Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 May 1;106(5):1573–1581. doi: 10.1083/jcb.106.5.1573

Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2 (MAP1.2)

PMCID: PMC2115037  PMID: 3372590

Abstract

This study characterizes effects of nerve growth factor (NGF) on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells. Past work showed that NGF significantly raises the relative levels of this phosphoprotein, designated MAP1.2, with a time course similar to that of neurite outgrowth. To study this in greater detail, MAP1.2 in PC12 cell lysates was resolved by SDS-PAGE in gels containing 3.25% acrylamide/4 M urea and identified by comigration with material immunoprecipitated from the lysates by MAP1 antibodies. Quantification by metabolic radiolabeling with [35S]methionine or by silver staining revealed a 3.0-3.5-fold increase in MAP1.2 levels relative to total cell protein after NGF treatment for 2 wk or longer. A partial increase was detectable after 3 d, but not after 2 h of NGF exposure. As measured by incorporation of [32P]phosphate, NGF had a dual effect on MAP1.2. Within 15 min to 2 h, NGF enhanced the incorporation of phosphate into MAP1.2 by two- to threefold relative to total cell phosphoproteins. This value slowly increased thereafter so that by 2 wk or more of NGF exposure, the average enhancement of phosphate incorporation per MAP1.2 molecule was over fourfold. The rapid action of NGF on MAP1.2 could not be mimicked by either epidermal growth factor, a permeant cAMP derivative, phorbol ester, or elevated K+, each of which alters phosphorylation of other PC12 cell proteins. SDS-PAGE revealed multiple forms of MAP1.2 which, based on the effects of alkaline phosphatase on their electrophoretic mobilities, differ, at least in part, in extent of phosphorylation. Before NGF treatment, most PC12 cell MAP1.2 is in more rapidly migrating, relatively poorly phosphorylated forms. After long-term NGF exposure, most is in more slowly migrating, more highly phosphorylated forms. The effects of NGF on the rapid phosphorylation of MAP1.2 and on the long-term large increase in highly phosphorylated MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation. Such roles may include effects on microtubule assembly, stability, and cross- linking and, possibly for the rapid effects, nuclear signaling.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

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

  1. Aletta J. M., Greene L. A. Sequential phosphorylation of chartin microtubule-associated proteins is regulated by the presence of microtubules. J Cell Biol. 1987 Jul;105(1):277–290. doi: 10.1083/jcb.105.1.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Binder L. I., Frankfurter A., Kim H., Caceres A., Payne M. R., Rebhun L. I. Heterogeneity of microtubule-associated protein 2 during rat brain development. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5613–5617. doi: 10.1073/pnas.81.17.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black M. M., Aletta J. M., Greene L. A. Regulation of microtubule composition and stability during nerve growth factor-promoted neurite outgrowth. J Cell Biol. 1986 Aug;103(2):545–557. doi: 10.1083/jcb.103.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bloom G. S., Luca F. C., Vallee R. B. Microtubule-associated protein 1B: identification of a major component of the neuronal cytoskeleton. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5404–5408. doi: 10.1073/pnas.82.16.5404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bloom G. S., Schoenfeld T. A., Vallee R. B. Widespread distribution of the major polypeptide component of MAP 1 (microtubule-associated protein 1) in the nervous system. J Cell Biol. 1984 Jan;98(1):320–330. doi: 10.1083/jcb.98.1.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
  9. DULBECCO R., VOGT M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954 Feb;99(2):167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Daniels M. P. Colchicine inhibition of nerve fiber formation in vitro. J Cell Biol. 1972 Apr;53(1):164–176. doi: 10.1083/jcb.53.1.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Drubin D. G., Feinstein S. C., Shooter E. M., Kirschner M. W. Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors. J Cell Biol. 1985 Nov;101(5 Pt 1):1799–1807. doi: 10.1083/jcb.101.5.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Egly J. M., Miyamoto N. G., Moncollin V., Chambon P. Is actin a transcription initiation factor for RNA polymerase B? EMBO J. 1984 Oct;3(10):2363–2371. doi: 10.1002/j.1460-2075.1984.tb02141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greene L. A., Drexler S. A., Connolly J. L., Rukenstein A., Green S. H. Selective inhibition of responses to nerve growth factor and of microtubule-associated protein phosphorylation by activators of adenylate cyclase. J Cell Biol. 1986 Nov;103(5):1967–1978. doi: 10.1083/jcb.103.5.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Greene L. A., Liem R. K., Shelanski M. L. Regulation of a high molecular weight microtubule-associated protein in PC12 cells by nerve growth factor. J Cell Biol. 1983 Jan;96(1):76–83. doi: 10.1083/jcb.96.1.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Huff K., End D., Guroff G. Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor. J Cell Biol. 1981 Jan;88(1):189–198. doi: 10.1083/jcb.88.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kuznetsov S. A., Rodionov V. I., Gelfand V. I., Rosenblat V. A. Microtubule-associated protein MAP1 promotes microtubule assembly in vitro. FEBS Lett. 1981 Dec 7;135(2):241–244. doi: 10.1016/0014-5793(81)80791-0. [DOI] [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. Lee K. Y., Seeley P. J., Müller T. H., Helmer-Matyjek E., Sabban E., Goldstein M., Greene L. A. Regulation of tyrosine hydroxylase phosphorylation in PC12 pheochromocytoma cells by elevated K+ and nerve growth factor. Evidence for different mechanisms of action. Mol Pharmacol. 1985 Aug;28(2):220–228. [PubMed] [Google Scholar]
  21. Leonard D. G., Ziff E. B., Greene L. A. Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells. Mol Cell Biol. 1987 Sep;7(9):3156–3167. doi: 10.1128/mcb.7.9.3156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lewis S. A., Sherline P., Cowan N. J. A cloned cDNA encoding MAP1 detects a single copy gene in mouse and a brain-abundant RNA whose level decreases during development. J Cell Biol. 1986 Jun;102(6):2106–2114. doi: 10.1083/jcb.102.6.2106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lewis S. A., Villasante A., Sherline P., Cowan N. J. Brain-specific expression of MAP2 detected using a cloned cDNA probe. J Cell Biol. 1986 Jun;102(6):2098–2105. doi: 10.1083/jcb.102.6.2098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. MORTON R. K. The substrate specificity and inhibition of alkaline phosphatases of cow's milk and calf intestinal mucosa. Biochem J. 1955 Oct;61(2):232–240. doi: 10.1042/bj0610232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Miller J. P., Beck A. H., Simon L. N., Meyer R. B., Jr Induction of hepatic tyrosine aminotransferase in vivo by derivatives of cyclic adenosine 3':5'-monophosphate. J Biol Chem. 1975 Jan 25;250(2):426–431. [PubMed] [Google Scholar]
  27. 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]
  28. Olmsted J. B. Microtubule-associated proteins. Annu Rev Cell Biol. 1986;2:421–457. doi: 10.1146/annurev.cb.02.110186.002225. [DOI] [PubMed] [Google Scholar]
  29. Riederer B., Matus A. Differential expression of distinct microtubule-associated proteins during brain development. Proc Natl Acad Sci U S A. 1985 Sep;82(17):6006–6009. doi: 10.1073/pnas.82.17.6006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Sato C., Nishizawa K., Nakayama T., Nose K., Takasaki Y., Hirose S., Nakamura H. Intranuclear appearance of the phosphorylated form of cytoskeleton-associated 350-kDa proteins in U1-ribonucleoprotein regions after growth stimulation of fibroblasts. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7287–7291. doi: 10.1073/pnas.83.19.7287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sherline P., Schiavone K. Immunofluorescence localization of proteins of high molecular weight along intracellular microtubules. Science. 1977 Dec 9;198(4321):1038–1040. doi: 10.1126/science.337490. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Vallee R. B., Davis S. E. Low molecular weight microtubule-associated proteins are light chains of microtubule-associated protein 1 (MAP 1). Proc Natl Acad Sci U S A. 1983 Mar;80(5):1342–1346. doi: 10.1073/pnas.80.5.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wiche G., Briones E., Koszka C., Artlieb U., Krepler R. Widespread occurrence of polypeptides related to neurotubule-associated proteins (MAP-1 and MAP-2) in non-neuronal cells and tissues. EMBO J. 1984 May;3(5):991–998. doi: 10.1002/j.1460-2075.1984.tb01918.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yamada K. M., Spooner B. S., Wessells N. K. Axon growth: roles of microfilaments and microtubules. Proc Natl Acad Sci U S A. 1970 Aug;66(4):1206–1212. doi: 10.1073/pnas.66.4.1206. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES