Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Using dephosphorylated neurofilament (NF) proteins as substrates, the kinase with a higher activity for the dephosphorylated NF-H than the phosphorylated form of NF-H was searched for in the porcine brain extract. Most NF-H kinase activity in the brain extract pelleted with microtubules. The NF-H kinase purified from a high salt extract of the microtubule pellets was composed of cdk5 and a 26 kDa protein, a fragment of the 35 kDa regulatory subunit of cdk5. In contrast to the association of the active kinase with microtubules, each of uncomplexed cdk5 and the 35 kDa regulatory subunit was differently distributed in the supernatant fraction and the pellet, respectively, by ultracentrifugation of the brain extract. Dephosphorylated forms of NF-H and NF-M became reactive to antibodies recognizing in vivo phosphorylation sites (SMI31, 34, and 36, JJ31 and 51) by phosphorylation with cdk5/p26. cdk5/p26 showed similar enzymatic properties to p34cdc2/cyclin B kinase; the substrate specificity and inhibition by a p34cdc2 kinase specific inhibitor, butyrolactone I. However, p34cdc2/cyclin B kinase was distinguished from cdk5/p26 by its binding to p13suc1 protein and by its reactivity to anti-p34cdc2 antibodies. In spite of similar enzymatic properties of cdk5/p26 and p34cdc2/cyclin B kinase, cdk5/p26 did not display M-phase promoting activity when assayed with a cell-free system of Xenopus egg extract.
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PMID:Porcine brain neurofilament-H tail domain kinase: its identification as cdk5/p26 complex and comparison with cdc2/cyclin B kinase. 755 15

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

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.
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PMID:Neuronal cyclin-dependent kinase-5 phosphorylation sites in neurofilament protein (NF-H) are dephosphorylated by protein phosphatase 2A. 776 48

Tau protein kinase II purified from a bovine brain tau protein fraction (Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T., and Imahori, K. (1992) J. Biol. Chem. 267, 10897-10901) was shown to have a similar substrate specificity to cdc2 kinase in that both phosphorylate neurofilament (NF) proteins. Tau protein kinase II recognized the dephosphorylated form of the heavy subunit of NF (NF-H) as a predominant substrate. The substrate was phosphorylated to the same extent with tau protein kinase II as with cdc2 kinase. Upon phosphorylation, the electrophoretic mobility of the NF-H on SDS-polyacrylamide gel electrophoresis changed to the position of the phosphorylated form. A synthetic peptide containing a KSPXK sequence was by far a better substrate for tau protein kinase II than that containing a KSPXX sequence, as was also observed with cdc2 kinase. NF-H lost its microtubule-associating ability upon phosphorylation with tau protein kinase II as well as with cdc2 kinase. Although anti-PSTAIR antibody (PSTAIR is an amino acid sequence commonly found in cdc2 and several cdc2-related kinases) failed to react with tau protein kinase II, tau protein kinase II bound to p13suc1-Sepharose beads (p13suc1 is a yeast protein known to bind to cdc2 kinase).
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PMID:Tau protein kinase II has a similar characteristic to cdc2 kinase for phosphorylating neurofilament proteins. 832 81

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.
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PMID:cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization. 834 7

Neuronal cdk5 can phosphorylate certain lys-ser-pro (KSP) motifs of neurofilaments and tau protein in the nervous system. We have immunoprecipitated the cdk5 from rat brain using a polyclonal antibody raised against the C-terminus of cdk5. The immunoprecipitate has phosphorylated a KSPXK peptide analog of NF-H, as well as histone H1 and a bacterially expressed rat NF-H protein. The kinase activity was inhibited by staurosporine, isopentanyladenine and olomoucine in a dose dependent manner. Kinetic studies indicated Ki values of 39 nM, 38 microM and 8 microM, respectively for staurosporine, isopentanyladenine and olomoucine. The inhibition by staurosporine was non-competitive with respect to phosphoryl acceptor acceptor substrates. Western blot analysis of the immunoprecipitate showed both cdk5 and p67 (Munc-18), a putative regulator molecule of the kinase. Addition of p67 fusion protein enhanced the kinase activity of the immunoprecipitate by 60% above the basal activity. P67 elevated Ki values for both staurosporine and olomoucine. The degree of inhibition at high concentrations of these inhibitors was unaltered by exogenous p67 indicating a lack of competitive interactions with p67. The high affinity of staurosporine for cdk5 suggests that cdk5 may be one of the targets for the neurotropic effect of staurosporine.
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PMID:Inhibition of neuronal cyclin-dependent kinase-5 by staurosporine and purine analogs is independent of activation by Munc-18. 872 73

An immunoprecipitation assay was used to identify protein kinases which are physically associated with neurofilaments (NF) in mouse brain extracts. Using this approach, we show that a cdc2-related kinase is associated with NF. The cdc2-related kinase was found to be distinct from cdk5 and the authentic cdc2 by a number of criteria. Firstly, it has a molecular mass on SDS-PAGE gels of 34 kDa, similar to that of cdc2, but differing from cdk5 (31 kDa). Secondly, it is not recognized by an antibody specific for cdk5. Thirdly, it is recognized by an antibody raised against the C-terminal region of authentic cdc2, but not by an antibody specific for the PSTAIRE motif. Using immunoblotting, we further show that the cdc2-related kinase copurifies with NF isolated from rat tissues. In vitro kinase assays further demonstrated that immunoprecipitated cdc2-related kinase phosphorylates recombinant NF-H protein. Phosphorylation of NF-H by the cdc2-like activity was not affected by 3 microM olomoucine but was inhibited by 10 microM of this kinase inhibitor. Phosphoamino acid analysis of in vitro phosphorylated NF-H indicates that the immunoprecipitated cdc2-related kinase phosphorylates serine residues.
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PMID:A cdc2-like kinase distinct from cdk5 is associated with neurofilaments. 879 43

Cyclin-dependent kinases (cdks), which regulate the cell division cycle, have also been found in postmitotic neurons. Cdk5, isolated from neural tissue, has been shown to phosphorylate neurofilaments (NFs). Instead of cyclins, however, other neuron-specific activators of cdk5 have been identified including a 67-kD protein (p67) which is identical to a syntaxin-binding protein (n-sec-1, Munc 18) that is thought to play a role in synaptic vesicle trafficking and transmitter release. These functions for p67 are not mutually exclusive since regulation of edk5 phosphorylation of cytoskeletal proteins may modulate axonal dynamics during growth, synaptogenesis and vesicle transport. To gain further insight into the role of p67 in neural tissue, we carried out a Western blot and immunohistochemical analysis of the developing rat cerebellum using antibodies to cdk5, p67, syntaxin and phosphorylated and nonphosphorylated neurofilaments. We assumed that spatiotemporal colocalizations of antigens might correlate with proposed functions for p67. The immunoblots showed that all antigens were developmentally regulated, and increased in expression from PN2 to the adult, with p67 and cdk5 showing a close temporal correlation. Immunohistochemically, p67 colocalized with cdk5 and P-NFH in selected fiber tracts, particularly those in the deep cerebellum. For the most part, p67 also showed strong colocalization patterns with syntaxin in regions of synaptogenesis throughout development such as the molecular layer and glomeruli of the inner nuclear layer. Finally, certain fiber tracts, the afferent fibers, climbing and mossy fibers and particularly the basket cell fibers that envelop and innervate Purkinje cell somata and dendrites, displayed colocalization of cdk5 and P-NFH without expressing any p67. Given the limitations of colocalization data in defining functional relationships, the results are consistent with the hypothesis that p67 is a multifunctional protein, its activity during cerebellum development dependent upon the neuronal phenotype, its location and its state of developmental maturation.
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PMID:Expression of p67 (Munc-18), Cdk5, P-NFH and syntaxin during development of the rat cerebellum. 909 32

Neurofilaments (NFs), the neuron-specific intermediate (i.e. approximately 10-nm diameter) filaments are major cytoskeletal components of most neurons. In a mature mammalian neuron, NFs are co-assembled from three subunits, NF-L (low), NF-M (medium), and NF-H (high), with molecular masses of 68, 95, and 115 kDa, respectively. Neurofilament proteins (NF-Ps), particularly, NF-H, are most extensively phosphorylated in large myelinated axons under normal conditions. This phosphorylation occurs on the serine residues of the lysine (Lys)-serine (Ser)-proline (Pro) (KSP) multiple amino acid repeats of the carboxy-terminal tail domain. Phosphorylation of KSP motifs affects physical, biochemical, and immunological properties of NF-H. For example, phosphorylation is thought to play a pivotal role in the maintenance of the neuronal cytoskeletal structure which influences the conduction velocity of the nerve fiber. The key components responsible for phosphorylation are not known. In this study, an identified cyclin-dependent kinase 5 (cdk5), isolated from nervous tissue, has been shown to phosphorylate the human NF-H (hNF-H) and affects its electrophoretic mobility. On the basis of the following observations, it is suggested that neuronal cdk5 (cdk5) phosphorylates KSPXK motifs in the human high molecular weight neurofilament (hNF-H) and affects its electrophoretic mobility. (1) A 14-mer synthetic peptide (KSPEKAKSPVKEEA) derived from hNF-H; (2) a bacterially expressed protein containing 14 KSPXK multiple repeats of hNF-H in C-terminal tail domain; and (3) a dephosphorylated hNF-H in neurofilament preparation are phosphorylated by cdk5. The decrease in molecular mass of hNF-H caused by dephosphorylated was completely recovered upon cdk5 phosphorylation. It is proposed that neuronal cdk5 regulates phosphorylation of the KSPXK motif in hNF-H and other cytoskeletal proteins with similar motifs in the nervous system.
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PMID:Phosphorylation of human high molecular weight neurofilament protein (hNF-H) by neuronal cyclin-dependent kinase 5 (cdk5). 931 98


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