Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Saccharomyces cerevisiae gene KIN28 is a member of the cyclin-dependent kinase (CDK) family. The Kin28 protein shares extensive sequence identity with the vertebrate CDK-activating kinase MO15 (Cdk7), which phosphorylates CDKs in vitro on a critical threonine residue. Kin28 and MO15 have recently been found to copurify with the transcription factor IIH (TFIIH) holoenzyme of yeast and human cells, respectively. Although TFIIH is capable of phosphorylating the C-terminal domain (CTD) of RNA polymerase II, it has been unclear whether Kin28 is the physiologically relevant CTD kinase or what role CTD phosphorylation plays in transcription. In this study, we used a thermosensitive allele of KIN28 and a hemagglutinin epitope-tagged Kin28 protein to investigate Kin28 function in transcription and in the cell cycle. We show that Kin28 acts as a positive regulator of mRNA transcription in vivo and possesses CTD kinase activity in vitro. However, Kin28 neither regulates the phosphorylation state of the yeast cell cycle CDK, Cdc28, nor possesses CDK-activating kinase activity in vitro. We conclude that Kin28 is a strong candidate for the physiological CTD kinase of S. cerevisiae and that Kin28 function is required for mRNA transcription.
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PMID:KIN28 encodes a C-terminal domain kinase that controls mRNA transcription in Saccharomyces cerevisiae but lacks cyclin-dependent kinase-activating kinase (CAK) activity. 776 Jul 96

Tat protein mediates transactivation of human immunodeficiency virus type 1 (HIV-1), which results in more-efficient transcript elongation. Since phosphorylation of C-terminal domain (CTD) of RNA polymerase II correlates with its enhanced processivity, we studied the properties of a Tat-associated CTD kinase derived from mitogenically stimulated human primary T lymphocytes (TTK). TTK binds to full-length Tat and specifically phosphorylates CTD and CDK2. This dual kinase activity is characteristic of CDK-activating kinase (CAK). The CTD kinase activity is induced upon mitogenic stimulation of primary T lymphocytes. Fractionation of T-cell lysate demonstrates that Tat-associated CTD kinase activity elutes in two peaks. About 60% of Tat-associated CTD kinase copurifies with CDK2 kinase activity and contains the CAK components CDK7 and cyclin H. The rest of Tat-associated kinase is free of CDK2 kinase activity and the CAK components and thus may represent a novel CTD kinase. The kinase activities of TTK are blocked by the adenosine analog 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) as well as by the kinase inhibitor H8 at concentrations known to block transcript elongation. Importantly, the Tat-associated kinase markedly induced CAK. We suggest that the mechanism of Tat-mediated processive transcription of the HIV-1 promoter includes a Tat-associated CAK activator.
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PMID:A human primary T-lymphocyte-derived human immunodeficiency virus type 1 Tat-associated kinase phosphorylates the C-terminal domain of RNA polymerase II and induces CAK activity. 931 22

The human immunodeficiency virus encodes the transcriptional transactivator Tat, which binds to the transactivation response (TAR) RNA stem-loop in the viral long terminal repeat (LTR) and increases rates of elongation rather than initiation of transcription by RNA polymerase II (Pol II). In this study, we demonstrate that Tat binds directly to the cyclin-dependent kinase 7 (CDK7), which leads to productive interactions between Tat and the CDK-activating kinase (CAK) complex and between Tat and TFIIH. Tat activates the phosphorylation of the carboxy-terminal domain (CTD) of Pol II by CAK in vitro. The ability of CAK to phosphorylate the CTD can be inhibited specifically by a CDK7 pseudosubstrate peptide that also inhibits transcriptional activation by Tat in vitro and in vivo. We conclude that the phosphorylation of the CTD by CAK is essential for Tat transactivation. Our data identify a cellular protein that interacts with the activation domain of Tat, demonstrate that this interaction is critical for the function of Tat, and provide a mechanism by which Tat increases the processivity of Pol II.
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PMID:The HIV transactivator TAT binds to the CDK-activating kinase and activates the phosphorylation of the carboxy-terminal domain of RNA polymerase II. 933 27

The activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop catalyzed by CDK-activating kinases (CAKs). Thus far no functional CAK homologue has been reported in plants. We screened an Arabidopsis cDNA expression library for complementation of a budding yeast CAK mutant. A cDNA, cak1At, was isolated that suppressed the CAK mutation in budding yeast, and it also complemented a fission yeast CAK mutant. cak1At encodes a protein related to animal CAKs. The CAK similarity was restricted to the conserved kinase domains, leading to classification of Cak1At as a distinct CDK in the phylogenetic tree. Immunoprecipitates with the anti-Cak1At antibody phosphorylated human CDK2 at the threonine residue (T160) within the T-loop and activated its activity to phosphorylate histone H1. Whereas CAKs in animals and fission yeast are involved in regulation of the cell cycle and basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II, Cak1At did not phosphorylate the CTD. An Arabidopsis CTD-kinase isolated separately from Cak1At was shown to interact with the yeast protein p13(suc1), but it had no CDK2-kinase activity. Therefore, the CTD of RNA polymerase II is probably phosphorylated by a Cdc2-related kinase distinct from Cak1At. cak1At is a single-copy gene in Arabidopsis and is highly expressed in proliferating cells of suspension cultures.
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PMID:A distinct cyclin-dependent kinase-activating kinase of Arabidopsis thaliana. 956 Feb 21

P-TEFb is required for the transition from abortive elongation into productive elongation and is capable of phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II. We cloned a cDNA encoding the large subunit of Drosophila P-TEFb and found the predicted protein contained a cyclin motif. We now name the large subunit cyclin T and the previously cloned small subunit (Zhu, Y. R., Peery, T., Peng, J. M., Ramanathan, Y., Marshall, N., Marshall, T., Amendt, B., Mathews, M. B., and Price, D. H. (1997) Genes Dev. 11, 2622-2632) cyclin-dependent kinase 9 (CDK9). Recombinant P-TEFb produced in baculovirus-transfected Sf9 cells exhibited 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-sensitive kinase activity similar to native P-TEFb. Kc cell nuclear extract depleted of P-TEFb failed to generate long DRB-sensitive transcripts, but this activity was restored upon addition of either native or recombinant P-TEFb. Like other CDKs, CDK9 is essentially inactive in the absence of its cyclin partner. P-TEFb containing a CDK9 mutation that knocked out the kinase activity did not function in transcription. Deletion of the carboxyl-terminal domain of cyclin T in P-TEFb reduced both the kinase and transcription activity to about 10%. The CDK-activating kinase in TFIIH was unable to activate the CTD kinase activity of P-TEFb.
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PMID:Identification of a cyclin subunit required for the function of Drosophila P-TEFb. 959 31

The activation of cyclin-dependent protein kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK). The R2 protein of rice is very similar to CAKs of animals and fission yeast at the amino acid level but phosphorylation by R2 has not yet been demonstrated. When R2 was overexpressed in a CAK-deficient mutant of budding yeast, it suppressed the temperature sensitivity of the mutation. Immunoprecipitates of rice proteins with the anti-R2 antibody phosphorylated human CDK2, one of the rice CDKs (Cdc2Os1), and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II of Arabidopsis. Mutational analysis indicated that R2 phosphorylated the threonine residue within the T-loop of CDK2 and Cdc2Os1. R2 was found mainly in two protein complexes which had molecular masses of 190 kDa and 70 kDa, respectively, whilst the CDK- and CTD-kinase activities associated with R2 were identified in a complex of 105 kDa. These results indicate that R2 is closely related to CAKs of animals and fission yeast in terms of its phosphorylation activity and, moreover, that this CAK of rice is distinct from a CAK of the dicotyledonous plant Arabidopsis.
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PMID:A rice homolog of Cdk7/MO15 phosphorylates both cyclin-dependent protein kinases and the carboxy-terminal domain of RNA polymerase II. 1003 78

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

The CDK9-cyclin T kinase complex, positive transcription elongation factor b (P-TEFb), stimulates the process of elongation of RNA polymerase (Pol) II during transcription of human immunodeficiency virus. P-TEFb associates with the human immunodeficiency virus Tat protein and with the transactivation response element to form a specific complex, thereby mediating efficient elongation. Here, we show that P-TEFb preferentially phosphorylates hSPT5 as compared with the carboxyl-terminal domain of RNA Pol II in vitro. Phosphorylation of hSPT5 by P-TEFb occurred on threonine and serine residues in its carboxyl-terminal repeat domains. In addition, we provide several lines of evidence that P-TEFb is a CDK-activating kinase (CAK)-independent kinase. For example, CDK9 was not phosphorylated by CAK, whereas CDK2-cyclin A kinase activity was dramatically enhanced by CAK. Therefore, it is likely that P-TEFb participates in regulation of elongation by RNA Pol II by phosphorylation of its substrates, hSPT5 and the CTD of RNA Pol II, in a CAK-independent manner.
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PMID:Positive transcription elongation factor B phosphorylates hSPT5 and RNA polymerase II carboxyl-terminal domain independently of cyclin-dependent kinase-activating kinase. 1114 67

Cyclin-dependent kinase (CDK)7-cyclin H, the CDK-activating kinase (CAK) and TFIIH-associated kinase in metazoans can be activated in vitro through T-loop phosphorylation or binding to the RING finger protein MAT1. Although the two mechanisms can operate independently, we show that in a physiological setting, MAT1 binding and T-loop phosphorylation cooperate to stabilize the CAK complex of Drosophila. CDK7 forms a stable complex with cyclin H and MAT1 in vivo only when phosphorylated on either one of two residues (Ser164 or Thr170) in its T-loop. Mutation of both phosphorylation sites causes temperature-dependent dissociation of CDK7 complexes and lethality. Furthermore, phosphorylation of Thr170 greatly stimulates the activity of the CDK7- cyclin H-MAT1 complex towards the C-terminal domain of RNA polymerase II without significantly affecting activity towards CDK2. Remarkably, the substrate-specific increase in activity caused by T-loop phosphorylation is due entirely to accelerated enzyme turnover. Thus phosphorylation on Thr170 could provide a mechanism to augment CTD phosphorylation by TFIIH-associated CDK7, and thereby regulate transcription.
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PMID:T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity. 1144 16

Cyclin-dependent kinases (CDKs) are the central components of eukaryotic cell cycle regulation. Phosphorylation of CDKs at a conserved threonine residue is required for their full activity and is mediated by a CDK-activating kinase (CAK). The CAK R2 from rice belongs to those CAKs that phosphorylate not only CDKs but also the C-terminal domain (CTD) of RNA polymerase II. We showed that R2 is a nuclear protein with increased expression and increased CTD kinase activity in S-phase. Increasing R2 abundance through a transgenic approach accelerated S-phase progression and overall growth rate in suspension cells. In planta, the CTD kinase activity of R2 was induced by a growth-promoting signal. R2 regulation, therefore, may constitute a plant-specific adaptive mechanism that is used to adjust the rate of cell proliferation in response to a changing environment.
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PMID:The rice cyclin-dependent kinase-activating kinase R2 regulates S-phase progression. 1182 8


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