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. 1991 Nov 1;115(3):809–819. doi: 10.1083/jcb.115.3.809

Signal transduction by nerve growth factor and fibroblast growth factor in PC12 cells requires a sequence of src and ras actions

PMCID: PMC2289191  PMID: 1717492

Abstract

We have investigated the roles of pp60c-src and p21c-ras proteins in transducing the nerve growth factor (NGF) and fibroblast growth factor (FGF) signals which promote the sympathetic neuronlike phenotype in PC12 cells. Neutralizing antibodies directed against either Src or Ras proteins were microinjected into fused PC12 cells. Each antibody both prevented and reversed NGF- or FGF-induced neurite growth, a prominent morphological marker for the neuronal phenotype. These data demonstrate the involvement of both pp60c-src and p21c-ras proteins in NGF and FGF actions in PC12 cells, and establish a physiological role for the pp60c- src tyrosine kinase in signal transduction pathways initiated by receptor tyrosine kinases in these cells. Additional microinjection experiments, using PC12 transfectants containing inducible v-src or ras oncogene activities, demonstrated a specific sequence of Src and Ras actions. Microinjection of anti-Ras antibody blocked v-src-induced neurite growth, but microinjection of anti-Src antibodies had no effect on ras oncogene-induced neurite growth. We propose that a cascade of Src and Ras actions, with Src acting first, is a significant feature of the signal transduction pathways for NGF and FGF. The Src-Ras cascade may define a functional cassette in the signal transduction pathways used by growth factors and other ligands whose receptors have diverse structures and whose range of actions on various cell types include mitogenesis and differentiation.

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Selected References

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  1. Alemà S., Casalbore P., Agostini E., Tatò F. Differentiation of PC12 phaeochromocytoma cells induced by v-src oncogene. Nature. 1985 Aug 8;316(6028):557–559. doi: 10.1038/316557a0. [DOI] [PubMed] [Google Scholar]
  2. Aroian R. V., Koga M., Mendel J. E., Ohshima Y., Sternberg P. W. The let-23 gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily. Nature. 1990 Dec 20;348(6303):693–699. doi: 10.1038/348693a0. [DOI] [PubMed] [Google Scholar]
  3. Bar-Sagi D., Feramisco J. R. Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation. Cell. 1985 Oct;42(3):841–848. doi: 10.1016/0092-8674(85)90280-6. [DOI] [PubMed] [Google Scholar]
  4. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  5. Bolen J. B., Veillette A. A function for the lck proto-oncogene. Trends Biochem Sci. 1989 Oct;14(10):404–407. doi: 10.1016/0968-0004(89)90288-0. [DOI] [PubMed] [Google Scholar]
  6. Borasio G. D., John J., Wittinghofer A., Barde Y. A., Sendtner M., Heumann R. ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons. Neuron. 1989 Jan;2(1):1087–1096. doi: 10.1016/0896-6273(89)90233-x. [DOI] [PubMed] [Google Scholar]
  7. Cai H., Szeberényi J., Cooper G. M. Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells. Mol Cell Biol. 1990 Oct;10(10):5314–5323. doi: 10.1128/mcb.10.10.5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cremins J., Wagner J. A., Halegoua S. Nerve growth factor action is mediated by cyclic AMP- and Ca+2/phospholipid-dependent protein kinases. J Cell Biol. 1986 Sep;103(3):887–893. doi: 10.1083/jcb.103.3.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Der C. J., Finkel T., Cooper G. M. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell. 1986 Jan 17;44(1):167–176. doi: 10.1016/0092-8674(86)90495-2. [DOI] [PubMed] [Google Scholar]
  10. Deshpande A. K., Kung H. F. Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins. Mol Cell Biol. 1987 Mar;7(3):1285–1288. doi: 10.1128/mcb.7.3.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  12. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feramisco J. R., Clark R., Wong G., Arnheim N., Milley R., McCormick F. Transient reversion of ras oncogene-induced cell transformation by antibodies specific for amino acid 12 of ras protein. Nature. 1985 Apr 18;314(6012):639–642. doi: 10.1038/314639a0. [DOI] [PubMed] [Google Scholar]
  14. Furth M. E., Davis L. J., Fleurdelys B., Scolnick E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gorman C., Padmanabhan R., Howard B. H. High efficiency DNA-mediated transformation of primate cells. Science. 1983 Aug 5;221(4610):551–553. doi: 10.1126/science.6306768. [DOI] [PubMed] [Google Scholar]
  16. Greene L. A., Burstein D. E., Black M. M. The role of transcription-dependent priming in nerve growth factor promoted neurite outgrowth. Dev Biol. 1982 Jun;91(2):305–316. doi: 10.1016/0012-1606(82)90037-9. [DOI] [PubMed] [Google Scholar]
  17. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Guerrero I., Pellicer A., Burstein D. E. Dissociation of c-fos from ODC expression and neuronal differentiation in a PC12 subline stably transfected with an inducible N-ras oncogene. Biochem Biophys Res Commun. 1988 Feb 15;150(3):1185–1192. doi: 10.1016/0006-291x(88)90754-1. [DOI] [PubMed] [Google Scholar]
  19. Hagag N., Halegoua S., Viola M. Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21. Nature. 1986 Feb 20;319(6055):680–682. doi: 10.1038/319680a0. [DOI] [PubMed] [Google Scholar]
  20. Halegoua S., Armstrong R. C., Kremer N. E. Dissecting the mode of action of a neuronal growth factor. Curr Top Microbiol Immunol. 1991;165:119–170. doi: 10.1007/978-3-642-75747-1_7. [DOI] [PubMed] [Google Scholar]
  21. Halegoua S. Changes in the phosphorylation and distribution of vinculin during nerve growth factor induced neurite outgrowth. Dev Biol. 1987 May;121(1):97–104. doi: 10.1016/0012-1606(87)90142-4. [DOI] [PubMed] [Google Scholar]
  22. Hall A. ras and GAP--who's controlling whom? Cell. 1990 Jun 15;61(6):921–923. doi: 10.1016/0092-8674(90)90054-i. [DOI] [PubMed] [Google Scholar]
  23. Hama T., Huang K. P., Guroff G. Protein kinase C as a component of a nerve growth factor-sensitive phosphorylation system in PC12 cells. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2353–2357. doi: 10.1073/pnas.83.8.2353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hempstead B. L., Martin-Zanca D., Kaplan D. R., Parada L. F., Chao M. V. High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor. Nature. 1991 Apr 25;350(6320):678–683. doi: 10.1038/350678a0. [DOI] [PubMed] [Google Scholar]
  25. Johnson D., Lanahan A., Buck C. R., Sehgal A., Morgan C., Mercer E., Bothwell M., Chao M. Expression and structure of the human NGF receptor. Cell. 1986 Nov 21;47(4):545–554. doi: 10.1016/0092-8674(86)90619-7. [DOI] [PubMed] [Google Scholar]
  26. Jove R., Mayer B. J., Iba H., Laugier D., Poirier F., Calothy G., Hanafusa T., Hanafusa H. Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters. J Virol. 1986 Dec;60(3):840–848. doi: 10.1128/jvi.60.3.840-848.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kalman D., Wong B., Horvai A. E., Cline M. J., O'Lague P. H. Nerve growth factor acts through cAMP-dependent protein kinase to increase the number of sodium channels in PC12 cells. Neuron. 1990 Mar;4(3):355–366. doi: 10.1016/0896-6273(90)90048-k. [DOI] [PubMed] [Google Scholar]
  28. Kaplan D. R., Hempstead B. L., Martin-Zanca D., Chao M. V., Parada L. F. The trk proto-oncogene product: a signal transducing receptor for nerve growth factor. Science. 1991 Apr 26;252(5005):554–558. doi: 10.1126/science.1850549. [DOI] [PubMed] [Google Scholar]
  29. Kaplan D. R., Martin-Zanca D., Parada L. F. Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF. Nature. 1991 Mar 14;350(6314):158–160. doi: 10.1038/350158a0. [DOI] [PubMed] [Google Scholar]
  30. Kaplan D. R., Morrison D. K., Wong G., McCormick F., Williams L. T. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 Apr 6;61(1):125–133. doi: 10.1016/0092-8674(90)90220-9. [DOI] [PubMed] [Google Scholar]
  31. Kazlauskas A., Ellis C., Pawson T., Cooper J. A. Binding of GAP to activated PDGF receptors. Science. 1990 Mar 30;247(4950):1578–1581. doi: 10.1126/science.2157284. [DOI] [PubMed] [Google Scholar]
  32. Klein R., Jing S. Q., Nanduri V., O'Rourke E., Barbacid M. The trk proto-oncogene encodes a receptor for nerve growth factor. Cell. 1991 Apr 5;65(1):189–197. doi: 10.1016/0092-8674(91)90419-y. [DOI] [PubMed] [Google Scholar]
  33. Korn L. J., Siebel C. W., McCormick F., Roth R. A. Ras p21 as a potential mediator of insulin action in Xenopus oocytes. Science. 1987 May 15;236(4803):840–843. doi: 10.1126/science.3554510. [DOI] [PubMed] [Google Scholar]
  34. Kypta R. M., Goldberg Y., Ulug E. T., Courtneidge S. A. Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell. 1990 Aug 10;62(3):481–492. doi: 10.1016/0092-8674(90)90013-5. [DOI] [PubMed] [Google Scholar]
  35. Lacal J. C., Aaronson S. A. Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein. Mol Cell Biol. 1986 Apr;6(4):1002–1009. doi: 10.1128/mcb.6.4.1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lee P. L., Johnson D. E., Cousens L. S., Fried V. A., Williams L. T. Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor. Science. 1989 Jul 7;245(4913):57–60. doi: 10.1126/science.2544996. [DOI] [PubMed] [Google Scholar]
  37. Levi-Montalcini R. The nerve growth factor 35 years later. Science. 1987 Sep 4;237(4819):1154–1162. doi: 10.1126/science.3306916. [DOI] [PubMed] [Google Scholar]
  38. Levy J. B., Dorai T., Wang L. H., Brugge J. S. The structurally distinct form of pp60c-src detected in neuronal cells is encoded by a unique c-src mRNA. Mol Cell Biol. 1987 Nov;7(11):4142–4145. doi: 10.1128/mcb.7.11.4142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Maher P. A. Nerve growth factor induces protein-tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6788–6791. doi: 10.1073/pnas.85.18.6788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mayer B. J., Jove R., Krane J. F., Poirier F., Calothy G., Hanafusa H. Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants. J Virol. 1986 Dec;60(3):858–867. doi: 10.1128/jvi.60.3.858-867.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. McCormick F. GAP as ras effector or negative regulator? Mol Carcinog. 1990;3(4):185–187. doi: 10.1002/mc.2940030404. [DOI] [PubMed] [Google Scholar]
  43. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  44. Mobley W. C., Schenker A., Shooter E. M. Characterization and isolation of proteolytically modified nerve growth factor. Biochemistry. 1976 Dec 14;15(25):5543–5552. doi: 10.1021/bi00670a019. [DOI] [PubMed] [Google Scholar]
  45. Molloy C. J., Bottaro D. P., Fleming T. P., Marshall M. S., Gibbs J. B., Aaronson S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. doi: 10.1038/342711a0. [DOI] [PubMed] [Google Scholar]
  46. Morrison D. K., Kaplan D. R., Escobedo J. A., Rapp U. R., Roberts T. M., Williams L. T. Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF beta-receptor. Cell. 1989 Aug 25;58(4):649–657. doi: 10.1016/0092-8674(89)90100-1. [DOI] [PubMed] [Google Scholar]
  47. Mulcahy L. S., Smith M. R., Stacey D. W. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature. 1985 Jan 17;313(5999):241–243. doi: 10.1038/313241a0. [DOI] [PubMed] [Google Scholar]
  48. Noda M., Ko M., Ogura A., Liu D. G., Amano T., Takano T., Ikawa Y. Sarcoma viruses carrying ras oncogenes induce differentiation-associated properties in a neuronal cell line. Nature. 1985 Nov 7;318(6041):73–75. doi: 10.1038/318073a0. [DOI] [PubMed] [Google Scholar]
  49. O'Lague P. H., Huttner S. L. Physiological and morphological studies of rat pheochromocytoma cells (PC12) chemically fused and grown in culture. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1701–1705. doi: 10.1073/pnas.77.3.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Potts W. M., Reynolds A. B., Lansing T. J., Parsons J. T. Activation of pp60c-src transforming potential by mutations altering the structure of an amino terminal domain containing residues 90-95. Oncogene Res. 1988;3(4):343–355. [PubMed] [Google Scholar]
  51. Radeke M. J., Misko T. P., Hsu C., Herzenberg L. A., Shooter E. M. Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature. 1987 Feb 12;325(6105):593–597. doi: 10.1038/325593a0. [DOI] [PubMed] [Google Scholar]
  52. Rausch D. M., Dickens G., Doll S., Fujita K., Koizumi S., Rudkin B. B., Tocco M., Eiden L. E., Guroff G. Differentiation of PC12 cells with v-src: comparison with nerve growth factor. J Neurosci Res. 1989 Sep;24(1):49–58. doi: 10.1002/jnr.490240108. [DOI] [PubMed] [Google Scholar]
  53. Rodaway A. R., Sternberg M. J., Bentley D. L. Similarity in membrane proteins. Nature. 1989 Dec 7;342(6250):624–624. doi: 10.1038/342624a0. [DOI] [PubMed] [Google Scholar]
  54. Rydel R. E., Greene L. A. Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal differentiation in cultures of PC12 cells. J Neurosci. 1987 Nov;7(11):3639–3653. doi: 10.1523/JNEUROSCI.07-11-03639.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Santos E., Nebreda A. R. Structural and functional properties of ras proteins. FASEB J. 1989 Aug;3(10):2151–2163. doi: 10.1096/fasebj.3.10.2666231. [DOI] [PubMed] [Google Scholar]
  56. Sassone-Corsi P., Der C. J., Verma I. M. ras-induced neuronal differentiation of PC12 cells: possible involvement of fos and jun. Mol Cell Biol. 1989 Aug;9(8):3174–3183. doi: 10.1128/mcb.9.8.3174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Schubert D., LaCorbiere M., Whitlock C., Stallcup W. Alterations in the surface properties of cells responsive to nerve growth factor. Nature. 1978 Jun 29;273(5665):718–723. doi: 10.1038/273718a0. [DOI] [PubMed] [Google Scholar]
  58. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Scolnick E. M. Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4725–4729. doi: 10.1073/pnas.83.13.4725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Smith M. R., DeGudicibus S. J., Stacey D. W. Requirement for c-ras proteins during viral oncogene transformation. Nature. 1986 Apr 10;320(6062):540–543. doi: 10.1038/320540a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Soriano P., Montgomery C., Geske R., Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. doi: 10.1016/0092-8674(91)90499-o. [DOI] [PubMed] [Google Scholar]
  61. Sugimoto Y., Noda M., Kitayama H., Ikawa Y. Possible involvement of two signaling pathways in induction of neuron-associated properties by v-Ha-ras gene in PC12 cells. J Biol Chem. 1988 Aug 25;263(24):12102–12108. [PubMed] [Google Scholar]
  62. Szeberényi J., Cai H., Cooper G. M. Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells. Mol Cell Biol. 1990 Oct;10(10):5324–5332. doi: 10.1128/mcb.10.10.5324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Thomas S. M., Hayes M., D'Arcangelo G., Armstrong R. C., Meyer B. E., Zilberstein A., Brugge J. S., Halegoua S. Induction of neurite outgrowth by v-src mimics critical aspects of nerve growth factor-induced differentiation. Mol Cell Biol. 1991 Sep;11(9):4739–4750. doi: 10.1128/mcb.11.9.4739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Togari A., Baker D., Dickens G., Guroff G. The neurite-promoting effect of fibroblast growth factor on PC12 cells. Biochem Biophys Res Commun. 1983 Aug 12;114(3):1189–1193. doi: 10.1016/0006-291x(83)90688-5. [DOI] [PubMed] [Google Scholar]
  65. Togari A., Dickens G., Kuzuya H., Guroff G. The effect of fibroblast growth factor on PC12 cells. J Neurosci. 1985 Feb;5(2):307–316. doi: 10.1523/JNEUROSCI.05-02-00307.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  67. VOGT M., DULBECCO R. Steps in the neoplastic transformation of hamster embryo cells by polyoma virus. Proc Natl Acad Sci U S A. 1963 Feb 15;49:171–179. doi: 10.1073/pnas.49.2.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wagner J. A. The fibroblast growth factors: an emerging family of neural growth factors. Curr Top Microbiol Immunol. 1991;165:95–118. doi: 10.1007/978-3-642-75747-1_6. [DOI] [PubMed] [Google Scholar]
  69. Yamaizumi M., Uchida T., Mekada E., Okada Y. Antibodies introduced into living cells by red cell ghosts are functionally stable in the cytoplasm of the cells. Cell. 1979 Dec;18(4):1009–1014. doi: 10.1016/0092-8674(79)90213-7. [DOI] [PubMed] [Google Scholar]

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