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H C Pant

Publications and source records attributed to H C Pant.

At least 19 recordsLinked to original sources
PubMed1995

Molecular cloning and chromosomal mapping of the mouse cyclin-dependent kinase 5 gene.

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neurons. In vitro, Cdk5 purified from the nervous tissue phosphorylates both high-molecular-weight neurofilament and microtubule-associated tau. The mouse gene encoding Cdk5 (Cdk5) was found to be 5 kb in length and divided into 12 exons. All of the exon-intron junctions matched the expected consensus sequence with the exception of the splice junction for intron 9, which has AT and AC dinucleotides instead of the usual GT and AG bordering sequence. In the 5'-flanking region of mouse Cdk5, several putative promoter elements were present, including AP1, Sp1, PuF, and TATA motifs. A metal regulatory element was also identified at position -207 to -201. Nucleotide sequence analysis of mouse Cdk5 showed high identity to the homologues of other vertebrate species, indicating that this kinase is highly conserved during evolution. Mouse Cdk5 was mapped to the centromeric region of mouse chromosome 5.

Amino Acid Sequence↗
PubMed1995

Expression of neurofilament proteins during development of the nervous system in the squid Loligo pealei.

The squid nervous system includes various brain ganglia, optic lobes (the visual center), and the stellate ganglia, the system of giant motor fibers responsible for rapid jet-propelled escape behavior. The large caliber of giant fibers is due, in part, to the accumulation of squid-specific neurofilaments (NFs) made up of a heavily phosphorylated NF 220 protein together with NF 70 and NF 60 subunits. Using antibodies prepared against known peptide sequences in these proteins, together with a mammalian-derived antibody that specifically recognizes phosphorylated squid NF 220, we studied the localization of NFs in adult tissues and during neural development. Immunoblot and immunohistochemical analyses showed that NFs were present in adult neural tissues, primarily in selected fibers, with giant axons showing the most robust expression. After the first neurons differentiated at stage 22, immunoblots showed NF 60- and NF 70-immunoreactive proteins at all stages. The NF 220 subunit, however, was not detected in immunoblots at any developmental stage. Phosphorylated NF 220 immunoreactivity, although absent in immunoblots, was first seen in selected fibers of the stellate ganglia at stage 25, increasing thereafter in all giant fibers until hatching (stage 30). The stellate ganglion is the first neural tissue to acquire a mature neurofilament complement (i.e., phosphorylated NF 220), shortly before the onset of jet-propelled escape behavior. The temporal pattern of expression of the NFs during development resembled that seen in vertebrates; i.e., the smaller NFs appeared before the larger subunit in most neural tissues. In the squid, the expression pattern seems to depend upon the post-transcriptional regulation of a single gene rather than upon transcriptional regulation of three independent genes as in vertebrates.

Animals↗
PubMed1995

Molecular characterization of a neuronal-specific protein that stimulates the activity of Cdk5.

Cyclin-dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and tau protein phosphorylation. This report describes the characterization of a 62-kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full-length cDNA was characterized coding for a 67.5-kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose-dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components.

Amino Acid Sequence↗
PubMed1995

Neuronal cyclin-dependent kinase-5 phosphorylation sites in neurofilament protein (NF-H) are dephosphorylated by protein phosphatase 2A.

Neurofilament (NF) protein [high molecular mass (NF-H)] is extensively phosphorylated in vivo. The phosphorylation occurs mainly in its characteristic KSP (Lys-Ser-Pro) repeat motifs. There are two major types of KSP motifs in the NF-H tail domain: KSPXKX and KSPXXX. Recent studies by two different laboratories have demonstrated the presence of a cdc2-like kinase [cyclin-dependent kinase-5 (cdk5)] in nervous tissue that selectively phosphorylates KSPXKX and XS/TXK motifs in NF-H and lysine-rich histone (H1). This article describes the identification of phosphatases dephosphorylating three different substrates: histone (H1), NF-H in a NF preparation, and a bacterially expressed C-terminal tail domain of NF-H, each containing KSPXKX repeats phosphorylated in vitro by cdk5. Among various phosphatases identified, protein phosphatase (PP) 2A from rabbit skeletal muscle appeared to be the most effective phosphatase in in vitro assays. Three phosphatase activity peaks--P1, P2, and P3--were partially purified from frozen rat spinal cord by ion exchange and size exclusion column chromatography and then characterized on the basis of biochemical, pharmacological, and immunochemical studies. One of the three peaks was identified as PP2A, whereas the others were mixtures of both PP2A and PP1. These three peaks could dephosphorylate cdk5-phosphorylated 32P-histone (H1), 32P-NF-H in the NF preparation, and 32P-NF-H tail fusion protein. These studies suggest the involvement of PP2A or a PP2A-like activity in the regulation of the phosphorylation state of KSPXKX motifs in NF-H.

Animals↗
PubMed1995

P13suc1 associates with a cdc2-like kinase in a multimeric cytoskeletal complex in squid axoplasm.

P13suc1 sepharose-conjugated beads were used to extract the kinases that phosphorylate neurofilaments in the squid giant axon. Using Western blots and in vitro kinase assays, we demonstrated the presence of an active cdc2-like kinase and its putative regulators such as cyclin E, p13, and p67 in axoplasm and a P13-axoplasm complex (P13-Ax). Protein kinase A (PKA) and casein kinase (CK) I and II were also found in the P13-Ax. Western blot analysis of the P13-Ax also demonstrated several axonal cytoskeletal components; e.g., neurofilaments (NFs; NF 60, 70, and 220), tubulin, actin, and microtubule-associated proteins. NF 220 and tubulin were phosphorylated by the kinases in the P13-Ax. To determine whether NFs bound directly to the P13 beads, or bound indirectly by association with cdc2 kinase, a washed, axon-derived neurofilament preparation that contained NFs, PKA, CKl, and tubulin, but no cdc2-like kinase, yielded no bound proteins after incubation with P13suc1. The wash supernatant from the neurofilament preparation, however, containing the cdc2-like kinase, did yield cytoskeletal components that bound to P13suc1. Moreover, a bacterial-expressed cdk5 associated with P13 beads was able to complex with selected cytoskeletal components in the washed neurofilament preparation. These data indicate that direct binding of P13 beads with a cdc2-like kinase could extract active multimeric complexes composed of axonal cytoskeletal proteins and kinases. Application of P13 chromatography may be useful in characterizing the network of functional interactions among cytoskeletal elements and protein kinases in neurons.

Animals↗
PubMed1994

Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protein.

The high-molecular-weight neurofilament protein (NF-H) is highly phosphorylated in vivo, with estimates as high as 16-51 mol of Pi/mol of protein. Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxy-terminal tail region of the molecule. Because the extent and site-specific patterns of tail domain phosphorylation are believed to modulate neurofilament structure and function, it becomes essential to identify the endogenous sites of phosphorylation. In this study, we have used selective proteolytic cleavage procedures, Pi determinations, microsequencing, and mass-spectral analysis to determine the endogenously phosphorylated sites in the NF-H tail isolated from rat spinal cord. Twenty Ser residues in NF-H carboxy-terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated. No detectable phosphorylation could be identified in any of the 11 "non-KSP" Ser residues that were examined. KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphorylated. In addition, a 27-kDa KSP-rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and found to contain between 30 and 35 mol of Pi/mol of protein. This domain appeared to be highly resistant to endoproteinase Glu-C digestion, although it contains a large number of glutamate residues. It could be proteolyzed, however, after dephosphorylation. This suggests that phosphorylation of the tail domain may contribute to neurofilament stability in vivo. A neuronal-derived protein kinase that specifically phosphorylates only KSPXKX motifs in neurofilaments has been reported. The presence of extensively phosphorylated KSPXXX repeats in NF-H in vivo suggests the existence of yet another, unidentified kinase(s) with specificity for KSPXXX motifs.

Alkaline Phosphatase↗
PubMed1993

cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization.

A protein kinase that phosphorylates a specific KSP sequence [K(S/T)PXK], which is abundant in high molecular weight neurofilament (NF) proteins, was identified and isolated from rat spinal cord. Characterization of this enzyme activity revealed a close relationship with p34cdc2 kinase with respect to its molecular mass (32.5 kDa by SDS/PAGE) and substrate specificities. It could phosphorylate a synthetic peptide analog of the simian virus 40 large tumor antigen, reportedly a specific substrate for p34cdc2 kinase. Histone (H1) and peptide analogs of the KSP sequence present in the C-terminal end of rat and mouse neurofilament proteins were phosphorylated. This kinase did not phosphorylate alpha-casein and peptide substrates of other known second messenger-dependent or -independent kinases. Dephosphorylated rat NF protein NF-H was strongly phosphorylated by the purified enzyme; NF proteins NF-M and native NF-H, but not NF-L, were slightly phosphorylated. Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate. On Western blots, the enzyme was immunoreactive with antibody against the C-terminal end of cdc2 kinase (mouse) and neuronal cdc2-like kinase from rat but not with an antibody against the conserved PSTAIRE region of the p34cdc2 kinase. The antibody against the C-terminal end of cdc2 kinase could immunoprecipitate (immunodeplete) the purified kinase activity. Since the adult nervous system is composed primarily of postmitotic cells, the present observations indicate a nonmitotic role for this cdc2-like kinase activity. The effective phosphorylation of NF-H by this kinase suggests a function in axonal structure.

Amino Acid Sequence↗
PubMed1993

Bovine neurofilament-enriched preparations contain kinase activity similar to casein kinase I--neurofilament phosphorylation by casein kinase I (CKI).

Neurofilament (NF)-enriched preparations from bovine spinal cord contain regulator-independent kinase activities that phosphorylate NF subunits as well as alpha-casein. CKI-7 (N-2-amino ethyl, 5-chloroisoquinoline, 8-sulfonamide), a specific inhibitor of casein kinase I (CKI), inhibits the phosphorylation of NF subunits in the neurofilament preparation. This inhibition occurs at a concentration range identical to concentrations where CKI-7 inhibits rabbit reticulocyte CKI phosphorylation of alpha-casein. Heparin, a specific inhibitor of casein kinase II (CKII), produced only 20% inhibition of 32P incorporation into NF subunits, and only at concentrations 5 to 10-fold higher than those required to inhibit CKII from reticulocytes. CKI from rabbit reticulocytes phosphorylated all three NF subunits (NF-H, NF-M and NF-L). Comparison of the tryptic phosphopeptide maps of NF-M, phosphorylated by the NF-associated kinase and CKI, indicates that the casein kinase I phosphorylates many of the peptides phosphorylated by the NF-associated kinase and this phosphorylation occurs at the carboxy terminal tail domain of the NF-M subunit. These studies suggest that the major independent kinase activity associated with NFs is CKI.

Amino Acid Sequence↗
PubMed1992

Neuronal cdc2-like kinase: a cdc2-related protein kinase with predominantly neuronal expression.

Recent studies have shown that there exists a family of protein kinases structurally and functionally related to the yeast cell cycle regulatory kinase cdc2 [Meyerson, M., Faha, B., Su, L.-K., Harlow, E. & Tsai, L.-H. (1991) Cold Spring Harbor Symp. Quant. Biol. 56, 177-186 and Meyerson, M., Enders, G. H., Wu, C.-L., Su, L.-K., Gorka, C., Nelson, C., Harlow, E. & Tsai, L.-H. (1992) EMBO J. 11, 2909-2917]. Two members of cdc2 family, p34cdc2 (also named cdk1) and cdk2, have been identified in mammalian cells. cdk1 kinase regulates the progression from G2 to M phase, and cdk2 kinase has been proposed to regulate the progression from G1 to S phase. In this work, we have cloned and structurally characterized a third member of the cdc2 kinase family with 58% amino acid sequence identity to mouse cdk1 and 61% identity to human cdk2. We call this kinase neuronal cdc2-like kinase (nclk) because, in contrast to either cdk1 or cdk2, nclk is expressed at high levels in terminally differentiated neurons no longer in the cell cycle. Previous studies have shown [Hisanaga, S., Kusubata, M., Okumura, E. & Kishimoto, T. (1991) J. Biol. Chem. 266, 21798-21803 and Guan, R. J., Hall, F. L. & Cohlberg, J. A. (1992) J. Neurochem. 58, 1365-1371] that cdk1 kinase, but not other structurally defined protein kinases, could phosphorylate the repeated Lys-Ser-Pro (KSP) motifs found in mammalian high and middle molecular mass neurofilament subunits in vitro, but the precise molecular nature of the endogenous neuronal KSP kinase has remained undefined. The structural similarity of nclk to cdk1 kinase and its high level of expression in terminally differentiated neurons suggest that nclk may play a role in the phosphorylation of the neurofilament KSP repeats in vivo, a function distinct from cell cycle regulation.

Amino Acid Sequence↗
PubMed1992

A high-molecular-weight squid neurofilament protein contains a lamin-like rod domain and a tail domain with Lys-Ser-Pro repeats.

Previous studies have shown that two low molecular-weight neurofilament (NF) proteins (NF-60 and NF-70) from the squid Loligo pealei are translated from mRNAs that are splice variants of a single squid NF gene. In this study, we report the isolation and characterization of cDNA clones encoding a high-molecular-weight squid NF protein (NF-220), the mRNA of which derives from the same squid NF gene. All three proteins are identical in their amino-terminal and lamin-like rod domains but differ in their carboxyl-terminal tail regions. In contrast to the short tail domains of NF-60 and NF-70, the NF-220 protein has a longer tail domain containing an acidic cluster of amino acids immediately followed by repeated copies of the sequence motif Lys-Ser-Pro. The Lys-Ser-Pro domain is similar to that of mammalian medium NF (NF-M) and high NF (NF-H) proteins, where the serines are highly phosphorylated. Except for these Lys-Ser-Pro motifs, there is surprisingly little structural similarity between the squid NF-220 protein and mammalian NF-M and NF-H proteins. Furthermore, the location of introns in squid NF-220 protein shows that it is more closely related to nuclear lamins and type III intermediate-filament proteins than to vertebrate NF proteins.

Amino Acid Sequence↗
PubMed1992

Association of cyclic-AMP-dependent protein kinase with neurofilaments.

Neurofilament preparations isolated from bovine spinal cord contain cyclic-AMP-dependent protein kinase (PKA) activity. Treatment of this preparation with cyclic AMP, to dissociate the regulatory subunit of the kinase from the catalytic subunit, resulted in retention of the kinase activity but loss of cyclic AMP regulation. This suggests that PKA is associated via its catalytic subunit with the neurofilament preparation. The association of exogenous PKA from bovine heart with the neurofilament preparation and with neurofilaments reconstituted from purified neurofilament proteins was also investigated. Either the free catalytic subunit or combinations of the catalytic and regulatory subunits of PKA were incubated with the preparations, and the degree of association was determined as the level of kinase activity that co-sediments with neurofilaments. The results indicate that the free catalytic subunit of PKA co-sediments with neurofilaments reconstituted from purified proteins. The regulatory subunit of PKA from bovine heart, when pre-mixed with the catalytic subunit, decreased the level of kinase that co-sediments with the neurofilament fraction in a dose-dependent manner. This effect of the regulatory subunit was reversed by inclusion of cyclic AMP in the incubation medium before centrifugation. The above findings suggest that the regulatory subunit, when attached to the catalytic subunit, has an inhibitory effect on its association with neurofilaments, with the implication that the association may be a cyclic-AMP-regulated event.

Animals↗
PubMed1991

Effect of cadmium on Ca2+ transport in brain microsomes.

The effect of Cd2+ on Ca2+ transport properties (uptake/release) in rat brain microsomes is examined by the tracer method using 45Ca2+. Cadmium ion (Cd2+) shows a dose-dependent inhibition of Ca(2+)-ATPase activity and consequently, exhibits a reduction in ATP-dependent Ca2+ uptake. In addition to this, Cd2+ also stimulates a rapid release of Ca2+ (t1/2 = 0.5 min) from the microsomes in a dose-dependent manner. The effect of Cd2+ is reversible by 1 mM cysteine or dithiothreitol (DTT). It is suggested that Cd2+ plays an important role in regulating the transmembrane flux of the cations in the microsomes. This effect is dramatically modulated by DTT suggesting a role of sulfhydryl groups in Ca(2+)-transport.

Animals↗
PubMed1991

Squid low molecular weight neurofilament proteins are a novel class of neurofilament protein. A nuclear lamin-like core and multiple distinct proteins formed by alternative RNA processing.

The primary structure of the major 60-kDa squid low molecular mass neurofilament protein (NF60) and a related 70-kDa neurofilament protein have been determined from cDNA clones isolated from a squid brain cDNA library. Structural analysis suggests that the squid NF60 and NF70 neurofilament genes and proteins are remarkably distinct from vertebrate neuronal intermediate filaments characterized previously. Both proteins are encoded on mRNAs generated by alternative RNA processing of the primary transcript of a single gene. Among the known intermediate filament proteins, NF60 and NF70 neurofilament proteins show highest similarity to an epithelial intermediate filament protein from Helix pomatia, a gastropod mollusk, and are less similar to vertebrate neurofilaments. The length of the alpha-helical rod domain in the NF60 and NF70 proteins was reminiscent of the vertebrate nuclear lamins, 6 heptads longer than is found in all known vertebrate cytoplasmic intermediate filaments, in particular the vertebrate neurofilaments. These distinct structural properties suggest that the vertebrate and invertebrate low molecular weight neurofilaments evolved independently from primordial intermediate filament proteins.

Amino Acid Sequence↗
PubMed1991

Principal neurofilament-associated protein kinase in squid axoplasm is related to casein kinase I.

A cytoskeletal extract of pure axoplasm, highly enriched with neurofilaments (ANF), was prepared from the giant axon of the squid. This ANF preparation also contained potent kinase activities which phosphorylated the Mr greater than 400,000 (high molecular weight) and Mr 220,000 squid neurofilament protein subunits. High salt (1 M) extraction of this ANF preparation solubilized most of the neurofilament proteins and kinase activities and gel filtration on an AcA 44 column separated these two components. The neurofilaments eluted in the void volume of the column while the kinase activities eluted in the 17-44-kDa range of the column. Two major kinase activities were measured in this peak of activity. One of these strongly phosphorylated the phosphate acceptor peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and was completely inhibited by the selective inhibitor of cAMP-dependent kinase Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- NH2 (Wiptide). Since addition of cAMP did not stimulate activity, this suggested that this kinase was a free catalytic subunit of cAMP-dependent kinase associated with the neurofilaments. The second kinase activity most effectively phosphorylated alpha-casein, and this activity was not affected by Wiptide. The alpha-casein phosphorylating activity (ANF kinase) was the principal activity responsible for neurofilament protein phosphorylation, and was not inhibited by various inhibitors against second messenger regulated kinases, suggesting it was related to the casein kinase family. Four lines of evidence indicate ANF kinase was similar to casein kinase I. These were: 1) the apparent molecular weight determined by gel filtration and the chromatographic elution profile on phosphocellulose column corresponded to casein kinase I; 2) heparin, an inhibitor of casein kinase II at 2-5 micrograms/ml, stimulated both ANF kinase and purified casein kinase I at these concentrations, while CKI-7, a relatively selective inhibitor of casein kinase I, inhibited ANF kinase in a comparable dose-response fashion; 3) purified casein kinase I strongly phosphorylated both ANF protein subunits (like ANF kinase) whereas casein kinase II was relatively ineffective; and 4) tryptic peptide maps of the HMW and Mr 220,000 neurofilament proteins after phosphorylation by ANF kinase or purified casein kinase I showed similar 32P-peptide patterns.

Animals↗
PubMed1991

Depolarization of synaptosomal membranes: a study of mechanism by which rhodamine 6G measures membrane potential.

The fluorescence intensity of Rhodamine 6G in synaptosomal suspensions has been measured to monitor the membrane potential changes in pre-synaptic nerve terminals. The fluorescence response of the dye was seen to be a function of potential-dependent partitioning of dye molecules between the synaptosomes and the extracellular medium. Binding of dye molecules to the hydrophobic regions of membranes results in the quenching of fluorescence. Upon depolarization of the synaptosomal membrane, the dye molecules are released from the cells. The effect of changing extracellular ionic composition was also studied. The membrane potential increased linearly with log of [K]0. The resting membrane potential in buffer containing 5 mM K+ was calculated to be -60 mV. Raising the extracellular Ca2+ and Mg2+ from 1.2 mM to 10 mM did not change the membrane potential. Ca2+ ionophore A23187, in the presence of Ca2+ was found to depolarize the membranes.

Animals↗
PubMed1990

Characterization of the distinctive neurofilament subunits of the soma and axon initial segments in the squid stellate ganglion.

The stellate ganglion, which gives rise to the giant axons of the squid, was dissected into two parts, one containing primarily cell bodies and the other axon initial segments. A neurofilament protein-enriched extract of each was prepared and compared biochemically and immunochemically with an axoplasmic neurofilament preparation and with the glial sheath that surrounds the axons. Both parts of the ganglion lacked the 220 kDa subunit of axoplasmic neurofilaments (NFs). However, they did contain a protein of about 190 kDa that reacted with the Pruss anti-intermediate filament antibody (aIFA; Pruss et al.: Cell 27:419-428, 1981), but not with a phosphorylation-dependent NF antibody (Cohen et al.: J Neurosci 7: 2056-2074, 1987). Dephosphorylation of the axoplasmic NF220 yielded a product that comigrated on two-dimensional (2D) gel electrophoresis with the 190 kDa ganglion protein, suggesting that the latter represented the incompletely phosphorylated precursor of NF220. The major low molecular weight aIFA-reactive species in the ganglion preparations was a polypeptide of about 65 kDa. A relatively small quantity of that polypeptide was also found in axoplasm and it comigrated in 2D gels with an aIFA-reactive polypeptide from the glial sheath. These results indicate that the site of modification of the 190 kDa NF precursor to the 220 kDa axonal form is probably at the point where the axon initial segments leave the ganglion, which is several mm distal to its site of synthesis in the cell body. Furthermore, the filament network of the axoplasm and possibly the cell bodies includes a glial-like intermediate filament protein in addition to the NF protein subunits.

Animals↗
PubMed1990

Characterization of neurofilament-associated protein kinase activities from bovine spinal cord.

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. 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 0.8 M KCl. 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. 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 as alpha-casein. Only the regulator-independent kinase activity in this fraction is responsible for the phosphorylation of neurofilament proteins. 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.

Animals↗