Properties of neurofilament protein kinase

Toru-Delbauffe, D.; Martineau, Pierre; Osty, Jeannine; Chantoux, Françoise; Francon, Jacques

doi:10.1042/bj2350283

Cited by 37 publications

(21 citation statements)

References 38 publications

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“…Several protein kinases can phosphorylate NFs, including cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase (16,17), but NF-M is the preferred substrate for these enzymes. A previously described NF-associated kinase (14) differs from the NF kinase that we have purified because it shows a much broader substrate specificity and does not phosphorylate NFs in the in vivo pattern. In contrast, the NF kinase that we have isolated shows a strong preference for NF-H, unlike any NF-associated enzyme activity previously reported, and has an activity that results in the characteristic in vivo NF phosphorylation pattern.…”

Section: Discussionmentioning

confidence: 73%

Resolution and purification of a neurofilament-specific kinase.

Wible¹,

Smith²,

Angelides³

1989

Proc. Natl. Acad. Sci. U.S.A.

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Both in vivo and in vitro, neuroframents (NFs) are among the most highly phosphorylated proteins known. The majority of the NF phosphorylation sites reside on the carboxyl-terminal tails of the proteins. We have isolated and characterized an effector-independent neurofflamentspecific protein kinase from bovine spinal cord that is associated with the NF complex and exhibits a marked substrate specificity for NF-H, the largest subunit of the NF triplet. This kinase activity emerges from a NF-conjugated affmity column coincident with a 67-kDa doublet on NaDodSO4/polyacrylamide gels and has a purity of >90%. The purified enzyme exclusively phosphorylates NF-H tails and is dependent on prior phosphorylation of this molecule. The enzyme is also not autophosphorylated. While the molecular properties and substrate specificities of the NF kinase distinguish it from cAMPdependent protein kinase, protein kinase C, Ca2+/calmodulin kinase, and casein kinases I and II, it exhibits certain properties similar to, but different from, the growth-associated histone HI kinase. The molecular properties and specific sequence requirements ofthe NF kinase suggest that this enzyme could play a pivotal role in the phosphorylation of NFs in normal and pathological states such as Alzheimer disease, where NFs are hvnernhnenharv Atd.Nerve cell intermediate filaments, or neurofilaments (NFs), are composed of three proteins of low (66 kDa; NF-L), medium (130 kDa; NF-M), and high (180 kDa; NF-H) molecular masses (1, 2). While the three NF subunits share a highly conserved a-helical rod region with other intermediate filament proteins, NF-H and NF-M differ in that they each possess a highly acidic carboxyl-terminal tail domain. NF-M and NF-H MATERIALS AND METHODS Chemicals. The following protease inhibitors at 1 pug/ml were included in all preparative buffers: pepstatin, leupeptin, N2-(p-tosyl)-L-lysine chloromethyl ketone, and L-1-tosylamido-2-phenylethyl chloromethyl ketone; and 0.2 mM phenylmethylsulfonyl fluoride was also added.Neurorflaments and Purified Subunits. NF-enriched fractions and purified subunits (NF-H, NF-M, and NF-L) were prepared from fresh bovine spinal cord as described (2).NF-enriched cytoskeletal proteins and resolved NF subunits were dephosphorylated by incubation with 2-20 units of Escherichia coli alkaline phosphatase per mg of NF protein for various times ranging from 5 min to 18 hr (6). Dephosphorylated subunits were brought to 50 mM EDTA, boiled, and dialyzed against 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes; pH 6.5) prior to assay.Phosphorylated NF-H was digested with chymotrypsin at an enzyme/NF-H ratio of 1:400 (wt/wt) for 15 min at 220C (9).Under these conditions NF-H is cleaved into two fragments, a 40-kDa rod and a 160-kDa tail. Microtubules (MT) and microtubule-associated proteins (MAPs) were prepared by using temperature-dependent cycles of assembly/disassembly (10 During the purification, the specific activity of the enzyme was monitored by assaying 1 ,ug of purified NF-H under the same condit...

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Section: Discussionmentioning

confidence: 73%

Resolution and purification of a neurofilament-specific kinase.

Wible¹,

Smith²,

Angelides³

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…Protein kinases that phosphorylate all three subunits of NF were partially purified by several other laboratories, although their molecular weights and structural characteristics are unknown. One of these, partially purified by Toru-Delbauffe and Pierre (47) and Toru-Delbauffe et al (48), has a high specificity for NFH and is insensitive to 5 mM Ca 2ϩ . Another one, purified by Dosemeci et al (49), has marked preference for casein rather than histone or NF as substrate.…”

Section: Pkn Phosphorylates the Head-rod Domain Of Each Subunit Of Nf-mentioning

confidence: 99%

PKN Associates and Phosphorylates the Head-Rod Domain of Neurofilament Protein

Mukai

Toshimori

Shibata

et al. 1996

Journal of Biological Chemistry

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“…Several previous biochemical studies have described protein kinase activities that copurify with preparations of NF proteins (for example, Julien et al, 1983;Toru-Delbauffe et al, 1986;Xiao and Monteiro, 1994). We have identified a major NF-associated kinase (NFAK) in chicken NF preparations that we partially purified and examined biochemically (Hollander and Bennett, 1992).…”

mentioning

confidence: 95%

Characterization of Additional Casein Kinase I Sites in the C‐Terminal “Tail” Region of Chicken and Rat Neurofilament‐M

Shaw¹,

Miller²,

Wang³

et al. 1997

Journal of Neurochemistry

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Abstract:In previous studies we have identified Ser 502, Ser528, and Ser534 as target sites in chicken neurofilament middle molecular mass protein (N F-M) for casein kinase I (CKI) in vitro and have shown that these sites are also phosphorylated in vivo. We now make use of a combination of molecular biological and protein chemical techniques to show that two additional in vivo phosphorylation sites in chicken NF-M, Ser464 and Ser471, can also be phosphorylated by CKI in vitro. These two sites are conserved in higher vertebrate NF-M molecules, and recombinant protein constructs containing the homologous rat NF-M peptides can be phosphorylated by CKI in vitro, suggesting that phosphorylation of these sites is conserved at least in higher vertebrates. The two new sites are adjacent to a conserved peptide sequence (VEE-IIEET-V) found once in higher vertebrate NF-M molecules and twice in lamprey NF-180. Variants of this sequence are also found in neurofilament low and high molecular mass proteins (NF-L and NF-H) and a-internexin, and in mammalian NF-L are known to be associated with in vivo phosphorylation sites. We speculate that CKI phosphorylation in general, and these sites in particular, may be important in neurofilament function.

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Properties of neurofilament protein kinase

Cited by 37 publications

References 38 publications

Resolution and purification of a neurofilament-specific kinase.

Resolution and purification of a neurofilament-specific kinase.

PKN Associates and Phosphorylates the Head-Rod Domain of Neurofilament Protein

Characterization of Additional Casein Kinase I Sites in the C‐Terminal “Tail” Region of Chicken and Rat Neurofilament‐M

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