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)

Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2-like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk-activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2-cdks complexes through phosphorylation of Thr161 (or its homologues).
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PMID:The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues. 834 51

Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40(MO15)/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Here we report that Cak1p and p40(MO15) have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40(MO15) preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40(MO15) only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21(CIP1), p27(KIP1), p57(KIP2), p16(INK4a), and p18(INK4c), could block phosphorylation by p40(MO15) but not phosphorylation by Cak1p. Our results demonstrate that although both Cak1p and p40(MO15) activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly. Structural and physiological implications of these findings will be discussed.
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PMID:Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities. 972 11

Eukaryotic cell cycles are controlled by the activities of cyclin-dependent kinases (cdks). The major cdk in budding yeast, Saccharomyces cerevisiae, is Cdc28p. Activation of Cdc28p requires phosphorylation on threonine 169 and binding to a cyclin. Thr-169 is phosphorylated by the cdk-activating kinase (CAK), Cak1p, which was recently identified as the physiological CAK in budding yeast. Here we present our further characterization of yeast Cak1p. We have found that Cak1p is dispersed throughout the cell as shown by immunofluorescence; biochemical subcellular fractionation confirmed that most of the Cak1p is found in the cytoplasm. Cak1p is a monomeric enzyme in crude yeast lysates. Mutagenesis of potential sites of activating phosphorylation had little effect on the activity of Cak1p in vitro or in vivo. Furthermore, Cak1p contains no posttranslational modifications detectable by two-dimensional isoelectric focusing. We found that Cak1p is a stable protein during exponential growth but that its expression decreases considerably when cells enter stationary phase. In contrast, Cak1p levels oscillate dramatically during meiosis, reflecting regulation at both the transcriptional and post-translational level. The localization and regulation of Cak1p are in contrast to those of the known vertebrate CAK, p40(MO15).
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PMID:Localization and regulation of the cdk-activating kinase (Cak1p) from budding yeast. 981 50

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.
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PMID:Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts. 989 Oct 57

Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2(Thr-160)) or to the mutant CDK2 (CDK2(Ser-160)). In the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes. In contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2(Ser-160). The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Cak1p. In fact, the k(cat) values for phosphorylation of CDK2(Ser-160) were significantly higher than for phosphorylation of CDK2(Thr-160), indicating that CDK2(Ser-160) is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2, either in HeLa cell extract or by purified PP2Cbeta. Combined with the more efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.
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PMID:The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine. 1093 29

Phosphorylation of human p34(cdc2) at Thr 161 seems to be necessary for its catalytic activity. CAK (cdk activating kinase) containing p40(MO15) from Xenopus egg extracts phosphorylates and activates p34(cdc2) in a cyclin dependent manner at Thr 161. We describe the cDNA sequence coding for human MO15, which predicts a serine/threonine kinase of 346 aa. Despite the high homology of 91% between the human and Xenopus proteins we observed a rather different mRNA distribution in adult tissues: In contrast to ubiquitously expressed human MO15-transcripts MO15-mRNA expression in Xenopus is restricted to oocytes indicating a different cellular role in these two phylogenetically distant species. By virtue of the homology to members of the family of cell cycle kinase genes we examined MO15 mRNA expression for its correlation to the proliferative activity of cells. Stimulation of lymphocytes showed MO15 mRNA expression to be independent of mitotic activity.
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PMID:Human and Xenopus mo15 messenger-RNA are highly conserved but show different patterns of expression in adult tissues. 2160 29