Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Oct 1;107(4):1531–1539. doi: 10.1083/jcb.107.4.1531

K-252a, a potent protein kinase inhibitor, blocks nerve growth factor- induced neurite outgrowth and changes in the phosphorylation of proteins in PC12h cells

PMCID: PMC2115256  PMID: 2844830

Abstract

Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. One of the most prominent and distinguishing features of neuronal differentiation is neurite outgrowth. The mechanism by which NGF causes the cells to elaborate neurites is unknown. This study shows that K-252a, a potent protein kinase inhibitor, blocks NGF-induced neurite outgrowth and the changes in protein phosphorylation elicited by NGF. In the experiment with intact cells phosphorylated with 32P-orthophosphoric acid, an exposure of PC12h cells to NGF (50 ng/ml) caused an increase in the phosphorylation of tyrosine hydroxylase and a 35,000-D protein and a decrease in a 36,500-D protein. Pretreatment of PC12h cells with K-252a (100 nM) inhibited the effects of NGF on the phosphorylation of these three proteins. In the phosphorylation of cell-free extracts with [gamma-32P] ATP, treatment of PC12h cells with NGF (50 ng/ml) caused a decrease in the phosphorylation of Nsp100. Pretreatment of the cells with K-252a (30 nM) almost completely blocked the NGF effect on the phosphorylation of Nsp100 elicited by subsequent treatment of the cells with NGF. Treatment of PC12h cells with NGF promoted outgrowth of neurites. The addition of K-252a (100 nM) into the culture almost completely blocked the generation of neurites elicited by NGF. Earlier studies demonstrated that NGF-induced neurite outgrowth in PC12 cells involves at least two components: the first of these is transcription-dependent and the second is transcription-independent. To determine the component on which K-252a acts, experiments were carried out on NGF-induced priming or regeneration of neurites. When K-252a was present in the priming step, NGF induced only actinomycin D-sensitive neurites, showing that K-252a interferes with the transcription-dependent actions of NGF. When already primed cells were treated with NGF, actinomycin D- resistant neurites were formed and these were blocked by K-252a, showing that the inhibitor interferes with the transcription- independent actions of NGF as well. Although the exact mechanism of inhibition of NGF-promoted neurite formation by K-252a is unknown, the most probable explanation is that both transcription-dependent and - independent components are involved in at least one step of the activation of some specific protein kinase(s) that can be suppressed by K-252a.

Full Text

The Full Text of this article is available as a PDF (4.3 MB).

Selected References

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

  1. Bocchini V., Angeletti P. U. The nerve growth factor: purification as a 30,000-molecular-weight protein. Proc Natl Acad Sci U S A. 1969 Oct;64(2):787–794. doi: 10.1073/pnas.64.2.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Burstein D. E., Greene L. A. Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6059–6063. doi: 10.1073/pnas.75.12.6059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burstein D. E., Seeley P. J., Greene L. A. Lithium ion inhibits nerve growth factor-induced neurite outgrowth and phosphorylation of nerve growth factor-modulated microtubule-associated proteins. J Cell Biol. 1985 Sep;101(3):862–870. doi: 10.1083/jcb.101.3.862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Dichter M. A., Tischler A. S., Greene L. A. Nerve growth factor-induced increase in electrical excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line. Nature. 1977 Aug 11;268(5620):501–504. doi: 10.1038/268501a0. [DOI] [PubMed] [Google Scholar]
  7. End D., Hanson M., Hashimoto S., Guroff G. Inhibition of the phosphorylation of a 1000,000-dalton soluble protein in whole cells and cell-free extracts of PC12 pheochromocytoma cells following treatment with nerve growth factor. J Biol Chem. 1982 Aug 25;257(16):9223–9225. [PubMed] [Google Scholar]
  8. End D., Tolson N., Hashimoto S., Guroff G. Nerve growth factor-induced decrease in the cell-free phosphorylation of a soluble protein in PC12 cells. J Biol Chem. 1983 May 25;258(10):6549–6555. [PubMed] [Google Scholar]
  9. 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]
  10. Greene L. A., Shooter E. M. The nerve growth factor: biochemistry, synthesis, and mechanism of action. Annu Rev Neurosci. 1980;3:353–402. doi: 10.1146/annurev.ne.03.030180.002033. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Hashimoto S., Ikeno T., Kuzuya H. Wheat germ agglutinin inhibits the effects of nerve growth factor on the phosphorylation of proteins in PC12h cells. J Neurochem. 1985 Sep;45(3):906–912. doi: 10.1111/j.1471-4159.1985.tb04079.x. [DOI] [PubMed] [Google Scholar]
  15. Hatanaka H. Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line. Brain Res. 1981 Oct 19;222(2):225–233. doi: 10.1016/0006-8993(81)91029-5. [DOI] [PubMed] [Google Scholar]
  16. Heumann R., Schwab M., Thoenen H. A second messenger required for nerve growth factor biological activity? Nature. 1981 Aug 27;292(5826):838–840. doi: 10.1038/292838a0. [DOI] [PubMed] [Google Scholar]
  17. Kase H., Iwahashi K., Nakanishi S., Matsuda Y., Yamada K., Takahashi M., Murakata C., Sato A., Kaneko M. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Commun. 1987 Jan 30;142(2):436–440. doi: 10.1016/0006-291x(87)90293-2. [DOI] [PubMed] [Google Scholar]
  18. Koizumi S., Contreras M. L., Matsuda Y., Hama T., Lazarovici P., Guroff G. K-252a: a specific inhibitor of the action of nerve growth factor on PC 12 cells. J Neurosci. 1988 Feb;8(2):715–721. doi: 10.1523/JNEUROSCI.08-02-00715.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Landreth G. E., Rieser G. D. Nerve growth factor- and epidermal growth factor-stimulated phosphorylation of a PC12 cytoskeletally associated protein in situ. J Cell Biol. 1985 Mar;100(3):677–683. doi: 10.1083/jcb.100.3.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Levi-Montalcini R., Angeletti P. U. Nerve growth factor. Physiol Rev. 1968 Jul;48(3):534–569. doi: 10.1152/physrev.1968.48.3.534. [DOI] [PubMed] [Google Scholar]
  22. Matsuda Y., Guroff G. Purification and mechanism of activation of a nerve growth factor-sensitive S6 kinase from PC12 cells. J Biol Chem. 1987 Feb 25;262(6):2832–2844. [PubMed] [Google Scholar]
  23. Matsuda Y., Nakanishi N., Dickens G., Guroff G. A nerve growth factor-sensitive S6 kinase in cell-free extracts from PC12 cells. J Neurochem. 1986 Dec;47(6):1728–1734. doi: 10.1111/j.1471-4159.1986.tb13081.x. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Nakanishi N., Guroff G. Nerve growth factor-induced increase in the cell-free phosphorylation of a nuclear protein in PC12 cells. J Biol Chem. 1985 Jun 25;260(12):7791–7799. [PubMed] [Google Scholar]
  26. Ozaki H., Ishihara H., Kohama K., Nonomura Y., Shibata S., Karaki H. Calcium-independent phosphorylation of smooth muscle myosin light chain by okadaic acid isolated from black sponge (Halichondria okadai). J Pharmacol Exp Ther. 1987 Dec;243(3):1167–1173. [PubMed] [Google Scholar]
  27. Rowland E. A., Müller T. H., Goldstein M., Greene L. A. Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells. J Biol Chem. 1987 Jun 5;262(16):7504–7513. [PubMed] [Google Scholar]
  28. 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]
  29. Thoenen H., Barde Y. A. Physiology of nerve growth factor. Physiol Rev. 1980 Oct;60(4):1284–1335. doi: 10.1152/physrev.1980.60.4.1284. [DOI] [PubMed] [Google Scholar]
  30. Togari A., Guroff G. Partial purification and characterization of a nerve growth factor-sensitive kinase and its substrate from PC12 cells. J Biol Chem. 1985 Mar 25;260(6):3804–3811. [PubMed] [Google Scholar]
  31. Vale R. D., Hosang M., Shooter E. M. Sialic acid residues on NGF receptors on PC12 cells. Dev Neurosci. 1985;7(1):55–64. doi: 10.1159/000112276. [DOI] [PubMed] [Google Scholar]
  32. Yamada K., Iwahashi K., Kase H. K252a, a new inhibitor of protein kinase C, concomitantly inhibits 40K protein phosphorylation and serotonin secretion in a phorbol ester-stimulated platelets. Biochem Biophys Res Commun. 1987 Apr 14;144(1):35–40. doi: 10.1016/s0006-291x(87)80471-0. [DOI] [PubMed] [Google Scholar]
  33. Yu M. W., Tolson N. W., Guroff G. Increased phosphorylation of specific nuclear proteins in superior cervical ganglia and PC12 cells in response to nerve growth factor. J Biol Chem. 1980 Nov 10;255(21):10481–10492. [PubMed] [Google Scholar]

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

RESOURCES