Skip to main content
NCBI home page
Cellular and Molecular Neurobiology logoLink to Cellular and Molecular Neurobiology
. 1995 Apr;15(2):269–281. doi: 10.1007/BF02073333

Phosphorylation and dephosphorylation of distinct isoforms of the heavy neurofilament protein NF-H

Roberto Chertoff 1, Lior Soussan 1, Hanno Roder 2, Daniel M Michaelson 1,✉
PMCID: PMC11563135  PMID: 8590456

Abstract

1. Previous immunohistochemical studies led to the suggestion that distinctly phosphorylated neurofilament isoforms exist in different types of neurons. We have recently examined this hypothesis by direct biochemical experiments, which revealed that the heavy neurofilament protein NF-H of bovine ventral root cholinergic neurons is more acidic and markedly more phosphorylated than that of bovine dorsal root neurons.

2. In the present study we employed this system to study the degree to which distinctly phosphorylated NF-H isoforms differ in the extents to which they can be phosphorylated and dephosphorylatedin vitro. This was performed utilizing alkaline phosphatase and protein kinase PK40ERK, which is specific to serines of Lys-Ser-Pro (KSP) repeats. The results obtained reveal that:

3. The more extensively phosphorylated ventral root NF-H is dephosphorylated more rapidly than dorsal root NF-H.

4. Ventral root NF-H and dorsal root NF-H in their native form are both poor substrates of PK40ERK.

5. Following dephosphorylation, ventral root and dorsal root NF-H are phosphorylated extensively and differentially by this kinase. Under these conditions, PK40ERK catalyzes the incorporation of, respectively, 4.2±1.3 and 2.8±0.6 mol of phosphate per molecule of ventral root NF-H and dorsal root NF-H. The ratio of phosphates incorporated into ventral root NF-H to those incorporated into dorsal root NF-H is 1.46±0.17.

6. These findings support the hypothesis that different classes of neurons contain distinctly phosphorylated neurofilaments and show that ventral root and dorsal root neurons are a useful model system for studying the distinct characteristics of neurofilament phosphorylation in different types of neurons.

Key words: neurofilaments, phosphorylation, ventral root neurons, dorsal root neurons

References

  1. Berglund, A. M., and Ryugo, D. K. (1991). Neurofilament antibodies and spinal ganglion neurons of the mammalian cochlea.J. Comp. Neurol.306393–408. [DOI] [PubMed] [Google Scholar]
  2. Campbell, M. J., and Morrison, J. H. (1989). Monoclonal antibody to neurofilament protein (SM132) labels a subpopulation of pyramidal neurons in the human amd monkey neocortex.J. Comp. Neurol.282191–205. [DOI] [PubMed] [Google Scholar]
  3. Caputo, C., Syzowski, L., Brunner, W., Scott, C., and Salama, A. (1989). Properties of several protein kinases that copurify with rat spinal cord neurofilaments.Biochim. Biophys. Acta1012299–307. [DOI] [PubMed] [Google Scholar]
  4. Carden, M. J., and Eagles, P. A. M. (1983). Neurofilaments from ox spinal nerves: Isolation, disassembly, reassembly and cross-linking properties.Biochem. J.215215–237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carden, M. J., Schlaepfer, W. W., and Lee, N. M.-Y. (1985). The structure, biochemical properties and immunogenecity of neurofilament peripheral regions are determined by phosphorylation state.J. Biol. Chem.2609805–9817. [PubMed] [Google Scholar]
  6. Carden, M. J., Trojanowski, J. Q., Schlaepfer, W. W., and Lee, N. M.-Y. (1987). Two stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns.J. Neurosci.273489–3504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clark, E. A., and Lee, V. M.-Y. (1991). Dynamics of mammalian high-molecular-weight neurofilament subunit phosphorylation in cultured rat sympathetic neurons.J. Neurosci. Res.30116–123. [DOI] [PubMed] [Google Scholar]
  8. Cohen, R. S., Pont, H. C., House, S., and Gainer, H. (1987). Biochemical and immunocytochemical characterization of the distribution of phosphorylated and non phosphorylated subunits in neurofilaments in squid giant axon and stellate ganglion.J. Neurosci.72056–2074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dahl, D. (1983). Immunohistochemical differences between neurofilaments in perikarya, dendrates and axons.Exp. Cell Res.149397–408. [DOI] [PubMed] [Google Scholar]
  10. Dahl, D., Labkovsky, B., and Bigmani, A. (1988). Neurofilament phosphorylation in axons and perikarya: Immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies.J. Comp. Neurol.271445–450. [DOI] [PubMed] [Google Scholar]
  11. Dahl, D., Gilad, L. H., Mazzini, L., and Bigmani, A. (1992). Effect of the substrate on neurofilament phosphorylation in mixed cultures on rat embryo spinal cord and dorsal root ganglia.Int. J. Dev. Neurosci.10(2):111–119. [DOI] [PubMed] [Google Scholar]
  12. de Waegh, S. M., Lee, V. M.-Y., and Brady, S. (1992). Local modulation of neurofilament phosphorylation axonal caliber and slow axonal transport by myelinating Schwann cells.Cell62451–463. [DOI] [PubMed] [Google Scholar]
  13. Durham, H. D. (1992). An antibody against hyperphosphorylated neurofilament protein collapses the neurofilament network in motor neurons but not in dorsal root ganglion cells.J. Neuropathol. Exp. Neurol.51(3):287–297. [DOI] [PubMed] [Google Scholar]
  14. Guan, R., Hall, F., and Cohlberg, J. (1992). Proline directed kinase (p34cdc2/p58cyclinA) phosphorylates bovine neurofilaments.J. Neurochem.581365–1371. [DOI] [PubMed] [Google Scholar]
  15. Harris, J., Ayyub, C., and Shaw, G. (1991). A molecular dissection of the carboxyterminal tails of the major neurofilament subunit NF-M and NF-H.J. Neurosci. Res.3047–62. [DOI] [PubMed] [Google Scholar]
  16. Hisanaga, S., Kusubata, M., Okumura, E., and Kishimoto, T. (1991). Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules.J. Biol. Chem.26621798–21803. [PubMed] [Google Scholar]
  17. Jones, S. M., and Williams, R. C. (1982). Phosphate content of mammalian neurofilaments.J. Biol. Chem.2579902–9905. [PubMed] [Google Scholar]
  18. Julien, J.-P., and Mushynski, W. E. (1982). Multiple phosphorylation sites in mammalian neurofilament polypeptides.J. Biol. Chem.25710467–10470. [PubMed] [Google Scholar]
  19. Kaufmann, E., Geisler, N., and Weber, K. (1984). SDS-PAGE strongly over-estimates the molecular mass of the neurofilament proteins.FEBS Lett.12081–84. [DOI] [PubMed] [Google Scholar]
  20. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature277680–685. [DOI] [PubMed] [Google Scholar]
  21. Lee, V. M.-Y., Carden, M. J., Schlaepfer, W. W., and Trojanowski, J. Q. (1987). Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats.J. Neurosci.73474–3488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee, V. M.-Y., Otvos, L., Carden, M., Hollosi, M., Dietzschold, B., and Lazzarini, R. (1988). Identification of the major multiphosphorylation site in mammalian neurofilaments.Proc. Natl. Acad. Sci. USA1851998–2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nixon, R. A., and Sihag, R. K. (1991). Neurofilament phosphorylation: A new look at regulation and function.Trends Neurosci.14501–506. [DOI] [PubMed] [Google Scholar]
  24. Robinson, P. A., and Anderton, B. H. (1988). Neurofilament probes—A review of neurofilament distribution and biology.Rev. Neurosci.21–41. [Google Scholar]
  25. Roder, H. M., and Ingram, V. M. (1991). Two novel kinases phosphorylate Tau and the KSP site of heavy neurofilament subunits in high stoichiometric ratios.J. Neurosci.113325–3343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roder, H. M., Eden, P. A., Vernon, N., and Ingram, M. (1993). Brain protein kinase PK40ERK converts TAU into a PHF-like form as found in Alzheimer's disease.Biochem. Biophys. Res. Comm.193639–647. [DOI] [PubMed] [Google Scholar]
  27. Soppet, D. R., Beasley, L. L., and Willard, M. B. (1992). Evidence for unequal crossing over in the evolution of the neurofilament polypeptide H.J. Biol. Chem.26717354–17361. [PubMed] [Google Scholar]
  28. Soussan, L., Barzilai, A., and Michaelson, D. M. (1994). Distinctly phosphorylated neurofilaments in different classes of neurons.J. Neurochem.62770–776. [DOI] [PubMed] [Google Scholar]
  29. Steinart, D. M., and Roop, D. R. (1988). Molecular biology of intermediate filaments.Annu. Rev. Biochem.575593–625. [DOI] [PubMed] [Google Scholar]
  30. Sternberger, L. A., and Sternberger, N. H. (1983). Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilamentsin situ.Proc. Natl. Acad. Sci. USA8061256–6130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Szaro, B. G., Whitnall, M. H., and Gainer, H. (1990). Phosphorylation dependent epitopes on neurofilament proteins and neurofilament densities in differ in axons in the corticospinal and primary sensory dorsal column tracts in the rat spinal cord.J. Comp. Neurol.302220–235. [DOI] [PubMed] [Google Scholar]
  32. Tokui, T., Yamauchi, T., Yano, T., Nishi, Y., Kusagawa, M., Yatani, R., and Inagaki, M. (1990). Ca2+-calmodulin-dependent protein kinase II phosphorylates various types of nonepithelial intermediate filament proteins.Biochem. Biophys. Res. Comm.169896–904. [DOI] [PubMed] [Google Scholar]
  33. Vickers, J. C., Costa, M., Vitadello, M., Dahl, D., and Marotta, C. A. (1990). Neurofilament protein-triplet immunoreactivity in distinct subpopulations of peptide containing neurons in the guinea-pig coeliac ganglion.Neuroscience39743–759. [DOI] [PubMed] [Google Scholar]
  34. Wible, B. A., Smith, K. E., and Angelides, K. J. (1989). Resolution and purification of a neurofilament-specific kinase.Proc. Natl. Acad. Sci. USA86720–724. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Cellular and Molecular Neurobiology are provided here courtesy of Springer

RESOURCES