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Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cdc2+ gene of Schizosaccharomyces pombe is homologous to the CDC28 gene of Saccharomyces cerevisiae. Both genes share limited homology with vertebrate protein kinases and have protein kinase activity. cdc2+ has been subjected to mutagenesis in vitro. A null allele of the gene, constructed by insertion of the S. cerevisiae LEU2 gene into a site within the gene, has a phenotype similar to that of many temperature-sensitive alleles of cdc2. Mutations within the predicted ATP-binding site and in a region which may be a site of phosphorylation result in loss of cdc2+ activity. A single substitution of Gly-146 to Asp-146 has been identified in cdc2-1w, a dominant activated allele of the gene. The four introns within the cdc2+ gene have been deleted. The resulting gene not only functions in fission yeast but also rescues cdc28(Ts) strains of S. cerevisiae, a property which is not shared by the genomic cdc2+ gene.
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PMID:Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe. 379 91

Progression through the cell cycle is catalyzed by cyclin-dependent kinases (CDKs) and is negatively controlled by CDK inhibitors (CDIs). We have isolated a new member of the p21CIP1/p27KIP1 CDI family and named it p57KIP2 to denote its apparent molecular mass and higher similarity to p27KIP1. Three distinct p57 cDNAs were cloned that differ at the start of their open reading frames and correspond to messages generated by the use of distinct splice acceptor sites. p57 is distinguished from p21 and p27 by its unique domain structure. Four distinct domains follow the heterogeneous amino-terminal region and include, in order, a p21/p27-related CDK inhibitory domain, a proline-rich (28% proline) domain, an acidic (36% glutamic or aspartic acid) domain, and a carboxy-terminal nuclear targeting domain that contains a putative CDK phosphorylation site and has sequence similarity to p27 but not to p21. Most of the acidic domain consists of a novel, tandemly repeated 4-amino acid motif. p57 is a potent inhibitor of G1- and S-phase CDKs (cyclin E-cdk2, cyclin D2-cdk4, and cyclin A-cdk2) and, to lesser extent, of the mitotic cyclin B-Cdc2. In mammalian cells, p57 localizes to the nucleus, associates with G1 CDK components, and its overexpression causes a complete cell cycle arrest in G1 phase. In contrast to the widespread expression of p21 and p27 in human tissues, p57 is expressed in a tissue-specific manner, as a 1.5-kb species in placenta and at lower levels in various other tissues and a 7-kb mRNA species observed in skeletal muscle and heart. The expression pattern and unique domain structure of p57 suggest that this CDI may play a specialized role in cell cycle control.
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PMID:Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. 772 83

Site-directed mutagenesis was used to remove a critical phosphorylation site, Thr-197, near the active site of the catalytic subunit of cAMP-dependent protein kinase. This residue is present in a number of protein kinases, and its phosphorylation largely influences catalytic activity. We changed Thr-197 to aspartic acid and alanine and measured the effects of these substitutions on the kinetic mechanism and inhibitor affinities. The mutants were expressed as the free catalytic subunit and as soluble fusion proteins of glutathione-S-transferase. The values for KATP and Kpeptide for all three mutants are raised by approximately 2 orders of magnitude relative to the wild-type enzyme. Viscosometric measurements indicate that elevations in Kpeptide are the result of reduced rates for phosphoryl transfer and not reduced substrate affinities. This implies that the loop that contains the phosphothreonine, the activation loop, does not reduce access to the substrate site as proposed for the inactive forms of cdk2 kinase [DeBont, H. L., et al. (1993) Nature 363, 595-602] and MAP kinase [Zhang, F., et al. (1994) Nature 367, 704-711]. The mutants associate slowly with the wild-type regulatory subunit, although the cAMP-free wild-type regulatory subunit inhibits the mutants stoichiometrically. A mutant regulatory subunit that binds cAMP poorly and rapidly inhibits the wild-type catalytic subunit does not inhibit the mutant proteins. These data suggest that the phosphothreonine region serves as a docking surface for the regulatory subunit in the holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase. 787 23

The induction of apoptosis by the Fas/APO-1 receptor is important for T-cell-mediated cytotoxicity and down-regulation of immune responses. Binding of Fas ligand to the Fas/APO-1 receptor transduces an apoptotic signal that requires activation of interleukin 1beta-converting enzyme (ICE) and CPP32beta, members of a family of cysteine proteases that are evolutionarily conserved determinants of cell death. We report here that Fas/APO-1-triggered apoptosis involves ICE-mediated activation of p34cdc2 kinase. Ligation of the Fas receptor resulted in the rapid stimulation of ICE proteolytic activity and activation of p34cdc2 kinase. Specific tetrapeptide inhibitors of ICE (Acetyl-Tyr-Val-Ala-Asp-chloromethylketone) or CPP32beta (Acetyl-Asp-Glu-Val-Asp-aldehyde) prevented the anti-Fas antibody-mediated activation of p34cdc2 and inhibited apoptosis. Inhibition of p34cdc2 activity by transient overexpression of a dominant-negative cdc2 construct or human WEE1 kinase inhibited Fas-mediated apoptosis. These results suggest that activation of p34cdc2 kinase is a critical determinant of cell death mediated by Fas and ICE family proteases.
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PMID:Requirement of p34cdc2 kinase for apoptosis mediated by the Fas/APO-1 receptor and interleukin 1beta-converting enzyme-related proteases. 884 Sep 58

abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of approximately 124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH2-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34(cdc2) and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.
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PMID:The Drosophila gene abnormal spindle encodes a novel microtubule-associated protein that associates with the polar regions of the mitotic spindle. 915 90

We have previously shown that a 20 amino acid peptide derived from the third ankyrin-like repeat of the p16CDKN2/INK4a (p16) tumour suppressor protein (residues 84-103 of the human p16 protein) can bind to cdk4 and cdk6 and inhibit cdk4-cyclin D1 kinase activity in vitro as well as block cell cycle progression through G1. Substitution of two valine residues corresponding to amino acids 95 and 96 (V95A and V96A) of the p16 peptide reduces the binding to cdk4 and cdk6 and increases its IC0.5 for kinase inhibition approximately threefold when linked to the Antennapedia homeodomain carrier sequence. The same mutations increase the IC0.5 approximately fivefold in the p16 protein. Substitution of aspartic acid 92 by alanine instead increases the binding of the peptide to cdk4 and cdk6 and the kinase inhibitory activity. The p16 peptide blocks S-phase entry in non-synchronized human HaCaT cells by approximately 90% at a 24 microM concentration. The V95A and V96A double substitution minimizes the cell cycle inhibitory capacity of the peptide whereas the D92A substitution increases its capacity to block cell cycle progression. A deletion series of the p16 derived peptide shows that a 10 residue peptide still retains cdk4-cyclin D1 kinase and cell cycle inhibitory activity. The p16 peptide inhibited S-phase entry in five cell lines tested, varying between 47-75%, but had only a limited (11%) inhibitory effect in the pRb negative Saos-2 cells at a concentration of 24 microM. Like the full length p16 protein, the p16 peptide does not inhibit cyclin E dependent cdk2 kinase activity in vitro. These data suggest that acute inhibition of CDK-cyclin D activity by a peptide derived from the INK4 family will stop cells in late G1 in a pRb dependent fashion.
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PMID:Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule. 948 4

Members of the 14-3-3 protein family bind the human intermediate filament protein keratin 18 (K18) in vivo, in a cell-cycle- and phosphorylation-dependent manner. We identified K18 Ser33 as an interphase phosphorylation site, which increases its phosphorylation during mitosis in cultured cells and regenerating liver, and as an in vitro cdc2 kinase phosphorylation site. Comparison of wild-type versus K18 Ser33-->Ala/Asp transfected cells showed that K18 Ser33 phosphorylation is essential for the association of K18 with 14-3-3 proteins, and plays a role in keratin organization and distribution. Mutation of another K18 major phosphorylation site (Ser52) or K18 glycosylation sites had no effect on the binding of K18 to 14-3-3 proteins. The K18 phospho-Ser33 motif is different from several 14-3-3-binding phosphomotifs already described. Antibodies that are specific to K18 phospho-Ser33 or phospho-Ser52 show that although Ser52 and Ser33 phosphorylated K18 molecules manifest partial colocalization, these phosphorylation events reside predominantly on distinct K18 molecules. Our results demonstrate a unique K18 phosphorylation site that is necessary but not sufficient for K18 binding to 14-3-3 proteins. This binding is likely to involve one or more mitotic events coupled to K18 Ser33 phosphorylation, and plays a role in keratin subcellular distribution. Physiological Ser52 or Ser33 phosphorylation on distinct K18 molecules suggests functional compartmentalization of these modifications.
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PMID:Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins. 952 13

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.
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PMID:Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation. 961 25

Several approaches have been used to study the interactions of the subunits of protein kinase CK2. The inactive mutant of CK2alpha that has Asp 156 mutated to Ala (CK2alphaA156) is able to bind the CK2beta subunit and to compete effectively in this binding with wild-type subunits alpha and alpha'. The interaction between CK2alphaA156 and CK2beta was also demonstrated by transfection of epitope-tagged cDNA constructs into COS-7 cells. Immunoprecipitation of epitope-tagged CK2alphaA156 coprecipitated the beta subunit and vice-versa. The assay of the CK2 activity of the extracts obtained from cells transiently transfected with these different subunits yielded some surprising results: The CK2 specific phosphorylating activity of these cells transfected with the inactive CK2alphaA156 was considerably higher than the control cells transfected with vectors alone. Assays of the immunoprecipitated CK2alphaA156 expressed in these cells, however, demonstrated that the mutant was indeed inactive. It can be concluded that transfection of the inactive CK2alphaA156 affects the endogenous activity of CK2. Transfection experiments with CK2alpha and beta subunits and CK2alphaA156 were also used to confirm the interaction of CK2 with the general CDK inhibitor p21WAF1/CIP1 co-transfected into these cells. Finally a search in the SwissProt databank for proteins with properties similar to those derived from the amino acid composition of CK2beta indicated that CK2beta is related to protein phosphatase 2A and to other phosphatases as well as to a subunit of some ion-transport ATPases.
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PMID:Interactions of protein kinase CK2 subunits. 1009 95

In Schizosaccharomyces pombe, wee1 encodes a tyrosine kinase that inhibits entry into mitosis by phophorylating Cdc2, the universal cyclin-dependent kinase (Cdk) that regulates the G2/M transition in all eukaryotic cells. A search for suppressors of the G2 arrest caused by overexpression of weel led to the isolation of a new allele of swo1 (named swo1-w1), the gene coding for chaperone Hsp90, which is required to stabilise Weel. The swo1-w1 allele carries a glycine to aspartic acid substitution at amino acid 155 that results in a partial loss of Hsp90 function. Cells bearing the swo1-w1 mutation in combination with the point mutation cdc2-33 or cdc2-M26 showed severe mitotic defects. Genetic interactions were not observed in combination with point mutations in other cdc genes, suggesting that Cdc2 specifically interacts with Hsp90. This synthetic lethal swo1-w1 cdc2-33 (or cdc2-M26) strain had normal levels of Cdc2 protein and histone H1 phosphorylation activity, indicating that Hsp90 is required to enable Cdc2 to interact with its mitotic substrates or regulators, rather than for its proper folding or stabilisation. In a wild-type background, swo1-w1 mutant cells were sensitive to temperature as well as to other stress agents, such as KCI, ethanol and formamide. Under these stressful growth conditions, the swo1-w1 cells displayed anaphase B arrest and aberrant septation patterns, indicating that a subset of proteins involved in mitosis and cytokinesis is highly dependent on chaperone Hsp90 for function.
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PMID:Genetic interactions between Hsp90 and the Cdc2 mitotic machinery in the fission yeast Schizosaccharomyces pombe. 1010 58


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