Summary
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Berglund, A. M., and Ryugo, D. K. (1991). Neurofilament antibodies and spinal ganglion neurons of the mammalian cochlea.J. Comp. Neurol. 306393–408.
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.
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. Acta 1012299–307.
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.
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.
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.
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.
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.
Dahl, D. (1983). Immunohistochemical differences between neurofilaments in perikarya, dendrates and axons.Exp. Cell Res. 149397–408.
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.
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.
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.Cell 62451–463.
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.
Guan, R., Hall, F., and Cohlberg, J. (1992). Proline directed kinase (p34cdc2/p58cyclinA) phosphorylates bovine neurofilaments.J. Neurochem. 581365–1371.
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.
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.
Jones, S. M., and Williams, R. C. (1982). Phosphate content of mammalian neurofilaments.J. Biol. Chem. 2579902–9905.
Julien, J.-P., and Mushynski, W. E. (1982). Multiple phosphorylation sites in mammalian neurofilament polypeptides.J. Biol. Chem. 25710467–10470.
Kaufmann, E., Geisler, N., and Weber, K. (1984). SDS-PAGE strongly over-estimates the molecular mass of the neurofilament proteins.FEBS Lett. 12081–84.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 277680–685.
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.
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. USA 1851998–2002.
Nixon, R. A., and Sihag, R. K. (1991). Neurofilament phosphorylation: A new look at regulation and function.Trends Neurosci. 14501–506.
Robinson, P. A., and Anderton, B. H. (1988). Neurofilament probes—A review of neurofilament distribution and biology.Rev. Neurosci. 21–41.
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.
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.
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.
Soussan, L., Barzilai, A., and Michaelson, D. M. (1994). Distinctly phosphorylated neurofilaments in different classes of neurons.J. Neurochem. 62770–776.
Steinart, D. M., and Roop, D. R. (1988). Molecular biology of intermediate filaments.Annu. Rev. Biochem. 575593–625.
Sternberger, L. A., and Sternberger, N. H. (1983). Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilamentsin situ.Proc. Natl. Acad. Sci. USA 8061256–6130.
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.
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.
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.Neuroscience 39743–759.
Wible, B. A., Smith, K. E., and Angelides, K. J. (1989). Resolution and purification of a neurofilament-specific kinase.Proc. Natl. Acad. Sci. USA 86720–724.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chertoff, R., Soussan, L., Roder, H. et al. Phosphorylation and dephosphorylation of distinct isoforms of the heavy neurofilament protein NF-H. Cell Mol Neurobiol 15, 269–281 (1995). https://doi.org/10.1007/BF02073333
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02073333