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)

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

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

Titin is a giant structural protein of striated muscle (M(r) approximately 3000 kDa) and single molecules span sarcomeres from the M- to Z-lines. We have cloned and sequenced the C-terminal region of the titin molecule, which is an integral part of M-lines and forms intimate contacts with the 165 and 190 kDa M-line proteins. In contrast to the regular motif patterns of the A-band portion of titin, the 5.7 kb of titin sequences from the M-line show a complex structure of immunoglobulin-C2 repeats, separated by unique interdomain insertion sequences. As a striking feature, one interdomain insertion comprises four KSP repeats analogous to the multi-phosphorylation repeats of neurofilament subunits H and M. In vitro phosphorylation assays with expressed titin KSP sequences detect high levels of titin KSP phosphorylating kinases in developing but not in differentiated muscle. Since this kinase activity can be depleted from myocyte extracts by antibodies against cdc2 kinase and p13suc1 beads, the titin KSP kinase is structurally related to cdc2 kinase. We suggest that titin C-terminal phosphorylation by SP-specific kinases is regulated during differentiation, and that this may control the assembly of M-line proteins into regular structures during myogenesis.
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PMID:Phosphorylation of KSP motifs in the C-terminal region of titin in differentiating myoblasts. 840 52

We have isolated a human homolog of Xenopus Eg5, a kinesin-related motor protein implicated in the assembly and dynamics of the mitotic spindle. We report that microinjection of antibodies against human Eg5 (HsEg5) blocks centrosome migration and causes HeLa cells to arrest in mitosis with monoastral microtubule arrays. Furthermore, an evolutionarily conserved cdc2 phosphorylation site (Thr-927) in HsEg5 is phosphorylated specifically during mitosis in HeLa cells and by p34cdc2/cyclin B in vitro. Mutation of Thr-927 to nonphosphorylatable residues prevents HsEg5 from binding to centrosomes, indicating that phosphorylation controls the association of this motor with the spindle apparatus. These results indicate that HsEg5 is required for establishing a bipolar spindle and that p34cdc2 protein kinase directly regulates its localization.
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PMID:Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. 854 3

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

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

Cyclin-dependent kinase 5 (CDK5), unlike other CDKs, is active only in neuronal cells where its neuron-specific activator p35 is present. However, it phosphorylates serines/threonines in S/TPXK/R-type motifs like other CDKs. The tail portion of neurofilament-H contains more than 50 KSP repeats, and CDK5 has been shown to phosphorylate S/T specifically only in KS/TPXK motifs, indicating highly specific interactions in substrate recognition. CDKs have been shown to have a high preference for a basic residue (lysine or arginine) as the n+3 residue, n being the location in the primary sequence of a phosphoacceptor serine or threonine. Because of the lack of a crystal structure of a CDK-substrate complex, the structural basis for this specific interaction is unknown. We have used site-directed mutagenesis ("charged to alanine") and molecular modeling techniques to probe the recognition interactions for substrate peptide (PKTPKKAKKL) derived from histone H1 docked in the active site of CDK5. The experimental data and computer simulations suggest that Asp86 and Asp91 are key residues that interact with the lysines at positions n+2 and/or n+3 of the substrates.
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PMID:Identification of substrate binding site of cyclin-dependent kinase 5. 1009 46

In mammalian and squid nervous systems, the phosphorylation of neurofilament proteins (NFs) seems to be topographically regulated. Although NFs and relevant kinases are synthesized in cell bodies, phosphorylation of NFs, particularly in the lys-ser-pro (KSP) repeats in NF-M and NF-H tail domains, seem to be restricted to axons. To explore the factors regulating the cellular compartmentalization of NF phosphorylation, we separated cell bodies (GFL) from axons in the squid stellate ganglion and compared the kinase activity in the respective lysates. Although total kinase activity was similar in each lysate, the profile of endogenous phosphorylated substrates was strikingly different. Neurofilament protein 220 (NF220), high-molecular-weight NF protein (HMW), and tubulin were the principal phosphorylated substrates in axoplasm, while tubulin was the principal GFL phosphorylated substrate, in addition to highly phosphorylated low-molecular-weight proteins. Western blot analysis showed that whereas both lysates contained similar kinases and cytoskeletal proteins, phosphorylated NF220 and HMW were completely absent from the GFL lysate. These differences were highlighted by P13(suc1) affinity chromatography, which revealed in axoplasm an active multimeric phosphorylation complex(es), enriched in cytoskeletal proteins and kinases; the equivalent P13 GFL complex exhibited six to 20 times less endogenous and exogenous phosphorylation activity, respectively, contained fewer cytoskeletal proteins and kinases, and expressed a qualitatively different cdc2-like kinase epitope, 34 kDa rather than 49 kDa. Cell bodies and axons share a similar repertoire of molecular consitutents; however, the data suggest that the cytoskeletal/kinase phosphorylation complexes extracted from each cellular compartment by P13 are fundamentally different.
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PMID:Topographic regulation of cytoskeletal protein phosphorylation by multimeric complexes in the squid giant fiber system. 1039 74

Paspalum notatum is a subtropical forage grass, which reproduces by either sexuality or aposporous gametophytic apomixis. The objective of this work was to identify and isolate mRNA transcripts differentially expressed during the development of the megagametophyte from spikelets of apomictic and sexual P. notatum. Crossing of a sexual mother plant with an apomictic pollen donor generated a progeny family segregating for reproductive mode. Individuals from this F1 family were cytoembryologically classified as sexual or apomictic. Spikelet mRNA compositions from both groups of plants were compared by differential display using an RNA-bulked procedure. Fifty primer combinations were assayed to generate nearly 2,500 total bands in the fingerprints. Three transcripts expressed at higher levels in apomictic plants (apo417, apo398, and apo396) were identified, isolated and cloned. Sequencing showed a high level of homology among the isolated clones. Analysis by RT-PCR Southern blots followed by densitometric studies confirmed that expression reached a level around 30 times higher in apomictic than in sexual individuals and was probably induced at early stages of the megagametophyte development. Genomic DNA from the parental and the F1 progeny plants showed 4-5 bands when hybridised with apo417 in Southern blots. Comparisons to the sequence data banks revealed no identities to genes of known function. However, a putative deduced 3' protein fragment showed homology to the well-characterised KSP multi-phosphorylation domain previously detected in several cdc2-regulated cytoskeletal proteins.
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PMID:Isolation of cDNA clones differentially expressed in flowers of apomictic and sexual Paspalum notatum. 1152 63

As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.
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PMID:Neurofilament tail phosphorylation: identity of the RT-97 phosphoepitope and regulation in neurons by cross-talk among proline-directed kinases. 1871 69


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