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)

Transcription factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). Here we report the identification of the Cdk-activating kinase (Cak) complex (Cdk7 and cyclin H) as a component of TFIIH after extensive purification of TFIIH by chromatography. We find that affinity-purified antibodies directed against cyclin H inhibit TFIIH-dependent transcription and that both cyclin H and Cdk7 antibodies inhibit phosphorylation of the CTD of the largest subunit of the RNAPII in the preinitiation complex. Cak is present in at least two distinct complexes, TFIIH and a smaller complex that is unable to phosphorylate RNAPII in the preinitiation complex. Both Cak complexes, as well as recombinant Cak, phosphorylate a CTD peptide. Finally, TFIIH was shown to phosphorylate both Cdc2 and Cdk2, suggesting that there could be a link between transcription and the cell cycle machinery.
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PMID:Cdk-activating kinase complex is a component of human transcription factor TFIIH. 753 95

We have isolated a cDNA from the cellular slime mold Dictyostelium discoideum encoding a protein that is 52% identical to the Xenopus Mo15 kinase and highly related to the equivalent proteins from human (52% identity), rice (52.7% identity), and yeast (47.6% identity). Mo15 is responsible for the activation of Cdc2 kinase and is itself a member of the large Cdc2-related family of protein kinases. The Dictyostelium protein is more related to the Xenopus Mo15 protein than it is to either the Dictyostelium Cdc2 or Crp proteins. Southern blot analysis of genomic V12-M2 DNA indicated that mo15 is present as a single copy gene that cross hybridizes with cdc2 at low stringency. Northern blot analysis of RNA from different stages of Dictyostelium development showed that mo15 is only expressed during vegetative cell growth.
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PMID:A Dictyostelium discoideum gene, which is highly related to mo15 from Xenopus, is expressed during growth but not during development. 766 15

Characterization of cdk (cyclin dependent kinases) substrates and studies of their regulation require purified enzymatic complexes of cdc2-related catalytic and cyclin regulatory subunits. We produced human Cdc2 kinase in the fission yeast Schizosaccharomyces pombe as a fusion protein with glutathione S-transferase (GST). The GST-human Cdc2p fusion protein was active in vivo since it rescued a temperature-sensitive allele of cdc2. The fusion protein was purified using a one-step chromatography procedure with glutathione-Sepharose and exhibited a catalytic activity in vitro. Yeast cyclin B and suc1 were found in association with GST-Cdc2. A 17-fold stimulation of GST-Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with the S. pombe cellular extract prior to affinity purification. This indicates that cyclin concentration is limiting in this overexpression system. These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for biochemical studies.
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PMID:Characterization of an active GST-human Cdc2 fusion protein kinase expressed in the fission yeast Schizosaccharomyces pombe: a new approach to the study of cell cycle control proteins. 772 98

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.
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PMID:Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity. 778 47

We have studied changes in cyclin A- and B1-dependent kinases during apoptosis induced in human promyelocytic leukemia (HL60) cells treated with the topoisomerase I inhibitor camptothecin. We found that cyclin B1/Cdc2 kinase activity transiently increases within 30 min after camptothecin treatment. This increase is followed by a rapid inactivation of the cyclin B1/Cdc2 kinase that is associated with Cdc2 tyrosine phosphorylation without any change in Cdc2 or cyclin B1 protein levels. The DNA polymerase inhibitor aphidicolin abrogates camptothecin-induced changes in cyclin B1/Cdc2 kinase activity, indicating that DNA replication-induced DNA damage is essential for both Cdc2 alterations and apoptosis activation. Apoptosis and the initial cyclin B1/Cdc2 kinase activation were amplified using synchronized S-phase cells, and cyclin A/cdk2 kinase did not change under these conditions. The same transient activation and subsequent inactivation of cyclin B1/Cdc2 kinase were observed after DNA damage by etoposide or bis-(2-chloroethyl)methylamine hydrochloride. These observations suggest that DNA damage promotes the transient and unscheduled stimulation of cyclin B1/Cdc2 kinase activity in HL60 cells prior to apoptosis.
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PMID:Unscheduled activation of cyclin B1/Cdc2 kinase in human promyelocytic leukemia cell line HL60 cells undergoing apoptosis induced by DNA damage. 781 49

The RNA polymerase II large subunit contains an essential carboxy-terminal domain (CTD) believed to be involved in the response to regulators during transcription initiation. The CTD is phosphorylated on a portion of RNA polymerase II molecules in vivo and it can be phosphorylated by the general transcription factor TFIIH in vitro. A highly purified TFIIH from rat liver has been described; this, like human and yeast TFIIH, contains associated CTD kinase and helicase activities. We report here that two polypeptides of the purified mammalian TFIIH are the MO15/Cdk7 kinase and cyclin H subunits of the Cdk-activating kinase Cak, previously identified as a positive regulator of Cdc2 and Cdk2. TFIIH and Cak preparations are each capable of phosphorylating recombinant CTD and recombinant Cdk2 proteins. The presence of Cak in TFIIH indicates that Cak may have roles in transcriptional regulation and in cell-cycle control.
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PMID:Association of Cdk-activating kinase subunits with transcription factor TFIIH. 788 50

We have identified a novel gene encoding a putative protein kinase from a Drosophila genomic library. The gene, about 2 kbp in length, consists of four exons and codes for a protein of 349 amino acid residues. The deduced sequence shows significant similarity to various kinases, especially to a subgroup of Ser/Thr kinases related to Cdc2 kinase; thus, the gene was termed Dcdrk (Drosophila cdc2-related kinase gene). Among the kinases examined, mammalian galactosyltransferase-associated 58 kDa protein kinase showed the highest homology (about 50% identity in the kinase domain) to Dcdrk kinase. Northern blot analysis revealed that the Dcdrk mRNA is expressed throughout development in nearly constant amounts. Moreover, a whole mount in situ hybridization experiment showed that the Dcdrk mRNA is ubiquitously distributed in almost all embryonic cells and tissues, suggesting a universal function of Dcdrk, possibly in cell cycle regulation.
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PMID:Identification of a novel Drosophila gene encoding a Cdc2-related protein kinase. 818 24

Cyclin-dependent, proline-directed protein kinases normally function to execute critical cell cycle transitions; abnormal expression and/or viral subversion of the positive (cyclins) and negative (Pic1) regulatory subunits may contribute to neoplastic transformation and tumorigenesis. In addition to the binding of regulatory subunits, the enzymatic activities of the cyclin-dependent kinases, Cdc2 and Cdk2, are tightly regulated by site-specific protein phosphorylation events. Recent studies have identified a critical phosphorylation site (Thr-161) located within kinase Subdomain VIII that is necessary for Cdc2 activation, and enzymatic activities capable of carrying out this heterologous phosphorylation event have been detected in both Xenopus oocytes and human somatic cells. In this report, we characterize by molecular cloning a human homologue of the Xenopus Cdk-activating kinase (Cak, encoded by MO15); the novel human gene is designated (HS)CAK1. While only 75% identity is observed at the nucleotide level, the deduced amino acid sequence encoded by (HS)CAK1 is approximately 87% identical to that of the Xenopus MO15 gene in corresponding regions. The catalytic domain of (HS)Cak1, defined by conserved kinase Subdomains I through XI, exhibits considerable homology with (HS)Cdc2, suggesting that this kinase cascade involves closely related enzymes. Immunological studies with anti-Cak antibodies confirm the presence of specific immunoreactivity in highly purified preparations of the human Cdc2-activating kinase. The molecular characterization of (HS)CAK1 should facilitate studies of its physiological regulation, as well as its potential utility as a target for therapeutic intervention in the treatment of proliferative disorders.
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PMID:Molecular cloning of the human CAK1 gene encoding a cyclin-dependent kinase-activating kinase. 820 56

It has been suggested that cell-type determination in Dictyostelium discoideum is dependent on the position of a cell in the cell cycle at the time of starvation. In order to understand the molecular basis of this phenomenon, we initiated studies on the cell cycle and have recently described the isolation of a Dictyostelium gene encoding a homolog of the Cdc2 kinase. We have been unable to isolate additional cdc2 genes from Dictyostelium using polymerase chain reaction technology, but have isolated a gene that is highly related to cdc2. The encoded product is a protein of 33 kDa that shares over 60% identity to the cell-cycle-dependent Cdc2 kinases. However, despite this high level of identity, the gene is not capable of complementing the temperature-sensitive cdc28 mutant of Saccharomyces cerevisiae. Furthermore, the gene product shares some characteristics with the recently described PCTAIRE proteins; it contains a PCTAIRE motif instead of the Cdc2 kinase conserved PSTAIRE sequence, it does not possess the conserved GDSEID sequence that is involved in the activation of the enzyme and it has a Ser in the position equivalent to Thr-161. However, the Dictyostelium protein exhibits a slightly higher level of identity to the Cdc2 kinases than to the PCTAIRE proteins and is smaller than any of the PCTAIRE proteins thus far identified. Since the gene product has characteristics of both Cdc2 kinases and PCTAIRE proteins we have designated the gene product Crp (Cdc2-Related PCTAIRE) kinase. The gene is expressed as two transcripts of 1.5 and 1.8 kb and the expression is developmentally regulated with low levels of mRNA in vegetative cells and significantly higher levels throughout the remainder of the differentiation process. These results suggest the possibility that the gene product is involved in Dictyostelium differentiation rather than growth. This report is the first evidence for a highly-related cdc2 gene in unicellular eukaryote. It also demonstrates for the first time that a unicellular eukaryote expresses a protein containing the PCTAIRE sequence.
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PMID:The isolation from a unicellular organism, Dictyostelium discoideum, of a highly-related cdc2 gene with characteristics of the PCTAIRE subfamily. 821 53

We report the distribution of phosphorylation sites in murine lamins A and C (A-type lamins) in vitro and in vivo followed by reverse-phase high-performance liquid chromatography and microsequencing of peptides spanning the almost complete lamin sequence. We show that two distinct protein kinases, cell-division-cycle-2 kinase (cdc2 kinase) and protein kinase C (PKC), phosphorylate murine A-type lamins at the non-alpha-helical amino- and carboxy-terminal domains in vitro and in vivo. Cdc2 kinase, but not PKC, is capable of inducing depolymerization of the nuclear lamina in permeabilized cells. Accordingly, lamins were proposed to be direct in vivo substrates of cdc2 kinase and PKC with different effects on the lamina dynamics. Analysis of the original A-type lamins revealed phosphorylation of residues Ser5 and Ser392. Residue Ser392 was substoichiometrically phosphorylated in the substrate and by cdc2 kinase in vitro. PKC phosphorylated peptides with its kinase-specific motifs surrounding Ser5, Thr199, Thr416, Thr480 and Ser625. In vivo, a mitosis-specific phosphorylation at the cdc2-kinase-specific phosphoacceptor site Ser392 and of the N-terminal peptide was identified. An interphase-specific phosphorylation at Ser525 matching the PKC consensus sequence and of peptides phosphorylated by unknown kinases was determined. The results lead us to propose that different cyclin-dependent kinase activities act as lamin kinases in mitosis and in interphase. Other kinases may cooperate with cdc2 kinase during reversible disassembly in mitosis and may modulate the supramolecular assembly of lamin filaments.
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PMID:Identification of novel phosphorylation sites in murine A-type lamins. 847 40


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