Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Mar 1;120(5):1263–1270. doi: 10.1083/jcb.120.5.1263

Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA

PMCID: PMC2119722  PMID: 8436593

Abstract

We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation- independent mRNA stabilization mechanism.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander K. A., Cimler B. M., Meier K. E., Storm D. R. Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein. J Biol Chem. 1987 May 5;262(13):6108–6113. [PubMed] [Google Scholar]
  2. Aloyo V. J., Zwiers H., Gispen W. H. Phosphorylation of B-50 protein by calcium-activated, phospholipid-dependent protein kinase and B-50 protein kinase. J Neurochem. 1983 Sep;41(3):649–653. doi: 10.1111/j.1471-4159.1983.tb04790.x. [DOI] [PubMed] [Google Scholar]
  3. Andreasen T. J., Luetje C. W., Heideman W., Storm D. R. Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes. Biochemistry. 1983 Sep 27;22(20):4615–4618. doi: 10.1021/bi00289a001. [DOI] [PubMed] [Google Scholar]
  4. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
  5. Basi G. S., Jacobson R. D., Virág I., Schilling J., Skene J. H. Primary structure and transcriptional regulation of GAP-43, a protein associated with nerve growth. Cell. 1987 Jun 19;49(6):785–791. doi: 10.1016/0092-8674(87)90616-7. [DOI] [PubMed] [Google Scholar]
  6. Benowitz L. I., Perrone-Bizzozero N. I. The expression of GAP-43 in relation to neuronal growth and plasticity: when, where, how, and why? Prog Brain Res. 1991;89:69–87. doi: 10.1016/s0079-6123(08)61716-1. [DOI] [PubMed] [Google Scholar]
  7. Benowitz L. I., Shashoua V. E., Yoon M. G. Specific changes in rapidly transported proteins during regeneration of the goldfish optic nerve. J Neurosci. 1981 Mar;1(3):300–307. doi: 10.1523/JNEUROSCI.01-03-00300.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Benowitz L. I., Yoon M. G., Lewis E. R. Transported proteins in the regenerating optic nerve: regulation by interactions with the optic tectum. Science. 1983 Oct 14;222(4620):185–188. doi: 10.1126/science.6194562. [DOI] [PubMed] [Google Scholar]
  9. Bernstein P., Ross J. Poly(A), poly(A) binding protein and the regulation of mRNA stability. Trends Biochem Sci. 1989 Sep;14(9):373–377. doi: 10.1016/0968-0004(89)90011-x. [DOI] [PubMed] [Google Scholar]
  10. Bjelfman C., Meyerson G., Cartwright C. A., Mellström K., Hammerling U., Påhlman S. Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells. Mol Cell Biol. 1990 Jan;10(1):361–370. doi: 10.1128/mcb.10.1.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brewer G., Ross J. Poly(A) shortening and degradation of the 3' A+U-rich sequences of human c-myc mRNA in a cell-free system. Mol Cell Biol. 1988 Apr;8(4):1697–1708. doi: 10.1128/mcb.8.4.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chiu R., Imagawa M., Imbra R. J., Bockoven J. R., Karin M. Multiple cis- and trans-acting elements mediate the transcriptional response to phorbol esters. Nature. 1987 Oct 15;329(6140):648–651. doi: 10.1038/329648a0. [DOI] [PubMed] [Google Scholar]
  13. Cleveland D. W. Gene regulation through messenger RNA stability. Curr Opin Cell Biol. 1989 Dec;1(6):1148–1153. doi: 10.1016/s0955-0674(89)80065-1. [DOI] [PubMed] [Google Scholar]
  14. Costello B., Meymandi A., Freeman J. A. Factors influencing GAP-43 gene expression in PC12 pheochromocytoma cells. J Neurosci. 1990 Apr;10(4):1398–1406. doi: 10.1523/JNEUROSCI.10-04-01398.1990. [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. DiFiglia M., Roberts R. C., Benowitz L. I. Immunoreactive GAP-43 in the neuropil of adult rat neostriatum: localization in unmyelinated fibers, axon terminals, and dendritic spines. J Comp Neurol. 1990 Dec 22;302(4):992–1001. doi: 10.1002/cne.903020421. [DOI] [PubMed] [Google Scholar]
  17. Doherty P., Mann D. A., Walsh F. S. Comparison of the effects of NGF, activators of protein kinase C, and a calcium ionophore on the expression of Thy-1 and N-CAM in PC12 cell cultures. J Cell Biol. 1988 Jul;107(1):333–340. doi: 10.1083/jcb.107.1.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Eisenstein R. S., Rosen J. M. Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level. Mol Cell Biol. 1988 Aug;8(8):3183–3190. doi: 10.1128/mcb.8.8.3183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. End D., Tolson N., Yu M. Y., Guroff G. Effects of 12-0-Tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: interactions with epidermal growth factor and nerve growth factor. J Cell Physiol. 1982 May;111(2):140–148. doi: 10.1002/jcp.1041110204. [DOI] [PubMed] [Google Scholar]
  20. Federoff H. J., Grabczyk E., Fishman M. C. Dual regulation of GAP-43 gene expression by nerve growth factor and glucocorticoids. J Biol Chem. 1988 Dec 25;263(36):19290–19295. [PubMed] [Google Scholar]
  21. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  22. Gispen W. H., Leunissen J. L., Oestreicher A. B., Verkleij A. J., Zwiers H. Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism. Brain Res. 1985 Mar 4;328(2):381–385. doi: 10.1016/0006-8993(85)91054-6. [DOI] [PubMed] [Google Scholar]
  23. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  24. Greene L. A., Aletta J. M., Rukenstein A., Green S. H. PC12 pheochromocytoma cells: culture, nerve growth factor treatment, and experimental exploitation. Methods Enzymol. 1987;147:207–216. doi: 10.1016/0076-6879(87)47111-5. [DOI] [PubMed] [Google Scholar]
  25. Hall F. L., Fernyhough P., Ishii D. N., Vulliet P. R. Suppression of nerve growth factor-directed neurite outgrowth in PC12 cells by sphingosine, an inhibitor of protein kinase C. J Biol Chem. 1988 Mar 25;263(9):4460–4466. [PubMed] [Google Scholar]
  26. Hoffman P. N. Expression of GAP-43, a rapidly transported growth-associated protein, and class II beta tubulin, a slowly transported cytoskeletal protein, are coordinated in regenerating neurons. J Neurosci. 1989 Mar;9(3):893–897. doi: 10.1523/JNEUROSCI.09-03-00893.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Karns L. R., Ng S. C., Freeman J. A., Fishman M. C. Cloning of complementary DNA for GAP-43, a neuronal growth-related protein. Science. 1987 May 1;236(4801):597–600. doi: 10.1126/science.2437653. [DOI] [PubMed] [Google Scholar]
  29. Machida C. M., Scott J. D., Ciment G. NGF-induction of the metalloproteinase-transin/stromelysin in PC12 cells: involvement of multiple protein kinases. J Cell Biol. 1991 Sep;114(5):1037–1048. doi: 10.1083/jcb.114.5.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Malter J. S. Identification of an AUUUA-specific messenger RNA binding protein. Science. 1989 Nov 3;246(4930):664–666. doi: 10.1126/science.2814487. [DOI] [PubMed] [Google Scholar]
  31. Matthies H. J., Palfrey H. C., Hirning L. D., Miller R. J. Down regulation of protein kinase C in neuronal cells: effects on neurotransmitter release. J Neurosci. 1987 Apr;7(4):1198–1206. doi: 10.1523/JNEUROSCI.07-04-01198.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mazzei G. J., Katoh N., Kuo J. F. Polymyxin B is a more selective inhibitor for phospholipid-sensitive Ca2+-dependent protein kinase than for calmodulin-sensitive Ca2+-dependent protein kinase. Biochem Biophys Res Commun. 1982 Dec 31;109(4):1129–1133. doi: 10.1016/0006-291x(82)91894-0. [DOI] [PubMed] [Google Scholar]
  33. Meiri K. F., Pfenninger K. H., Willard M. B. Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones. Proc Natl Acad Sci U S A. 1986 May;83(10):3537–3541. doi: 10.1073/pnas.83.10.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mizuno N., Matsuoka I., Kurihara K. Possible involvements of intracellular Ca2+ and Ca2+ -dependent protein phosphorylation in cholinergic differentiation of clonal rat pheochromocytoma cells (PC12) induced by glioma-conditioned medium and retinoic acid. Brain Res Dev Brain Res. 1989 Nov 1;50(1):1–10. doi: 10.1016/0165-3806(89)90123-5. [DOI] [PubMed] [Google Scholar]
  35. Montz H. P., Davis G. E., Skaper S. D., Manthorpe M., Varon S. Tumor-promoting phorbol diester mimics two distinct neuronotrophic factors. Brain Res. 1985 Nov;355(1):150–154. doi: 10.1016/0165-3806(85)90015-x. [DOI] [PubMed] [Google Scholar]
  36. Nedivi E., Basi G. S., Akey I. V., Skene J. H. A neural-specific GAP-43 core promoter located between unusual DNA elements that interact to regulate its activity. J Neurosci. 1992 Mar;12(3):691–704. doi: 10.1523/JNEUROSCI.12-03-00691.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nelson R. B., Routtenberg A. Characterization of protein F1 (47 kDa, 4.5 pI): a kinase C substrate directly related to neural plasticity. Exp Neurol. 1985 Jul;89(1):213–224. doi: 10.1016/0014-4886(85)90277-8. [DOI] [PubMed] [Google Scholar]
  38. Neve R. L., Finch E. A., Bird E. D., Benowitz L. I. Growth-associated protein GAP-43 is expressed selectively in associative regions of the adult human brain. Proc Natl Acad Sci U S A. 1988 May;85(10):3638–3642. doi: 10.1073/pnas.85.10.3638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Neve R. L., Ivins K. J., Benowitz L. I., During M. J., Geller A. I. Molecular analysis of the function of the neuronal growth-associated protein GAP-43 by genetic intervention. Mol Neurobiol. 1991;5(2-4):131–141. doi: 10.1007/BF02935542. [DOI] [PubMed] [Google Scholar]
  40. Neve R. L., Perrone-Bizzozero N. I., Finklestein S., Zwiers H., Bird E., Kurnit D. M., Benowitz L. I. The neuronal growth-associated protein GAP-43 (B-50, F1): neuronal specificity, developmental regulation and regional distribution of the human and rat mRNAs. Brain Res. 1987 Jul;388(2):177–183. doi: 10.1016/s0006-8993(87)80012-4. [DOI] [PubMed] [Google Scholar]
  41. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  42. Pandey N. B., Marzluff W. F. The stem-loop structure at the 3' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability. Mol Cell Biol. 1987 Dec;7(12):4557–4559. doi: 10.1128/mcb.7.12.4557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pennypacker K. R., Kincaid R. L., Polli J. W., Billingsley M. L. Expression of calmodulin-dependent phosphodiesterase, calmodulin-dependent protein phosphatase, and other calmodulin-binding proteins in human SMS-KCNR neuroblastoma cells. J Neurochem. 1989 May;52(5):1438–1448. doi: 10.1111/j.1471-4159.1989.tb09191.x. [DOI] [PubMed] [Google Scholar]
  44. Perrone-Bizzozero N. I., Benowitz L. I. Expression of a 48-kilodalton growth-associated protein in the goldfish retina. J Neurochem. 1987 Feb;48(2):644–652. doi: 10.1111/j.1471-4159.1987.tb04141.x. [DOI] [PubMed] [Google Scholar]
  45. Perrone-Bizzozero N. I., Finklestein S. P., Benowitz L. I. Synthesis of a growth-associated protein by embryonic rat cerebrocortical neurons in vitro. J Neurosci. 1986 Dec;6(12):3721–3730. doi: 10.1523/JNEUROSCI.06-12-03721.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rouault T. A., Tang C. K., Kaptain S., Burgess W. H., Haile D. J., Samaniego F., McBride O. W., Harford J. B., Klausner R. D. Cloning of the cDNA encoding an RNA regulatory protein--the human iron-responsive element-binding protein. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7958–7962. doi: 10.1073/pnas.87.20.7958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Seykora J. T., Ravetch J. V., Aderem A. Cloning and molecular characterization of the murine macrophage "68-kDa" protein kinase C substrate and its regulation by bacterial lipopolysaccharide. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2505–2509. doi: 10.1073/pnas.88.6.2505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shaw G., Kamen R. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell. 1986 Aug 29;46(5):659–667. doi: 10.1016/0092-8674(86)90341-7. [DOI] [PubMed] [Google Scholar]
  49. Shea T. B., Perrone-Bizzozero N. I., Beermann M. L., Benowitz L. I. Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis. J Neurosci. 1991 Jun;11(6):1685–1690. doi: 10.1523/JNEUROSCI.11-06-01685.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Shyu A. B., Greenberg M. E., Belasco J. G. The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. Genes Dev. 1989 Jan;3(1):60–72. doi: 10.1101/gad.3.1.60. [DOI] [PubMed] [Google Scholar]
  51. Simonet W. S., Ness G. C. Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA in rat liver. Glucocorticoids block the stabilization caused by thyroid hormones. J Biol Chem. 1989 Jan 5;264(1):569–573. [PubMed] [Google Scholar]
  52. Skene J. H. Axonal growth-associated proteins. Annu Rev Neurosci. 1989;12:127–156. doi: 10.1146/annurev.ne.12.030189.001015. [DOI] [PubMed] [Google Scholar]
  53. Skene J. H., Jacobson R. D., Snipes G. J., McGuire C. B., Norden J. J., Freeman J. A. A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes. Science. 1986 Aug 15;233(4765):783–786. doi: 10.1126/science.3738509. [DOI] [PubMed] [Google Scholar]
  54. Skene J. H., Willard M. Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems. J Cell Biol. 1981 Apr;89(1):96–103. doi: 10.1083/jcb.89.1.96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Skene J. H., Willard M. Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells. J Cell Biol. 1981 Apr;89(1):86–95. doi: 10.1083/jcb.89.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Strittmatter S. M., Valenzuela D., Kennedy T. E., Neer E. J., Fishman M. C. G0 is a major growth cone protein subject to regulation by GAP-43. Nature. 1990 Apr 26;344(6269):836–841. doi: 10.1038/344836a0. [DOI] [PubMed] [Google Scholar]
  57. Van Hooff C. O., Holthuis J. C., Oestreicher A. B., Boonstra J., De Graan P. N., Gispen W. H. Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells. J Cell Biol. 1989 Mar;108(3):1115–1125. doi: 10.1083/jcb.108.3.1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Vyas S., Faucon Biguet N., Mallet J. Transcriptional and post-transcriptional regulation of tyrosine hydroxylase gene by protein kinase C. EMBO J. 1990 Nov;9(11):3707–3712. doi: 10.1002/j.1460-2075.1990.tb07583.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wilson T., Treisman R. Removal of poly(A) and consequent degradation of c-fos mRNA facilitated by 3' AU-rich sequences. Nature. 1988 Nov 24;336(6197):396–399. doi: 10.1038/336396a0. [DOI] [PubMed] [Google Scholar]
  60. Yankner B. A., Benowitz L. I., Villa-Komaroff L., Neve R. L. Transfection of PC12 cells with the human GAP-43 gene: effects on neurite outgrowth and regeneration. Brain Res Mol Brain Res. 1990 Jan;7(1):39–44. doi: 10.1016/0169-328x(90)90071-k. [DOI] [PubMed] [Google Scholar]
  61. Yen T. J., Gay D. A., Pachter J. S., Cleveland D. W. Autoregulated changes in stability of polyribosome-bound beta-tubulin mRNAs are specified by the first 13 translated nucleotides. Mol Cell Biol. 1988 Mar;8(3):1224–1235. doi: 10.1128/mcb.8.3.1224. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES