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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 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

The activity of the N-terminal activation function AF-1 of RAR alpha1 is abrogated upon mutation of a phosphorylatable serine residue (Ser-77). Recombinant RAR alpha was phosphorylated by a variety of proline-directed protein kinases in vitro. However, only the coexpression of cdk7 stimulated Ser-77 phosphorylation in vivo and enhanced transactivation by RAR alpha, but not by a S77A RAR mutant. Both free CAK (cdk7, cyclin H, MAT1) and the CAK-containing general transcription factor TFIIH phosphorylated Ser-77 in vitro. Furthermore RAR alpha bound free CAK and purified TFIIH in vitro, and RAR alpha-TFIIH complexes could be isolated from HeLa nuclear extracts. These findings represent the first example of activation of a transactivator through binding to and phosphorylation by a general transcription factor.
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PMID:Stimulation of RAR alpha activation function AF-1 through binding to the general transcription factor TFIIH and phosphorylation by CDK7. 923 Mar 6

TFIIH is a general transcription factor for RNA polymerase II that in addition is involved in DNA excision repair. TFIIH is composed of eight or nine subunits and we show that at least four of them, namely cdk7, cyclin H, MAT1, and p62 are localized in the coiled body, a distinct subnuclear structure that is transcription dependent and highly enriched in small nuclear ribonucleoproteins. Although coiled bodies do not correspond to sites of transcription, in vivo incorporation of bromo-UTP shows that they are surrounded by transcription foci. Immunofluorescence analysis using antibodies directed against the essential repair factors proliferating cell nuclear antigen and XPG did not reveal labeling of the coiled body in either untreated cells or cells irradiated with UV light, arguing that coiled bodies are probably not involved in DNA repair mechanisms. The localization of cyclin H in the coiled body was predominantly detected during the G1 and S-phases of the cell cycle, whereas in G2 coiled bodies were very small or not detected. Finally, both cyclin H and cdk7 did not colocalize with P80 coilin after disruption of the coiled body, indicating that these proteins are specifically targeted to the small nuclear ribonucleoprotein-containing domain.
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PMID:The cdk7-cyclin H-MAT1 complex associated with TFIIH is localized in coiled bodies. 924 2

cdk7 started its life rather anonymously as a kinase called MO15, identified during a search for cDNA's which encode protein kinases related to cdc2. For several years its function remained obscure, but during the last 18 months MO15 has revealed itself as the catalytic subunit of cdk activating kinase, associating with at least two other subunits including a new cyclin, cyclin H. MO15(cdk7) has therefore been established paradoxically as both a new member and a regulator of the cyclin dependent kinase family. New evidence now suggests that cdk7 is also involved in the processes of transcription initiation and DNA repair, associating with the general transcription factor TFIIH. The engima of cdk7 is likely to remain for a while yet, and perhaps even more surprises are in store.
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PMID:The regulation and functions of cdk7. 955 66

Cell cycle progression is regulated by cyclin-dependent kinases (cdks). The activity of cdks is tightly controlled by several mechanisms, including binding of subunits to cdks (cyclins and inhibitors), and phosphorylation events. This review focuses on the activating phosphorylation of cdks by an enzyme termed cdk-activating kinase (CAK). Two classes of CAKs have been identified: monomeric Cak1p from budding yeast and the p40MO15 (cdk7)/cyclin H/MAT1 complex from vertebrates. Cak1p is the physiological CAK in budding yeast and localizes to the cytoplasm. p40MO15(cdk7)/cyclin H/MAT1 localizes to the nucleus, is a subunit of the general transcription factor IIH and activates cdks as well as phosphorylates several components of the transcriptional machinery. Functions, substrate specificities, regulation, localization, effects on cdk structure and involvement in transcription are compared for Cak1p and p40MO15(cdk7).
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PMID:The cdk-activating kinase (CAK): from yeast to mammals. 1018 87

Cyclin-dependent kinase (CDK)-activating kinases (CAKs) carry out essential activating phosphorylations of CDKs such as Cdc2 and Cdk2. The catalytic subunit of mammalian CAK, MO15/Cdk7, also functions as a subunit of the general transcription factor TFIIH. However, these functions are split in budding yeast, where Kin28p functions as the kinase subunit of TFIIH and Cak1p functions as a CAK. We show that Kin28p, which is itself a CDK, also contains a site of activating phosphorylation on Thr-162. The kinase activity of a T162A mutant of Kin28p is reduced by approximately 75 to 80% compared to that of wild-type Kin28p. Moreover, cells containing kin28(T162A) and a conditional allele of TFB3 (the ortholog of the mammalian MAT1 protein, an assembly factor for MO15 and cyclin H) are severely compromised and display a significant further reduction in Kin28p activity. This finding provides in vivo support for the previous biochemical observation that MO15-cyclin H complexes can be activated either by activating phosphorylation of MO15 or by binding to MAT1. Finally, we show that Kin28p is no longer phosphorylated on Thr-162 following inactivation of Cak1p in vivo, that Cak1p can phosphorylate Kin28p on Thr-162 in vitro, and that this phosphorylation stimulates the CTD kinase activity of Kin28p. Thus, Kin28p joins Cdc28p, the major cell cycle Cdk in budding yeast, as a physiological Cak1p substrate. These findings indicate that although MO15 and Cak1p constitute different forms of CAK, both control the cell cycle and the phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II by TFIIH.
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PMID:Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p. 1037 27

TATA-binding protein (TBP) is a key general transcription factor required for transcription by all three nuclear RNA polymerases. Although it has been intensively analyzed in vitro and in Saccharomyces cerevisiae, in vivo studies of vertebrate TBP have been limited. We applied gene-targeting techniques using chicken DT40 cells to generate heterozygous cells with one copy of the TBP gene disrupted. Such TBP-heterozygous (TBP-Het) cells showed unexpected phenotypic abnormalities, resembling those of cells with delayed mitosis: a significantly lower growth rate, larger size, more G2/-M- than G1-phase cells, and a high proportion of sub-G1, presumably apoptotic, cells. Further evidence for delayed mitosis in TBP-Het cells was provided by the differential effects of several cell cycle-arresting drugs. To determine the cause of these defects, we first examined the status of cdc2 kinase, which regulates the G2/M transition, and unexpectedly observed more hyperphosphorylated, inactive cdc2 in TBP-Het cells. Providing an explanation for this, mRNA and protein levels of cdc25B, the trigger cdc2 phosphatase, were significantly and specifically reduced. These properties were all due to decreased TBP levels, as they could be rescued by expression of exogeneous TBP, including, in most but not all cases, a mutant form lacking the species-specific N-terminal domain. Our results indicate that small changes in TBP concentration can have profound effects on cell growth in vertebrate cells.
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PMID:Heterozygous disruption of the TATA-binding protein gene in DT40 cells causes reduced cdc25B phosphatase expression and delayed mitosis. 1125 92

The histidine triad superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhit-related hydrolases, and a branch of galactose-1-phosphate uridylyltransferase (GalT)-related transferases. Although substrates of Fhit and GalT are known and consequences of mutations in Aprataxin, Fhit, and GalT are known, good substrates had not been reported for any member of the Hint branch, and mutational consequences were unknown for Hint orthologs, which are the most ancient and widespread proteins in the Hint branch and in the histidine triad superfamily. Here we show that rabbit and yeast Hint hydrolyze the natural product adenosine-5'-monophosphoramidate (AMPNH(2)) in an active-site-dependent manner at second order rates exceeding 1,000,000 m(-1) s(-1). Yeast strains constructed with specific loss of the Hnt1 active site fail to grow on galactose at elevated temperatures. Loss of Hnt1 enzyme activity also leads to hypersensitivity to mutations in Ccl1, Tfb3, and Kin28, which constitute the TFIIK kinase subcomplex of general transcription factor TFIIH and to mutations in Cak1, which phosphorylates Kin28. The target of Hnt1 regulation in this pathway was shown to be downstream of Cak1 and not to affect stability of Kin28 monomers. Functional complementation of all Hnt1 phenotypes was provided by rabbit Hint, which is only 22% identical to yeast Hnt1 but has very similar adenosine monophosphoramidase activity.
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PMID:Adenosine monophosphoramidase activity of Hint and Hnt1 supports function of Kin28, Ccl1, and Tfb3. 1180 11

The transcriptional activity of nuclear retinoic acid receptors (RARs), which act as RAR/retinoid X receptor (RXR) heterodimers, depends on two activation functions, AF-1 and AF-2, which are targets for phosphorylations and synergize for the activation of retinoic acid target genes. The N-terminal AF-1 domain of RARalpha is phosphorylated at S77 by the cyclin-dependent kinase (cdk)-activating kinase (CAK) subcomplex (cdk7/cyclin H/MAT1) of the general transcription factor TFIIH. Here, we show that phosphorylation of S77 governing the transcriptional activity of RARalpha depends on cyclin H binding at a RARalpha region that encompasses loop 8-9 and the N-terminal tip of helix 9 of the AF-2 domain. We propose a model in which the structural constraints of this region control the architecture of the RAR/RXR/TFIIH complex and therefore the efficiency of RARalpha phosphorylation by cdk7. To our knowledge, this study provides the first example of a cooperation between the AF-2 and AF-1 domains of RARs through a kinase complex.
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PMID:Cyclin H binding to the RARalpha activation function (AF)-2 domain directs phosphorylation of the AF-1 domain by cyclin-dependent kinase 7. 1627 22

In metazoans, cyclin-dependent kinase 7 (CDK7) has essential roles in both the cell-division cycle and transcription, as a CDK-activating kinase (CAK) and as a component of the general transcription factor TFIIH, respectively. Controversy over its double duty has been resolved, but questions remain. First, how does CDK7 achieve the dual substrate specificity necessary to perform both roles? Second, is there a deeper connection implied by the dichotomy of CDK7 function, for example similar mechanisms controlling cell division and gene expression, and/or actual coordination of the two processes? Enzymological studies have revealed solutions to the unusual substrate recognition problem, and there is evidence that the distinct functions of CDK7 can be regulated independently. Finally, despite divergence in their wiring, the CAK-CDK networks of budding yeast, fission yeast and metazoans all link transcriptional regulation with operation of the cell-cycle machinery. This connection might help to ensure that mRNAs encoding effectors of cell division are expressed at the right time in the cycle.
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PMID:Secrets of a double agent: CDK7 in cell-cycle control and transcription. 1628 May 50


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