  • Vogel W, Lammers R, Huang J, Ullrich A. Activation of a phosphotyrosine phosphatase by tyrosine phosphorylation. Science. 1993 Mar 12;259(5101):1611–1614. [PubMed]
  • Walton GM, Chen WS, Rosenfeld MG, Gill GN. 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]
  • Wells A, Bishop JM. Genetic determinants of neoplastic transformation by the retroviral oncogene v-erbB. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7597–7601. [PMC free article] [PubMed]
  • Wells A, Welsh JB, Lazar CS, Wiley HS, Gill GN, Rosenfeld MG. Ligand-induced transformation by a noninternalizing epidermal growth factor receptor. Science. 1990 Feb 23;247(4945):962–964. [PubMed]
  • Welsh JB, Gill GN, Rosenfeld MG, Wells A. A negative feedback loop attenuates EGF-induced morphological changes. J Cell Biol. 1991 Aug;114(3):533–543. [PMC free article] [PubMed]
  • Wennström S, Siegbahn A, Yokote K, Arvidsson AK, Heldin CH, Mori S, Claesson-Welsh L. Membrane ruffling and chemotaxis transduced by the PDGF beta-receptor require the binding site for phosphatidylinositol 3' kinase. Oncogene. 1994 Feb;9(2):651–660. [PubMed]
  • Wood ER, McDonald OB, Sahyoun N. Quantitative analysis of SH2 domain binding. Evidence for specificity and competition. J Biol Chem. 1992 Jul 15;267(20):14138–14144. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...