Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II plays critical roles in the initiation, elongation and processing of primary transcripts. These activities are at least partially regulated by the phosphorylation of the CTD by three cyclin-dependent protein kinases (CDKs), namely CDK7, CDK8 and CDK9. In this study, we systematically compared the phosphorylation of different recombinant CTD substrates by recombinant CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1 kinases. We showed that CDK7, CDK8 and CDK9 produce different patterns of phosphorylation of the CTD. CDK7/CycH/MAT1 generates mostly hyperphosphorylated full-length and truncated CTD peptides, while CDK8/CycC and CDK9/CycT1 generate predominantly hypophosphorylated peptides. Total activity towards different parts of the CTD also differs between the three kinases; however, these differences did not correlate with their ability to hyperphosphorylate the substrates. The last 10 repeats of the CTD can act as a suppressor of the activity of the kinases in the context of longer peptides. Our results indicate that the three kinases possess different biochemical properties that could reflect their actions in vivo.
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PMID:Three cyclin-dependent kinases preferentially phosphorylate different parts of the C-terminal domain of the large subunit of RNA polymerase II. 1500 12

The HEXIM1 protein inhibits the kinase activity of P-TEFb (CDK9/cyclin T) to suppress RNA polymerase II transcriptional elongation in a process that specifically requires the 7SK snRNA, which mediates the interaction of HEXIM1 with P-TEFb. In an attempt to define the sequence requirements for HEXIM1 to interact with 7SK and inactivate P-TEFb, we have identified the first 18 amino acids within the previously described nuclear localization signal (NLS) of HEXIM1 as both necessary and sufficient for binding to 7SK in vivo and in vitro. This 7SK-binding motif was essential for HEXIM1's inhibitory action, as the HEXIM1 mutants with this motif replaced with a foreign NLS failed to interact with 7SK and P-TEFb and hence were unable to inactivate P-TEFb. The 7SK-binding motif alone, however, was not sufficient to inhibit P-TEFb. A region C-terminal to this motif was also required for HEXIM1 to associate with P-TEFb and suppress P-TEFb's kinase and transcriptional activities. The 7SK-binding motif in HEXIM1 contains clusters of positively charged residues reminiscent of the arginine-rich RNA-binding motif found in a wide variety of proteins. Part of it is highly homologous to the TAR RNA-binding motif in the human immunodeficiency virus type 1 (HIV-1) Tat protein, which was able to restore the 7SK-binding ability of a HEXIM1 NLS substitution mutant. We propose that a similar RNA-protein recognition mechanism may exist to regulate the formation of both the Tat-TAR-P-TEFb and the HEXIM1-7SK-P-TEFb ternary complexes, which may help convert the inactive HEXIM1/7SK-bound P-TEFb into an active one for Tat-activated and TAR-dependent HIV-1 transcription.
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PMID:A human immunodeficiency virus type 1 Tat-like arginine-rich RNA-binding domain is essential for HEXIM1 to inhibit RNA polymerase II transcription through 7SK snRNA-mediated inactivation of P-TEFb. 1516 77

The HIV transcriptional activator Tat enhances the processivity of RNA polymerase II by recruiting the CyclinT1/CDK9 complex to the TAR RNA element. In addition, Tat synergizes with the histone acetyltransferase p300 and is acetylated by p300 at a single lysine residue (K50) in the TAR RNA binding domain. We have recently reported that this post-translational modification is necessary for the interaction and transcriptional synergy of Tat with the transcriptional coactivator PCAF. We have further studied the relevance of Tat acetylation during HIV transcription and generated antibodies specific for acetylated Tat (AcTat). Microinjection of anti-AcTat antibodies inhibited Tat-mediated transactivation in cells. Similarly, the specific p300 inhibitor Lys-CoA and short inhibitory RNAs specific for p300 suppressed Tat transcriptional activity. Full-length synthetic AcTat bound to TAR RNA and CyclinT1 with high affinity, but formation of the Tat-TAR-CyclinT1 ternary complex was inhibited when K50 was acetylated. Our data collectively show that Tat acetylation by p300 defines a critical step in Tat transactivation that serves to disrupt the Tat/TAR/CyclinT1 complex and helps in recruiting PCAF to the elongating RNA polymerase II.
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PMID:Tat acetylation: a regulatory switch between early and late phases in HIV transcription elongation. 1517 Dec 54

The macrophage is an important cell type in the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection. Macrophages both support viral replication and are capable of attracting and activating lymphocytes, thus rendering CD4+ T lymphocytes highly permissive for infection. The viral Tat protein, whose function is mediated by the cellular cyclin T1 protein complexed with CDK9, is required for efficient transcription of the integrated HIV-1 provirus by RNA polymerase II. Cyclin T1 expression is highly regulated during macrophage differentiation, and this has important implications for HIV-1 replication. In monocytes isolated from healthy blood donors, cyclin T1 protein expression is low and is induced to high levels within the first few days of differentiation by a post-transcriptional mechanism. After 1-2 weeks of macrophage differentiation, however, cyclin T1 expression is shut off. Treatment of macrophages with lipopolysaccharide (LPS) can re-induce cyclin T1, indicating that the activation status of macrophages can regulate cyclin T1 expression. Recent results indicate that HIV-1 infection is able to induce cyclin T1 expression in macrophages. Future studies of cyclin T1 regulation in macrophages may suggest means of manipulating expression of this crucial cellular co-factor for therapeutic benefit in HIV-1 infected individuals.
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PMID:HIV-1 infection and regulation of Tat function in macrophages. 1518 43

The positive transcription elongation factor b (P-TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P-TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P-TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK-dependent HEXIM1 association to purified P-TEFb and subsequent CDK9 inhibition. Yeast three-hybrid tests and gel-shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNA-recognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152-155). Data from yeast two-hybrid and pull-down assay on GST fusion proteins converge to a direct binding of P-TEFb to the HEXIM1 C-terminal domain (aa 181-359). Consistently, point mutations in an evolutionarily conserved motif (aa 202-205) were found to suppress P-TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA-binding domain of HEXIM1 mediates its association with 7SK and that P-TEFb then enters the complex through association with HEXIM1.
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PMID:Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK9/cyclin T) inhibitor. 1520 69

The family of Cyclin-Dependent Kinases (CDKs) can be subdivided into two major functional groups based on their roles in cell cycle and/or transcriptional control. This review is centered on CDK9, which is activated by T-type cyclins and cyclin K generating distinct Positive-Transcription Elongation Factors termed P-TEFb. P-TEFb positively regulates transcriptional elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNA pol II), as well as negative elongation factors, which block elongation by RNA pol II shortly after the initiation of transcription. Work over the past few years has led to a dramatic increase in our understanding of how productive transcriptional elongation occurs. This review will briefly describe the mechanisms regulating the activity of T-type cyclin/CDK9 complexes and discuss how these complexes regulate gene expression. For further information, the reader is directed to excellent existing reviews on transcriptional elongation and HIV transcription.
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PMID:Cellular control of gene expression by T-type cyclin/CDK9 complexes. 1527 98

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) promotes tumor progression through activation of matrix metalloproteinase (MMP) activity. MMP-9 is a gelatinase secreted by both cancer cells and surrounding stromal cells, and it contributes to TNF-alpha-stimulated tumor invasion and metastasis. Cyclin-dependent kinase 9 (CDK9), the catalytic component of positive transcription elongation factor-b, phosphorylates serine 2 residues in the C-terminal domain of RNA polymerase II for productive transcription elongation and is up-regulated upon exposure to various stresses. This study investigated roles of CDK9 in TNF-alpha-stimulated MMP-9 expression in human lung adenocarcinoma cells. CDK9 activity was inhibited using three different strategies, including the CDK9 pharmacological inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a dominant-negative CDK9, and a CDK9-specific small interfering RNA. All three approaches reduced TNF-alpha-mediated accumulation of MMP-9 in the conditioned media as demonstrated by gelatin zymography. In contrast, transforming growth factor-beta1-induced accumulation of MMP-2 was unaffected by DRB. Expression of the MMP-9 gene was examined using reverse transcription real time PCR and using a transient transfection assay to evaluate MMP-9 promoter activity. DRB reduced the TNF-alpha-induced increase in MMP-9 mRNA levels but did not effect transforming growth factor-beta1-induced MMP-2 mRNA expression. Consistently DRB and dominant-negative CDK9 completely abrogated TNF-alpha-stimulated human MMP-9 promoter activity. TNF-alpha did not regulate expression or localization of CDK9 or its regulatory partner Cyclin T. However, TNF-alpha stimulated CDK9 binding to Cyclin T and MMP-9 gene occupancy by both CDK9 and the serine 2-phosphorylated form of RNA polymerase II. Our findings indicate that CDK9 mediates TNF-alpha-induced MMP-9 transcription. Disruption of TNF-alpha signaling using CDK9 inhibitors could serve as a potential therapeutic strategy against tumor invasion and metastasis.
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PMID:Cyclin-dependent kinase 9 is required for tumor necrosis factor-alpha-stimulated matrix metalloproteinase-9 expression in human lung adenocarcinoma cells. 1552 90

HIV-1 Tat binds human CyclinT1 and recruits the CDK9/P-TEFb complex to the viral TAR RNA in a step that links RNA polymerase II (RNAPII) C-terminal domain (CTD) Ser 2 phosphorylation with transcription elongation. Previous studies have suggested a connection between Tat and pre-mRNA splicing factors. Here we show that the splicing-associated c-Ski-interacting protein, SKIP, is required for Tat transactivation in vivo and stimulates HIV-1 transcription elongation, but not initiation, in vitro. SKIP associates with CycT1:CDK9/P-TEFb and Tat:P-TEFb complexes in nuclear extracts and interacts with recombinant Tat:P-TEFb:TAR RNA complexes in vitro, indicating that it may act through nascent RNA to overcome pausing by RNAPII. SKIP also associates with U5snRNP proteins and tri-snRNP110K in nuclear extracts, and facilitates recognition of an alternative Tat-specific splice site in vivo. The effects of SKIP on transcription elongation, binding to P-TEFb, and splicing are mediated through the SNW domain. HIV-1 Tat transactivation is accompanied by the recruitment of P-TEFb, SKIP, and tri-snRNP110K to the integrated HIV-1 promoter in vivo, whereas the U5snRNPs associate only with the transcribed coding region. These findings suggest that SKIP plays independent roles in transcription elongation and pre-mRNA splicing.
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PMID:A human splicing factor, SKIP, associates with P-TEFb and enhances transcription elongation by HIV-1 Tat. 1590 9

The Ms;CDKC;1 kinase is structurally similar to those cyclin-dependent kinases (CDKs) that are not involved directly in cell cycle regulation. The presence of a PITAIRE motif in Ms;CDKC;1 suggests that it interacts with cyclins different from known PSTAIRE/PPTALRE kinase regulatory subunits. Here we demonstrate that a Medicago CYCLINT (CYCT) protein is a specific interactor of Ms;CDKC;1 and the interaction between these two proteins gives rise to an active kinase complex that localizes to the nucleus and phosphorylates the carboxy-terminal YSPTSPS heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase II in vitro. Mutation of Ser to Ala at position 5 within the heptapeptide repeat abolishes substrate phosphorylation by the Ms;CDKC;1 kinase complex. Furthermore, our data show that addition of the Medicago CDKC;1-CYCT;1 heterodimer completely restored the transcriptional activity of a HeLa nuclear extract depleted of endogeneous CDK9 kinase complexes. Together, these results indicate that the Medicago CDKC;1-CYCT;1 complex is a positive regulator of transcription in plants and has a role similar to the CDK9/cyclin T complex of human positive transcription elongation factor P-TEFb.
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PMID:The Medicago CDKC;1-CYCLINT;1 kinase complex phosphorylates the carboxy-terminal domain of RNA polymerase II and promotes transcription. 1594 95

Seliciclib (CYC202, R-roscovitine) is a cyclin-dependent kinase (CDK) inhibitor that competes for the ATP binding site on the kinase. It has greatest activity against CDK2/cyclin E, CDK7/cyclin H, and CDK9/cyclin T. Seliciclib induces apoptosis from all phases of the cell cycle in tumor cell lines, reduces tumor growth in xenografts in nude mice and is currently in phase II clinical trials. This study investigated the mechanism of cell death in multiple myeloma cells treated with seliciclib. In myeloma cells treated in vitro, seliciclib induced rapid dephosphorylation of the carboxyl-terminal domain of the large subunit of RNA polymerase II. Phosphorylation at these sites is crucial for RNA polymerase II-dependent transcription. Inhibition of transcription would be predicted to exert its greatest effect on gene products where both mRNA and protein have short half-lives, resulting in rapid decline of the protein levels. One such gene product is the antiapoptotic factor Mcl-1, crucial for the survival of a range of cell types including multiple myeloma. As hypothesized, following the inhibition of RNA polymerase II phosphorylation, seliciclib caused rapid Mcl-1 down-regulation, which preceded the induction of apoptosis. The importance of Mcl-1 was confirmed by short interfering RNA, demonstrating that reducing Mcl-1 levels alone was sufficient to induce apoptosis. These results suggest that seliciclib causes myeloma cell death by disrupting the balance between cell survival and apoptosis through the inhibition of transcription and down-regulation of Mcl-1. This study provides the scientific rationale for the clinical development of seliciclib for the treatment of multiple myeloma.
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PMID:Seliciclib (CYC202, R-Roscovitine) induces cell death in multiple myeloma cells by inhibition of RNA polymerase II-dependent transcription and down-regulation of Mcl-1. 1595 89


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