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)

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 the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) is an important step in transcription and the positive transcription elongation factor b (P-TEFb) has been proposed to facilitate elongation at many genes. The P-TEFb contains a catalytic subunit (Cdk9) that, in association with a cyclin subunit (cyclinT1), has the ability to phosphorylate the CTD substrate in vitro. Here, we demonstrate that cyclinT1/Cdk9-mediated transcription requires CTD-containing RNAPII, suggesting that the CTD is the major target of the cyclinT1/Cdk9 complex in vivo. Unlike Cdk7 and Cdk8, two other cyclin-dependent kinases that are capable of phosphorylating the CTD in vitro, we found that only the Cdk9 activates gene expression in a catalysis-dependent manner. Finally, unlike cyclinT1 and T2, we found that the targeted recruitment to promoter DNA of cyclinK (a recently described alternative partner of Cdk9) does not stimulate transcription in vivo. Collectively, our data strongly indicate that the P-TEFb kinase subunits cyclinT/Cdk9 are specifically involved in transcription and the CTD domain of RNAPII is the major functional target of this complex in vivo.
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PMID:Transcriptional activity of positive transcription elongation factor b kinase in vivo requires the C-terminal domain of RNA polymerase II. 1097 44

In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available. To improve research concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo.
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PMID:A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells. 1136 49

The double-stranded RNA (dsRNA)-activated protein kinase PKR (protein kinase dsRNA-dependent) plays an important role in the regulation of protein synthesis by phosphorylating the alpha-subunit of eukaryotic initiation factor 2. Through this activity, PKR is thought to mediate the antiviral and antiproliferative actions of interferon. Here, we show that the human T cell leukemia Jurkat cells express an alternatively spliced form of PKR with a deletion of exon 7 (PKRDeltaE7), resulting in a truncated protein that retains the two dsRNA-binding motifs. PKRDeltaE7 exhibits a dominant negative function by inhibiting both PKR autophosphorylation and eukaryotic initiation factor 2 alpha-subunit phosphorylation in vitro and in vivo. Reverse transcriptase-polymerase chain reaction assays showed that PKRDeltaE7 is expressed in a broad range of human tissues at variable levels. Interestingly, expression of PKRDeltaE7 is higher in Jurkat cells than in normal peripheral blood mononuclear cells, raising the possibility of a role in cell proliferation and/or transformation. Thus, expression of alternatively spliced forms of PKR may represent a novel mechanism of PKR autoregulation with important implications in the control of cell proliferation.
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PMID:Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR. 1127 90

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO(4). This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.
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PMID:Three RNA polymerase II carboxyl-terminal domain kinases display distinct substrate preferences. 1127 2

TAK/P-TEFb is an elongation factor for RNA polymerase II-directed transcription that is thought to function by phosphorylating the C-terminal domain of the largest subunit of RNA polymerase II. TAK/P-TEFb is composed of Cdk9 and cyclin T and serves as the cellular cofactor for the human immunodeficiency virus transactivator Tat protein. In this study, we examined the subcellular distribution of Cdk9 and cyclin T1 using high resolution immunofluorescence microscopy and found that Cdk9 and cyclin T1 localized throughout the non-nucleolar nucleoplasm, with increased signal present at numerous foci. Both Cdk9 and cyclin T1 showed only limited colocalization with different phosphorylated forms of RNA polymerase II. However, significant colocalization with antibodies to several splicing factors that identify nuclear 'speckles' was observed for Cdk9 and especially for cyclin T1. The pattern of Cdk9 and cyclin T1 distribution was altered in cells treated with transcription inhibitors. Transient expression of cyclin T1 deletion mutants indicated that a region in the central portion of cyclin T1 is important for accumulation at speckles. Furthermore, cyclin T1 proteins that accumulated at speckles were capable of recruiting Cdk9 and the HIV Tat protein to this compartment in overexpression experiments. These results suggest that cyclin T1 functions to recruit its binding partners to nuclear speckles and raises the possibility that nuclear speckles are a site of TAK/P-TEFb function.
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PMID:The Cdk9 and cyclin T subunits of TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions. 1128 25

The translation of mRNA in eukaryotic cells is regulated by amino acids through multiple mechanisms. One such mechanism involves activation of mTOR (Fig. 1). mTOR controls a myriad of downstream effectors, including RNA polymerase I, S6K1, 4E-BP1, and eEF2 kinase. In yeast, and probably in higher eukaryotes, mTOR signals through Tap42p/alpha 4 to regulate protein phosphatases. Through phosphorylation of Tap42p/alpha 4, mTOR abrogates dephosphorylation of the downstream effectors by PP2 A and/or PP6, resulting in their increased phosphorylation. Although at this time still speculative, in vitro results using mTOR immunoprecipitates suggest that mTOR, or an associated kinase, may also be directly involved in phosphorylating some effectors. Enhanced RNA polymerase I activity results in increased transcription of rDNA genes, whereas increased S6K1 activity promotes preferential translation of TOP mRNAs, such as those encoding ribosomal proteins. Together, stimulated RNA polymerase I and S6K1 activities enhance ribosome biogenesis, increasing the translational capacity of the cell. Phosphorylation of 4E-BP1 prohibits its association with eIF4E, allowing eIF4E to bind to eIF4G and form the active eIF4F complex. Increased eIF4F formation preferentially stimulates translation of mRNAs containing long, highly-structured 5' UTRs. Finally, amino acids cause inhibition of the eEF2 kinase, resulting in an increase in the proportion of eEF2 in the active, dephosphorylated form. By inhibiting eEF2 phosphorylation, amino acids may not only stimulate translation elongation, but may also prevent activation of GCN2 by enhancing the rate of removal of deacylated tRNA from the P-site on the ribosome; a potential activator of GCN2. GCN2 may also be regulated directly by the accumulation of deacylated-tRNA caused by treatment with inhibitors of tRNA synthetases or in cells incubated in the absence of essential amino acids. However, because the Km of the tRNA synthetases for amino acids is well above the amino acid concentrations found in plasma of fasted animals, such a mechanism may not be operative in mammals in vivo. Activation of GCN2 results in increased phosphorylation of the alpha-subunit of eIF2, which in turn causes inhibition of eIF2B. Thus, by preventing activation of GCN2, amino acids preserve eIF2B activity, which promotes translation of all mRNAs, i.e., global protein synthesis is enhanced.
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PMID:Regulation of translation initiation by amino acids in eukaryotic cells. 1157 65

We identify and characterize several phosphorylated forms of the hSpt5 subunit of the DRB sensitivity-inducing factor (DSIF). A 175-kDa phosphorylated form of hSpt5 is bound to nuclei of interphase HeLa cells. This form is rapidly dephosphorylated when cultured cells are exposed to various drugs belonging to distinct chemical families. All these compounds are known to inhibit the protein kinase Cdk9, which phosphorylates in vitro hSpt5 and Rpb1, the largest subunit of RNA polymerase II. The efficiency to promote the dephosphorylation of both proteins matches their capacity to inhibit purified Cdk9 kinase, suggesting that Cdk9 is the major kinase phosphorylating hSpt5 and Rpb1 in vivo. We show that Cdk9 phosphorylates both the CTR1 and the CTR2 domains of recombinant hSpt5. These domains contain numerous serine-proline and threonine-proline residues similar to those found in the carboxyl-terminal domain (CTD) of Rpb1. The structural homology between hSpt5 CTRs and the Rpb1 CTD is further highlighted by the presence on both proteins of a phosphoepitope recognized by the monoclonal antibody CC-3. Of particular interest, the peptidyl-prolyl isomerase Pin1 interacts with Cdk9-phosphorylated hSpt5. Cdk9 dependent phosphorylation of Rpb1 and hSpt5 followed by Pin1 interaction might thus contribute to the regulation of transcription, pre-mRNA maturation, and the dynamics of these proteins in interphase and mitosis.
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PMID:The peptidyl-prolyl isomerase Pin1 interacts with hSpt5 phosphorylated by Cdk9. 1157 23

The human positive transcription elongation factor P-TEFb, consisting of a CDK9/cyclin T1 heterodimer, functions as both a general and an HIV-1 Tat-specific transcription factor. P-TEFb activates transcription by phosphorylating RNA polymerase (Pol) II, leading to the formation of processive elongation complexes. As a Tat cofactor, P-TEFb stimulates HIV-1 transcription by interacting with Tat and the transactivating responsive (TAR) RNA structure located at the 5' end of the nascent viral transcript. Here we identified 7SK, an abundant and evolutionarily conserved small nuclear RNA (snRNA) of unknown function, as a specific P-TEFb-associated factor. 7SK inhibits general and HIV-1 Tat-specific transcriptional activities of P-TEFb in vivo and in vitro by inhibiting the kinase activity of CDK9 and preventing recruitment of P-TEFb to the HIV-1 promoter. 7SK is efficiently dissociated from P-TEFb by treatment of cells with ultraviolet irradiation and actinomycin D. As these two agents have been shown to significantly enhance HIV-1 transcription and phosphorylation of Pol II (refs 6,7,8), our data provide a mechanistic explanation for their stimulatory effects. The 7SK/P-TEFb interaction may serve as a principal control point for the induction of cellular and HIV-1 viral gene expression during stress-related responses. Our studies demonstrate the involvement of an snRNA in controlling the activity of a Cdk-cyclin kinase.
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PMID:The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcription. 1171 32

CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties. It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells. Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts. Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants. Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes. Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2. Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors. The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2. CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII. CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts. Such evolutionary conservation suggests that this control may be of fundamental importance.
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PMID:CK2 forms a stable complex with TFIIIB and activates RNA polymerase III transcription in human cells. 1199 11


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