Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the polypeptide, from an apparent molecular mass of 116 kDa to 125 kDa. Treatment with either bacterial or potato phosphatase reverses the mobility shift and abolishes enzyme activity. Incubation of maturing X. laevis eggs with radioactive inorganic phosphate and subsequent immunoprecipitation demonstrate that the PARP protein is phosphorylated in vivo. We show that maturation-promoting factor (Cyclin B/cdc2) cannot itself be responsible for the phosphorylation and activation of PARP in maturing X. laevis eggs. Together, these results demonstrate that the enzyme activity of PARP in X. laevis oocytes and eggs is regulated by post-translational, covalent phosphorylation.
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PMID:Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation. 923 Jan 39

We have found that ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor in vivo in a number of cell lines, including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator, cyclin H, and cdk7, and abolished by expression of the cdk inhibitor, p27(KIP1), or by the expression of a dominant negative catalytically inactive cdk2 mutant. ER is phosphorylated between amino acids 82 and 121 in vitro by the cyclin A/cdk2 complex and incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A in vivo. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity.
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PMID:Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex. 929 75

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been known to bind to the pleckstrin homology domain and the phosphotyrosine-binding domain as well as actin-binding proteins, and to regulate their functions. We have tried to find new PtdIns(4,5)P2-binding proteins and to clarify the physiological effects of PtdIns(4,5)P2 on their function. We report here that histones H1 and H3 are PtdIns(4,5)P2-binding proteins which were identified using antibodies specific to PtdIns(4,5)P2, H1, and H3. This binding was further confirmed by extracting PtdIns(4,5)P2 from purified histone H1 and H3. Furthermore, the binding site of PtdIns(4,5)P2 in histone H1 was found in the carboxyl-terminal 103 amino acids. It was also shown that the amounts of PtdIns(4,5)P2 bound to H1 decrease when histone H1 is phosphorylated by protein kinase C but not by protein kinase A or cdc2 kinase, in vitro. The protein kinase C phosphorylation site is localized close to the PtdIns(4,5)P2-binding site, suggesting that phosphorylation of histone H1 by protein kinase C interferes stereostructurally with PtdIns(4,5)P2 binding. We further noticed that PtdIns(4,5)P2 binding to H1 counteracts the histone H1-mediated repression of basal transcription by RNA polymerase II in a Drosophila transcription system in vitro. Phosphatidylinositol 4-phosphate and phosphatidylinositol 3,4,5-trisphosphate affect this transcription activity more weakly than PtdIns(4,5)P2, but PtdIns and other acidic lipids have no effect on this activity. These data indicate that PtdIns(4,5)P2 bound to nuclear protein histone H1 may contribute to the regulation of transcription in eukaryotic cells.
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PMID:Phosphatidylinositol 4,5-bisphosphate reverses the inhibition of RNA transcription caused by histone H1. 949 95

Recent work has shown that high molecular weight neurofilament (NF) proteins are phosphorylated in their carboxy-terminal tail portion by the enzyme cyclin-dependent kinase 5 (CDK-5). The tail domain of neurofilaments contains 52 tripeptide repeats, viz. Lys-Ser-Pro, which mainly exist as KSPXK and KSPXXX motifs (X = amino acid). CDK-5 specifically phosphorylates the serine residues within the KSPXK sites. We probed the structural basis for this type of substrate selectivity by studying the conformation of synthetic peptides containing either KSPXK or KSPXXX repeats designed from native neurofilament sequences. Synthetic peptides with KSPXK repeats were phosphorylated on serine with a recombinant CDK-5/p25 complex whereas those with KSPXXX repeats were unreactive in this system. Circular dichroism (CD) studies in 50% TFE/H2O revealed a predominantly helical conformation for the KSPXXX-containing peptides, whereas the CD spectra for KSPXK-containing peptides indicated the presence of a high population of extended structures in water and 50% TFE solutions. However, detailed NMR analysis of one such peptide which included two such KSPXK repeats suggested a turn-like conformation encompassing the first KSPXK repeat. Restrained molecular dynamics calculations yielded an unusually stable, folded structure with a double "S"-like bend incorporating the central residues of the peptide. The data suggest that a transient reverse turn or loop-type structure may be a requirement for CDK-5-promoted phosphate transfer to neurofilament-specific peptide segments.
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PMID:Site-specific phosphorylation of Lys-Ser-Pro repeat peptides from neurofilament H by cyclin-dependent kinase 5: structural basis for substrate recognition. 953 91

Development of the rat embryo is arrested at the 2-cell stage in vitro in the presence of inorganic phosphate (Pi). Rat embryos were affected by exposure to 1.19 mM KH2PO4 in modified hamster embryo culture medium-1 at the late 2-cell stage only. When exposure durations were 6 h, embryos whose exposure timings were prior to cleavage had a reduced rate of development to the blastocyst stage (2-8%) when compared with embryos with no exposure to Pi (97%, P < 0.05). When exposure durations were 18 h, all embryos were arrested at the 2- to 4-cell stage. These timings would correspond to the G2 to M phase of the second cell cycle. Maturation-promoting factor (MPF), which is regulated by a phosphorylation cascade, controls cell division, and its kinase activity is necessary in order for the cell to enter the M phase. However, the histone H1 kinase activity levels and the patterns of the state of phosphorylation of cdc2 were the same in blocked and non-blocked embryos. Because MPF was active in blocked embryos, the developmental block in rat 2-cell embryos caused by phosphate was not due to MPF activity or its phosphorylation cascade.
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PMID:Effect of phosphate on the second cleavage division of the rat embryo. 955 46

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

It is now well established that progression through the eukaryotic cell cycle is controlled by oscillations in the activity of cyclin- dependent kinases (CDKs). In many cases, however, the physiological substrate(s) of CDKs are unknown. The Saccharomyces cerevisiae PHO5 gene encodes a secreted acid phosphatase which is induced in response to phosphate starvation. The PHO5 gene is activated by the Pho4p transcription factor, which itself is negatively regulated through phosphorylation by the products of PHO80 and PHO85. Pho80p and Pho85p are homologous to cyclins and CDKs, respectively, and the Pho80p/Pho85p heterodimer satisfies the biochemical definition of a cyclin/CDK. In the present study, several reporter genes were expressed in S. cerevisiae from promoters which are activated by the transcription factor Pho4p, thereby generating yeast strains which exhibit quantifiable phenotypes that reflect the activity of a specific cyclin/CDK. Positive genetic selections for inhibition of cyclin/CDK function were characterized using the E. coli neo and yeast LEU2 genes. Chromosomal disruptions of the yeast PHO80 and PHO85 genes were constructed and conditions for complementation by plasmid-borne genes were defined. Complementation is achieved at very low levels of expression of both Pho80p and Pho85p. High-level expression of Pho80p results in aberrant PHO5 promoter regulation, characterized by failure to derepress in low-phosphate medium. Genes encoding hybrid CDKs in which regions of Pho85p were replaced with the homologous region of human Cdk2 were constructed, and tested for function in S. cerevisiae by complementation of the pho85 chromosomal gene disruption. Hybrid proteins in which more than two-thirds of the molecule were derived from human Cdk2 retained Pho85p function with respect to high-phosphate repression of the PHO5 promoter. The hybrid proteins require the PHO80 gene product for this function. A hybrid human-yeast CDK in which a single amino acid is deleted, within a nonapeptide sequence which is perfectly conserved in Pho85p and human Cdk2, retains full function. These results demonstrate that, within the context of the conserved structure of CDKs, considerable primary sequence variability can be introduced without loss of the cyclin-dependent function of the CDK.
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PMID:Function of hybrid human-yeast cyclin-dependent kinases in Saccharomyces cerevisiae. 982 36

We obtained evidence that Rho-associated kinase (Rho-kinase) phosphorylates desmin, the myogenic intermediate filament protein, with approximately 2 mol phosphate per mole of desmin in vitro. Desmin phosphorylated by Rho-kinase lost the potential to form 10-nm filaments. Thr-16, Thr-75, and Thr-76 on desmin proved to be the major phosphorylation sites for Rho-kinase. All these sites are located within the head domain and are different from the reported phosphorylation sites of protein kinase. A, protein kinase C, and cdc2 kinase. We are entertaining the notion that Rho-kinase may regulate filament structures of desmin by site-specific phosphorylation.
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PMID:Rho-associated kinase phosphorylates desmin, the myogenic intermediate filament protein, at unique amino-terminal sites. 987 13

The nuclear pore complex mediates macromolecular transport between the nucleus and cytoplasm. Many nuclear pore components (nucleoporins) are modified by both phosphate and O-linked N-acetylglucosamine (O-GlcNAc). Among its many functions, protein phosphorylation plays essential roles in cell cycle progression. The role of O-GlcNAc addition is unknown. Here, levels of nucleoporin phosphorylation and glycosylation during cell cycle progression are examined. Whereas nuclear pore glycoproteins are phosphorylated in a cell-cycle-dependent manner, levels of O-GlcNAc remain constant. The major nucleoporin p62 can be phosphorylated in vitro by protein kinase A and glycogen synthase kinase (GSK)-3alpha but not by cyclin B/cdc2 or GSK-3beta. The consensus sites of these kinases resemble sites which can be glycosylated by O-GlcNAc transferase. These data are consistent with a model that O-GlcNAc limits nucleoporin hyperphosphorylation during M-phase and hastens the resumption of regulated nuclear transport at the completion of cell division.
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PMID:Phosphorylation and glycosylation of nucleoporins. 1037 98

Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated in vivo on Thr(35) and Ser(67). Thr(35) is phosphorylated by cAMP-dependent protein kinase (A kinase), and Thr(35)-phosphorylated I-1 inhibits PP1. Until now the kinase that phosphorylates Ser(67) had not been identified and the physiological role of Ser(67) phosphorylation was unknown. In this study we detected a high level of kinase activity in brain extract when a glutathione S-transferase (GST) fusion I-1 mutant containing an Ala substituted for Thr(35) [GST-I-1(T35A)] was used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein kinase (NCLK) in the brain extract could not be separated from each other by a series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated with anti-NCLK antibody from kinase-active column fractions. Purified NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of I-1). HPLC phosphopeptide mapping, amino acid sequencing, and site-directed mutagenesis determined that NCLK phosphorylates Ser(67) of I-1. NCLK-phosphorylated I-1 and I-1(T35A) inhibited PP1 with IC(50) values approximately 9.5 and 13. 8 nM, respectively. When compared, A kinase-phosphorylated I-1 was only approximately 1.2 times more inhibitory than NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential in vivo I-1 kinase and that Thr(35) and Ser(67) phosphorylation independently activate I-1.
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PMID:Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor. 1081 8


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