Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The carboxyl-terminal regions of neurofilament high (NF-H) and middle (NF-M) molecular weight proteins have been suggested to be phosphorylated in vivo by a p34cdc2-like protein kinase, on the basis of the in vivo phosphorylation site motif and in vitro phosphorylation of the proteins by p34cdc2 kinase (Hisanaga, S.I., Kusubata, M., Okumura, E. and Kishimoto, T. (1991) J. Biol. Chem. 266, 21798-21803). A novel proline-directed protein kinase previously identified and purified from bovine brain has been found in this study to phosphorylate NF-H and NF-M at sites identical to those phosphorylated by HeLa cell p34cdc2 kinase. The proline-directed kinase is composed of a 33-kDa and a 25-kDa subunit. The 33-kDa kinase subunit was partially sequenced, and degenerate oligonucleotide primers corresponding to the amino acid sequence information were used to clone the subunit by polymerase chain reaction (PCR). Two overlapping PCR products comprised a complete open reading frame of 292 amino acids. The sequence contains all features of a protein kinase, suggesting that the 33-kDa peptide represents the catalytic subunit of the kinase. The 33-kDa subunit shows high and approximately equal homology to human p34cdc2 and human cdk2, with about 58 and 59% amino acid identity, respectively. These results suggest that the brain kinase represents a new category of the cdc2 family, and that some members of the cdc2 kinase family may have major functions unrelated to cell cycle control.
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PMID:Brain proline-directed protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2. 146 4

Proline-directed protein kinase (PDPK), a complex of p34cdc2 and p58cyclin A, phosphorylates bovine neurofilaments (NFs) in vitro. Incubation of intact filaments with PDPK led to strong labeling of the heavy (NF-H) and middle (NF-M) molecular weight NF proteins and weaker labeling of the low molecular weight protein (NF-L). All three proteins were phosphorylated in solution, with the best substrate being NF-H. Proteins that had been dephosphorylated by enzymatic treatment were better substrates than native proteins--as many as 6 mol of phosphate were incorporated per mole of NF-H. Partial proteolytic cleavage experiments combined with two-dimensional peptide mapping indicated that NF-H and NF-M were phosphorylated predominantly in the tail domains, with some phosphate also appearing in the heads. Soluble NF-L is phosphorylated on the head domain peptide L-3, whereas NF-L within intact filaments is phosphorylated only on the tail domain peptide L-1. Phosphorylation does not lead to filament disassembly. A possible role for PDPK in NF phosphorylation in vivo is discussed.
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PMID:Proline-directed protein kinase (p34cdc2/p58cyclin A) phosphorylates bovine neurofilaments. 154 71

A highly purified preparation of protein kinase FA (where FA is the activating factor for phosphatase 1)/glycogen synthase kinase 3 from rabbit muscle readily phosphorylated bovine neurofilaments. All three neurofilament proteins, the high, middle, and low molecular proteins (NF-H, NF-M, and NF-L), were phosphorylated when intact filaments were incubated with the kinase. Experiments with individual proteins showed that NF-M was the best substrate. At protein concentrations of 0.13 mg/ml, the initial rate of NF-M phosphorylation was 30% of that observed for glycogen synthase. Km values were 0.24 mg/ml (7 x 10(-7) M tetramer) for glycogen synthase and 0.10 mg/ml (5 x 10(-7) M dimer) for NF-M. Vmax values were 0.36 mumol/min/mg for glycogen synthase and 0.035 mumol/min/mg for NF-M. Dephosphorylated NF-M was phosphorylated only half as much as native NF-M; this is consistent with the known substrate specificity of the kinase. The possible involvement of FA/GSK-3 in the phosphorylation of neurofilaments in vivo is discussed.
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PMID:Phosphorylation of bovine neurofilament proteins by protein kinase FA (glycogen synthase kinase 3). 185 Jul 42

We sought the mammalian neurofilament tail domain-specific kinase. Several well known kinases including cAMP-dependent protein kinase, protein kinase C, Ca(2+)-calmodulin-dependent protein kinase II, casein kinase I, and casein kinase II phosphorylated the high (NF-H) and middle molecular mass subunit (NF-M) of bovine neurofilaments, but they did not reduced the electrophoretic mobility of the dephosphorylated form of NF-M and NF-H by phosphorylation nor was the amount of phosphorylation increased by dephosphorylation of NF proteins, indicating that the phosphorylation sites by these kinases are not major in vivo phosphorylation sites at the tail domain. In contrast, cdc2 kinase phosphorylated specifically the dephosphorylated form of NF-H. 4 mol of phosphates were incorporated per mol of NF-H and this phosphorylation returned the electrophoretic mobility of the dephosphorylated form of NF-H to the position of the isolated, fully phosphorylated form of NF-H. Furthermore, the phosphorylation by cdc2 kinase dissociated the binding of dephosphorylated NF-H to microtubules. Phosphorylation sites were located at the carboxyl-terminal tail domain. The KSPXK motif, but not KSPXX, in the repetitive sequence was suggested to be the phosphorylation site by using synthetic peptides.
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PMID:Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules. 193 2

Neurofilaments are composed of 3 polypeptides designated NF-H, NF-M, and NF-L, all of which are subject to posttranslational phosphorylation. It has been suggested that phosphorylation of the NF-L polypeptide can influence the assembly of NF-L into filaments, but the sites at which NF-L is phosphorylated are unknown. To locate these phosphorylation sites, we have identified phosphopeptides of NF-L by labeling them with 32P both in vitro and in cultured neurons and also by observing their change in chromatographic behavior after they have been treated with phosphatase. We report here that serine 473, in the carboxy-terminal tail domain of NF-L, is a major substrate in vitro for protein kinases endogenous to a crude cytoskeleton-containing fraction. Moreover, serine 473 is a major phosphorylation site in vivo; in neurofilaments isolated from rat spinal cord, approximately 73% of serine 473 was phosphorylated, and accounted for at least one-third of the total phosphate associated with NF-L. The identification of this phosphorylation site in NF-L provides a criterion for identifying the protein kinase that phosphorylates NF-L and raises the question of its function.
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PMID:Identification of serine 473 as a major phosphorylation site in the neurofilament polypeptide NF-L. 216 86

Phosphorylation of c-Myb has been implicated in the regulation of the binding of c-Myb to DNA. We show that murine c-Myb is phosphorylated at Ser-11 and -12 in vivo and that these sites can be phosphorylated in vitro by casein kinase II (CKII), analogous to chicken c-Myb. An efficient method to study DNA binding properties of full-length c-Myb and Myb mutants under nondenaturing conditions was developed. It was found that a Myb mutant in which Ser-11 and -12 were replaced with Ala (Myb Ala-11/12), wild-type c-Myb, and Myb Asp-11/12 bound to the A site of the mim-1 promoter with decreasing affinities. In agreement with this finding, Myb Ala-11/12 transactivated better than wild-type c-Myb and Myb Asp-11/12 on the mim-1 promoter or a synthetic Myb-responsive promoter. Similar observations were made for the myeloid-specific neutrophil elastase promoter. The presence of NF-M or an NF-M-like activity abolished partially the differences seen with the Ser-11/12 mutants, suggesting that the reduced DNA binding due to negative charge at positions 11 and 12 can be compensated for by NF-M. Since no direct interaction of c-Myb and NF-M was observed, we propose that the cooperativity is mediated by a third factor. Our data offer two possibilities for how casein kinase II phosphorylation can influence c-Myb function: first, by reducing c-Myb DNA binding and thereby influencing transactivation, and second, by enhancing the apparent cooperativity between c-Myb and NF-M or an NF-M-like activity.
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PMID:Casein kinase II phosphorylation site mutations in c-Myb affect DNA binding and transcriptional cooperativity with NF-M. 756 49

We have identified and purified from bovine brain a novel protein kinase which catalyzes in vitro phosphorylation of neurofilament proteins NF-H and NF-M and tau proteins at sites implicating the enzyme in the regulation of neurocytoskeleton dynamics and in Alzheimer pathology. The protein kinase displays a phosphorylation site specificity similar or identical to the cell cycle regulatory kinase, cdc2 kinase. The purified kinase is a heterodimer of a cdc2-like catalytic subunit, called cdk5, and a 25 kDa regulatory subunit. The regulatory subunit is essential for kinase activity, and it is derived from a 35 kDa protein, p35 by proteolysis. Northern blot analysis of tissue distribution indicates that cdk5 is widely distributed but especially rich in brain, whereas p35 expression is only found in brain. The protein kinase is therefore termed neuronal cdc2-like kinase. The neuron-specificity of the enzyme appears to be conferred by the regulatory subunit. During cell division, cdc2 kinase is regulated by complex phosphorylation mechanisms involving a network of specific protein kinases. Some of these kinases or their homologs have been found in mammalian brains and they may be involved in the regulation of neuronal cdc2-like kinase.
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PMID:Structure, function, and regulation of neuronal Cdc2-like protein kinase. 756 36

In order to characterize the phosphorylation of neurofilaments (NF) in intact neurons, we examined the ability of several protein kinase inhibitors to interfere with the incorporation 32P into individual NF polypeptides of sensory neurons in culture. We also examined their effect on the post-translational mobility shift on SDS-PAGE that accompanies phosphorylation of newly synthesized NF-M. Several agents known to inhibit cyclic nucleotide-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases (H7, HA1004, trifluoperizine, sphingosine) had no effect on the phosphorylation of any NF polypeptide, in either assay. In contrast, two broadly active protein kinase inhibitors, staurosporine and K252a, inhibited the incorporation of 32P into NF-M by 60-70% and also blocked the post-translational mobility shift. They had no effect on NF-L. The action of staurosporine and K252a was identical to that of 25 mM LiCl. Proteolytic cleavage and phosphopeptide mapping of 32P-labeled NF-M from control and treated cultures revealed that the phosphorylation of only one subset of phosphopeptides was affected by staurosporine, K252a, and LiCl. These were contained within a single chymotryptic fragment of the NF-M tail segment, probably containing most of the 17 repeats of a KXXS/TP motif. The phosphorylation of another subset of phosphopeptides was insensitive to these inhibitors. They were contained within a different chymotryptic fragment of the tail segment which contains a KSD and four KSP potential phosphorylation sites. This differential sensitivity to protein kinase inhibitors distinguishes two different types of effector-independent kinases that phosphorylate, in vivo, different sites within the NF-M tail.
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PMID:Differential sensitivity to inhibitors discriminates between two types of kinases responsible for in vivo phosphorylation of different sites in the carboxy-terminal tail of chicken neurofilament-M. 780 6

In this investigation we studied developmentally regulated endogenous protein kinase activity in cytoskeletal proteins in the cerebral cortex of rats and the effect of early malnutrition imposed on dams on the pattern of 32P incorporation into the cytoskeleton of pups. Our results indicated that in vitro incorporation was maximum in 7-day-old pups for both normal and malnourished groups, decreasing with development, and reaching minimum values in adult animals. However, 32P incorporation into NF-M and tubulin was significantly lower in 7-day-old malnourished pups than in normal pups.
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PMID:Developmentally regulated in vitro phosphorylation of cytoskeletal proteins of the cerebral cortex of normal and malnourished rats. 786 15

Phosphorylation of neurofilament-L protein (NF-L) by the catalytic subunit of cAMP-dependent protein kinase (A-kinase) inhibits the reassembly of NF-L and disassembles filamentous NF-L. The effects of phosphorylation by A-kinase on native neurofilaments (NF) composed of three distinct subunits: NF-L, NF-M, and NF-H, however, have not yet been described. In this paper, we examined the effects of phosphorylation of NF proteins by A-kinase on both native and reassembled filaments containing all three NF subunits. In the native NF, A-kinase phosphorylated each NF subunit with stoichiometries of 4 mol/mol for NF-L, 6 mol/mol for NF-M, and 4 mol/mol for NF-H. The extent of NF-L phosphorylation in the native NF was nearly the same as that of purified NF-L. However, phosphorylation did not cause the native NFs to disassemble into oligomers, as was the case for purified NF-L. Instead, partial fragmentation was detected in sedimentation experiments and by electron microscopic observations. This is probably not due to the presence of the three NF subunits in NF or to differences in phosphorylation sites because reassembled NF containing all three NF subunits were disassembled into oligomeric forms by phosphorylation with A-kinase and the phosphorylation by A-kinase occurred at the head domain of NF-L whether NF were native or reassembled. Disassembling intermediates of reassembled NF containing all three NF subunits were somewhat different from disassembling intermediates of NF-L. Thinning and loosening of filaments was frequently observed preceding complete disassembly. From the fact that the thinning was also observed in the native filaments phosphorylated by A-kinase, it is reasonable to propose the native NF is fragmented through a process of thinning that is stimulated by phosphorylation in the head domain of the NF subunits.
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PMID:Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M, and NF-H by the catalytic subunit of cAMP-dependent protein kinase. 801 2


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