Summary
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1.
A neurofilament-enriched preparation from bovine spinal cord contains endogenous protein kinases that phosphorylate high, middle, and low molecular weight neurofilament subunits (NF-H, NF-M, and NF-L), as well as certain other endogenous and exogenous substrates.
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2.
Most of this associated kinase activity can be separated from the neurofilament subunits and the bulk of the protein by extraction of the neurofilament preparation with 0.8M KC1. Assays using specific exogenous substrates, activators, and inhibitors for known kinases reveal significant levels of Ca2+-calmodulin-dependent, cyclic nucleotide-dependent, Ca2+-phosphatidylserine diglyceride-dependent, and regulator-independent kinase activities in the high-salt extract.
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3.
Fractionation of the salt extract on a gel filtration column resolves a regulator-independent kinase activity identified by its ability to phosphorylate purified NF-M. This preparation can phosphorylate all three neurofilament proteins either in purified form or in the assembled form, as well asα-casein. Only the regulator-independent kinase activity in this fraction is responsible for the phosphorylation of neurofilament proteins.
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4.
While this partially purified kinase activity does not show a strong substrate specificity between the three neurofilament subunits, the phosphorylation pattern it produces upon incubation with salt-extracted neurofilaments is similar to the regulator-independent phosphorylation pattern found in the original neurofilament preparation and, thus, represents a useful starting point for the further purification of this neurofilament-associated kinase activity.
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References
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72248–254.
Caputo, C. B., Sygowski, L. A., Brunner, W. F., Scott, C. W., and Salama, A. I. (1989). Properties of several protein kinases that copurify with rat spinal cord neurofilaments.Biochim. Biophys. Acta 1012299–307.
Carden, M. J., Schlaepfer, W. W., and Lee, V. M.-Y. (1985). The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state.J. Biol. Chem. 2609805–9817.
Geisler, N., Vandekerckhove, J., and Weber, K. (1987). Location and sequence characterization of the major phosphorylation sites of the high molecular mass neurofilament proteins M and H.FEBS Lett. 221403–407.
Georges, E., Lefebvre, S., and Mushinsky, W. E. (1986). Dephosphorylation of neurofilaments by exogenous phosphatases has no effect on reassembly of subunits.J. Neurochem. 47477–483.
Glass, D. B., and Krebs, E. G. (1982). Phosphorylation by guanosine 3′,5′-monophosphate-dependent protein kinase of synthetic peptide analogs of a site phosphorylated in histone H2B.J. Biol. Chem. 2571196–1200.
Glicksman, M. A., Soppet, D., and Willard, M. B. (1987). Posttranslational modification of neurofilament polypeptides in rabbit retina.J. Neurobiol. 1867–196.
Hathaway, G. M., Lubben, T. H., and Traugh, J. A. (1980). Inhibition of casein kinase II by heparin.J. Biol. Chem. 2558038–8041.
Itarte, E., Mor, M. A., Salavert, A., Pena, J. M., Bertomeu, J. F., and Guinovart, J. J. (1981). Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol.Biochim. Biophys. Acta 658334–347.
Julien, J.-P., and Mushynski, W. E. (1982). Multiple phosphorylation sites in mammalian neurofilament polypeptides.J. Biol. Chem. 25710467–10470.
Julien, J.-P., Smoluk, G. D., and Mushynski, W. E. (1983). Characteristics of the protein kinase activity associated with rat neurofilament preparations.Biochim. Biophys. Acta 75525–31.
Kemp, B. (1976). Synthetic peptide substrates of the cAMP-dependent protein kinase.Fed. Proc. 351384 (abstract).
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4.Nature 227680–685.
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.
Oblinger, M. M. (1987). Characterization of posttranslational processing of the mammalian high-molecular weight neurofilament proteinin vivo.J. Neurosci. 72510–2521.
O'Brian, C. A., Lawrence, D. S., Kaiser, E. T., and Weinstein, I. B. (1984). Protein kinase C phosphorylates the synthetic peptide Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val in the presence of phospholipid plus either Ca2+ or a phorbol ester tumor promoter.Biochem. Biophys. Res. Commun. 124296–302.
Pant, H. C., Gallant, P. E., and Gainer, H. (1986). Characterization of a cyclic nucleotide- and calcium-independent neurofilament protein kinase activity in axoplasm from the squid giant axon.J. Biol. Chem. 2612958–2977.
Pearson, R. B., Woodgett, J. R., Cohen, P., and Kemp, B. E. (1985). Substrate specificity of a multifunctional calmodulin-dependent protein kinase.J. Biol. Chem. 26014471–14476.
Peterson, G. L. (1977). A simplification of the protein assay method of Lowryet al. which is more generally applicable.Anal. Biochem. 83346–356.
Roskoski, J. R. (1983). Assays of protein kinase.Methods Enzymol. 99 3–6.
Runge, M. S., El-Maghrabi, M. R., Claus, T. H., Pilkis, S. J., and Williams, R. C. (1981). A MAP-2 stimulated protein kinase activity associated with neurofilaments.Biochemistry 20175–180.
Scott, D., Smith, K. E., O'Brien, B. J., and Angelides, K. J. (1985). Characterization of mammalian neurofilament triplet proteins.J. Biol. Chem. 26010736–10747.
Shaw, G. (1989). Identification of previously unrecognized sequence motifs at the extreme carboxyterminus of the neurofilament subunit NF-M.Biochem. Biophys. Res. Commun. 162294–299.
Shecket, G., and Lasek, R. J. (1982). Neurofilament protein phosphorylation.J. Biol. Chem. 2574788–4795.
Sihag, R. K., and Nixon, R. A. (1989).In vivo phosphorylation of distinct domains of the 70-Kilodalton neurofilament subunit involves different protein kinases.J. Biol. Chem. 264457–464.
Sihag, R. K., Jeng, A. Y., and Nixon, R. A. (1988). Phosphorylation of neurofilament proteins by protein kinase C.FEBS Lett. 233181–185.
Tokutake, S. (1984). Complete separation of the triplet components of neurofilament by DE-52 column chromatography depends upon urea concentration.Anal. Biochem. 140203–207.
Toru-Delbauffe, D., and Pierre, M. (1983). A rat brain kinase phosphorylating specifically neurofilaments.FEBS Lett. 162230–234.
Toru-Delbauffe, D., Pierre, M., Osty, J., Chantoux, F., and Francon, J. (1986). Properties of neurofilament protein kinase.Biochem. J. 235283–289.
Vallano, M. L., Buckholz, T. M., and DeLorenzo, R. J. (1985). Phosphorylation of neurofilament proteins by endogenous calcium/calmodulin-dependent protein kinase.Biochem. Biophys. Res. Commun. 130957–963.
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.
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Dosemeci, A., Floyd, C.C. & Pant, H.C. Characterization of neurofilament-associated protein kinase activities from bovine spinal cord. Cell Mol Neurobiol 10, 369–382 (1990). https://doi.org/10.1007/BF00711181
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DOI: https://doi.org/10.1007/BF00711181