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. 1991 Nov 1;115(3):795–808. doi: 10.1083/jcb.115.3.795

ras isoprenylation is required for ras-induced but not for NGF-induced neuronal differentiation of PC12 cells

PMCID: PMC2289192  PMID: 1918164

Abstract

We have used compactin, an inhibitor of mevalonate biosynthesis, to block p21ras posttranslational modification and membrane association in PC12 cells. Previous studies have demonstrated a requirement for isoprenylation for mitogenic effects of activated p21ras in mammalian cells and for function of RAS gene products in yeast. Immunoprecipitation of [35S]methionine-labeled p21ras from PC12 cell homogenates confirmed that the processed p21ras species is missing from compactin-treated PC12 cells. Immunoprecipitation from particulate and cytosolic fractions of PC12 cells confirmed that compactin blocks p21ras membrane association: p21ras is confined to the cytosol fraction. Induction of neuronal differentiation and ornithine decarboxylase (ODCase) transcription by oncogenic p21N-ras does not occur in compactin-treated cells indicating that activity of oncogenic p21N-ras expressed in PC12 cells is abolished by compactin treatment. Thus, p21ras isoprenylation or association with the membrane appears to be required for early responses and neuronal differentiation attributable to p21ras activation. In contrast, blockade of p21ras isoprenylation and membrane association by compactin treatment did not significantly reduce PC12 cell responses to NGF. Responses examined included rapid phosphorylation of tyrosine hydroxylase, rapid induction of ODCase expression, survival in serum-free medium and neuronal differentiation. Compactin blocked growth factor-induced rapid changes in cell surface morphology but did so whether this response was induced by NGF or by EGF. These results indicate that functional p21ras is not necessary for responses to NGF which in turn implies that if a ras- dependent NGF signal transduction pathway exists, as has been previously suggested, at least one additional ras-independent pathway must also be present.

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Selected References

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  1. Alberts A. W., Chen J., Kuron G., Hunt V., Huff J., Hoffman C., Rothrock J., Lopez M., Joshua H., Harris E. Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3957–3961. doi: 10.1073/pnas.77.7.3957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderegg R. J., Betz R., Carr S. A., Crabb J. W., Duntze W. Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component. J Biol Chem. 1988 Dec 5;263(34):18236–18240. [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. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  6. Brown M. S., Goldstein J. L. Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth. J Lipid Res. 1980 Jul;21(5):505–517. [PubMed] [Google Scholar]
  7. Burstein D. E., Greene L. A. Nerve growth factor has both mitogenic and antimitogenic activity. Dev Biol. 1982 Dec;94(2):477–482. doi: 10.1016/0012-1606(82)90364-5. [DOI] [PubMed] [Google Scholar]
  8. Buss J. E., Sefton B. M. Direct identification of palmitic acid as the lipid attached to p21ras. Mol Cell Biol. 1986 Jan;6(1):116–122. doi: 10.1128/mcb.6.1.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  10. Casey P. J., Solski P. A., Der C. J., Buss J. E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8323–8327. doi: 10.1073/pnas.86.21.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. Clarke S., Vogel J. P., Deschenes R. J., Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4643–4647. doi: 10.1073/pnas.85.13.4643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Connolly J. L., Green S. A., Greene L. A. Comparison of rapid changes in surface morphology and coated pit formation of PC12 cells in response to nerve growth factor, epidermal growth factor, and dibutyryl cyclic AMP. J Cell Biol. 1984 Feb;98(2):457–465. doi: 10.1083/jcb.98.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Connolly J. L., Greene L. A., Viscarello R. R., Riley W. D. Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor. J Cell Biol. 1979 Sep;82(3):820–827. doi: 10.1083/jcb.82.3.820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Endo A., Kuroda M., Tanzawa K. Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by ML-236A and ML-236B fungal metabolites, having hypocholesterolemic activity. FEBS Lett. 1976 Dec 31;72(2):323–326. doi: 10.1016/0014-5793(76)80996-9. [DOI] [PubMed] [Google Scholar]
  17. Finegold A. A., Schafer W. R., Rine J., Whiteway M., Tamanoi F. Common modifications of trimeric G proteins and ras protein: involvement of polyisoprenylation. Science. 1990 Jul 13;249(4965):165–169. doi: 10.1126/science.1695391. [DOI] [PubMed] [Google Scholar]
  18. Fukada Y., Takao T., Ohguro H., Yoshizawa T., Akino T., Shimonishi Y. Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding. Nature. 1990 Aug 16;346(6285):658–660. doi: 10.1038/346658a0. [DOI] [PubMed] [Google Scholar]
  19. Goldstein J. L., Brown M. S. Regulation of the mevalonate pathway. Nature. 1990 Feb 1;343(6257):425–430. doi: 10.1038/343425a0. [DOI] [PubMed] [Google Scholar]
  20. Green S. H., Rydel R. E., Connolly J. L., Greene L. A. PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor. J Cell Biol. 1986 Mar;102(3):830–843. doi: 10.1083/jcb.102.3.830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Greene L. A. Nerve growth factor prevents the death and stimulates the neuronal differentiation of clonal PC12 pheochromocytoma cells in serum-free medium. J Cell Biol. 1978 Sep;78(3):747–755. doi: 10.1083/jcb.78.3.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Greene L. A., Volonté C., Chalazonitis A. Purine analogs inhibit nerve growth factor-promoted neurite outgrowth by sympathetic and sensory neurons. J Neurosci. 1990 May;10(5):1479–1485. doi: 10.1523/JNEUROSCI.10-05-01479.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Guerrero I., Wong H., Pellicer A., Burstein D. E. Activated N-ras gene induces neuronal differentiation of PC12 rat pheochromocytoma cells. J Cell Physiol. 1986 Oct;129(1):71–76. doi: 10.1002/jcp.1041290111. [DOI] [PubMed] [Google Scholar]
  25. Gunning P. W., Landreth G. E., Bothwell M. A., Shooter E. M. Differential and synergistic actions of nerve growth factor and cyclic AMP in PC12 cells. J Cell Biol. 1981 May;89(2):240–245. doi: 10.1083/jcb.89.2.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gutierrez L., Magee A. I., Marshall C. J., Hancock J. F. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J. 1989 Apr;8(4):1093–1098. doi: 10.1002/j.1460-2075.1989.tb03478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Halegoua S., Patrick J. Nerve growth factor mediates phosphorylation of specific proteins. Cell. 1980 Nov;22(2 Pt 2):571–581. doi: 10.1016/0092-8674(80)90367-0. [DOI] [PubMed] [Google Scholar]
  29. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  30. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  31. Hashimoto S. K-252a, a potent protein kinase inhibitor, blocks nerve growth factor-induced neurite outgrowth and changes in the phosphorylation of proteins in PC12h cells. J Cell Biol. 1988 Oct;107(4):1531–1539. doi: 10.1083/jcb.107.4.1531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jackson J. H., Cochrane C. G., Bourne J. R., Solski P. A., Buss J. E., Der C. J. Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3042–3046. doi: 10.1073/pnas.87.8.3042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Maher P. A. Role of protein tyrosine phosphorylation in the NGF response. J Neurosci Res. 1989 Sep;24(1):29–37. doi: 10.1002/jnr.490240106. [DOI] [PubMed] [Google Scholar]
  37. Maltese W. A., Defendini R., Green R. A., Sheridan K. M., Donley D. K. Suppression of murine neuroblastoma growth in vivo by mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J Clin Invest. 1985 Nov;76(5):1748–1754. doi: 10.1172/JCI112165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Maltese W. A. Induction of differentiation in murine neuroblastoma cells by mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Biochem Biophys Res Commun. 1984 Apr 30;120(2):454–460. doi: 10.1016/0006-291x(84)91275-0. [DOI] [PubMed] [Google Scholar]
  39. Maltese W. A., Robishaw J. D. Isoprenylation of C-terminal cysteine in a G-protein gamma subunit. J Biol Chem. 1990 Oct 25;265(30):18071–18074. [PubMed] [Google Scholar]
  40. Maltese W. A., Sheridan K. M. Differentiation of neuroblastoma cells induced by an inhibitor of mevalonate synthesis: relation of neurite outgrowth and acetylcholinesterase activity to changes in cell proliferation and blocked isoprenoid synthesis. J Cell Physiol. 1985 Dec;125(3):540–558. doi: 10.1002/jcp.1041250326. [DOI] [PubMed] [Google Scholar]
  41. Maltese W. A., Sheridan K. M. Isoprenylated proteins in cultured cells: subcellular distribution and changes related to altered morphology and growth arrest induced by mevalonate deprivation. J Cell Physiol. 1987 Dec;133(3):471–481. doi: 10.1002/jcp.1041330307. [DOI] [PubMed] [Google Scholar]
  42. Maltese W. A., Sheridan K. M., Repko E. M., Erdman R. A. Post-translational modification of low molecular mass GTP-binding proteins by isoprenoid. J Biol Chem. 1990 Feb 5;265(4):2148–2155. [PubMed] [Google Scholar]
  43. McTigue M., Cremins J., Halegoua S. Nerve growth factor and other agents mediate phosphorylation and activation of tyrosine hydroxylase. A convergence of multiple kinase activities. J Biol Chem. 1985 Jul 25;260(15):9047–9056. [PubMed] [Google Scholar]
  44. Michaeli T., Field J., Ballester R., O'Neill K., Wigler M. Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae. EMBO J. 1989 Oct;8(10):3039–3044. doi: 10.1002/j.1460-2075.1989.tb08454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. 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]
  48. Repko E. M., Maltese W. A. Post-translational isoprenylation of cellular proteins is altered in response to mevalonate availability. J Biol Chem. 1989 Jun 15;264(17):9945–9952. [PubMed] [Google Scholar]
  49. 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]
  50. Schafer W. R., Kim R., Sterne R., Thorner J., Kim S. H., Rine J. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science. 1989 Jul 28;245(4916):379–385. doi: 10.1126/science.2569235. [DOI] [PubMed] [Google Scholar]
  51. Seeley P. J., Rukenstein A., Connolly J. L., Greene L. A. Differential inhibition of nerve growth factor and epidermal growth factor effects on the PC12 pheochromocytoma line. J Cell Biol. 1984 Feb;98(2):417–426. doi: 10.1083/jcb.98.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sigmund O., Naor Z., Anderson D. J., Stein R. Effect of nerve growth factor and fibroblast growth factor on SCG10 and c-fos expression and neurite outgrowth in protein kinase C-depleted PC12 cells. J Biol Chem. 1990 Feb 5;265(4):2257–2261. [PubMed] [Google Scholar]
  53. Stimmel J. B., Deschenes R. J., Volker C., Stock J., Clarke S. Evidence for an S-farnesylcysteine methyl ester at the carboxyl terminus of the Saccharomyces cerevisiae RAS2 protein. Biochemistry. 1990 Oct 16;29(41):9651–9659. doi: 10.1021/bi00493a021. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Thomson T. M., Green S. H., Trotta R. J., Burstein D. E., Pellicer A. Oncogene N-ras mediates selective inhibition of c-fos induction by nerve growth factor and basic fibroblast growth factor in a PC12 cell line. Mol Cell Biol. 1990 Apr;10(4):1556–1563. doi: 10.1128/mcb.10.4.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tocco M. D., Contreras M. L., Koizumi S., Dickens G., Guroff G. Decreased levels of nerve growth factor receptor on dexamethasone-treated PC12 cells. J Neurosci Res. 1988 Aug;20(4):411–419. doi: 10.1002/jnr.490200403. [DOI] [PubMed] [Google Scholar]
  57. Trotta R. J., Thomson T. M., Lacal J. C., Pellicer A., Burstein D. E. Potentiation of oncogenic N-ras-induced neurite outgrowth and ornithine decarboxylase activity by phorbol dibutyrate and protein kinase inhibitor H-8. J Cell Physiol. 1990 Apr;143(1):68–78. doi: 10.1002/jcp.1041430109. [DOI] [PubMed] [Google Scholar]
  58. Volonté C., Rukenstein A., Loeb D. M., Greene L. A. Differential inhibition of nerve growth factor responses by purine analogues: correlation with inhibition of a nerve growth factor-activated protein kinase. J Cell Biol. 1989 Nov;109(5):2395–2403. doi: 10.1083/jcb.109.5.2395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Willingham M. C., Pastan I., Shih T. Y., Scolnick E. M. Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1980 Apr;19(4):1005–1014. doi: 10.1016/0092-8674(80)90091-4. [DOI] [PubMed] [Google Scholar]
  60. Willumsen B. M., Norris K., Papageorge A. G., Hubbert N. L., Lowy D. R. Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus. EMBO J. 1984 Nov;3(11):2581–2585. doi: 10.1002/j.1460-2075.1984.tb02177.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wolda S. L., Glomset J. A. Evidence for modification of lamin B by a product of mevalonic acid. J Biol Chem. 1988 May 5;263(13):5997–6000. [PubMed] [Google Scholar]

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