Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The metazoan transcription elongation factor P-TEFb (CDK-9/cyclin T) is essential for HIV transcription, and is recruited by some cellular activators. P-TEFb promotes elongation in vitro by overcoming pausing that requires the SPT-4/SPT-5 complex, but considerable evidence indicates that SPT-4/SPT-5 facilitates elongation in vivo. Here we used RNA interference to investigate P-TEFb functions in vivo, in the Caenorhabditis elegans embryo. We found that P-TEFb is broadly essential for expression of early embryonic genes. P-TEFb is required for phosphorylation of Ser 2 of the RNA Polymerase II C-terminal domain (CTD) repeat, but not for most CTD Ser 5 phosphorylation, supporting the model that P-TEFb phosphorylates CTD Ser 2 during elongation. Remarkably, although heat shock genes are cdk-9-dependent, they can be activated when spt-4 and spt-5 expression is inhibited along with cdk-9. This observation suggests that SPT-4/SPT-5 has an inhibitory function in vivo, and that mutually opposing influences of P-TEFb and SPT-4/SPT-5 may combine to facilitate elongation, or insure fidelity of mRNA production. Other genes are not expressed when cdk-9, spt-4, and spt-5 are inhibited simultaneously, suggesting that these genes require P-TEFb in an additional mechanism, and that they and heat shock genes are regulated through different P-TEFb-dependent elongation pathways.
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PMID:CDK-9/cyclin T (P-TEFb) is required in two postinitiation pathways for transcription in the C. elegans embryo. 1218 67

RNA polymerase II (pol II) is subject to an early elongation delay induced by negative factors Spt5/Spt4 and NELF, which is overcome by the positive factor P-TEFb (Cdk9/cyclin T), a protein kinase that phosphorylates the pol II C-terminal domain (CTD) and the transcription elongation factor Spt5. Although the rationale for this arrest and restart is unclear, recent studies suggest a connection to mRNA capping, which is coupled to transcription elongation via physical and functional interactions between the cap-forming enzymes, the CTD-PO(4), and Spt5. Here we identify a novel interaction between fission yeast RNA triphosphatase Pct1, the enzyme that initiates cap formation, and Schizosaccharomyces pombe Cdk9. The C-terminal segment of SpCdk9 comprises a Pct1-binding domain distinct from the N-terminal Cdk domain. We show that the Cdk domain interacts with S. pombe Pch1, a homolog of cyclin T, and that the purified recombinant SpCdk9/Pch1 heterodimer can phosphorylate both the pol II CTD and the C-terminal domain of S. pombe Spt5. We provide genetic evidence that SpCdk9 and Pch1 are functional orthologs of the Saccharomyces cerevisiae CTD kinase Bur1/Bur2, a putative yeast P-TEFb. Mutations of the kinase active site and the regulatory T-loop of SpCdk9 abolish its activity in vivo. Deleting the C-terminal domain of SpCdk9 causes a severe growth defect. We suggest a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest. This elongation checkpoint may avoid wasteful rounds of transcription of uncapped pre-mRNAs.
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PMID:Interactions between fission yeast Cdk9, its cyclin partner Pch1, and mRNA capping enzyme Pct1 suggest an elongation checkpoint for mRNA quality control. 1247 73

Schizosaccharomyces pombe Cdk9/Pch1 protein kinase is a functional ortholog of the essential Saccharomyces cerevisiae Bur1/Bur2 kinase and a putative ortholog of metazoan P-TEFb (Cdk9/cyclin T). SpCdk9/Pch1 phosphorylates of the carboxyl-terminal domain (CTD) of the S. pombe transcription elongation factor Spt5, which consists of 18 tandem repeats of a nonapeptide of consensus sequence 1TPAWNSGSK9. We document the divalent cation dependence and specificity of SpCdk9/Pch1, its NTP dependence and specificity, the dependence of Spt5-CTD phosphorylation on the number of tandem nonamer repeats, and the specificity for phosphorylation of the Spt5-CTD on threonine at position 1 within the nonamer element. SpCdk9/Pch1 also phosphorylates the CTD heptaptide repeat array of the largest subunit of S. pombe RNA polymerase II (consensus sequence YSPTSPS) and does so exclusively on serine. SpCdk9/Pch1 catalyzes autophosphorylation of the kinase and cyclin subunits of the kinase complex. The distribution of phosphorylation sites on SpCdk9 (86% Ser(P), 11% Thr(P), 3% Tyr(P)) is distinct from that on Pch1 (2% Ser(P), 98% Thr(P)). We conducted a structure-guided mutational analysis of SpCdk9, whereby a total of 29 new mutations of 12 conserved residues were tested for in vivo function by complementation of a yeast bur1Delta mutant. We identified many lethal and conditional mutations of side chains implicated in binding ATP and the divalent cation cofactor, phosphoacceptor substrate recognition, and T-loop dynamics. We surmise that the lethality of the of T212A mutation in the T-loop reflects an essential phosphorylation event, insofar as the conservative T212S change rescued wild-type growth; the phosphomimetic T212E change rescued growth at 30 degrees C; and the effects of mutating the T-loop threonine were phenocopied by mutations in the three conserved arginines predicted to chelate the phosphate on the T-loop threonine.
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PMID:Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 protein kinase: Spt5 phosphorylation, autophosphorylation, and mutational analysis. 1290 90

Cyclin-dependent kinases (CDKs) and their related pathways represent some of the most attractive targets in the development of anticancer therapeutics. Among a variety of CDK inhibitors under development, flavopiridol, UCN-01, CYC202, and BMS-387032 are undergoing clinical evaluation based on evidence of preclinical antitumor activity. Flavopiridol exerts multiple effects in tumor cells, including inhibition of multiple CDKs, transcriptional inhibition secondary to disruption of P-TEFb (CDK9/cyclin T), induction of apoptosis, and antiangiogenesis. UCN-01 was initially developed as a protein kinase C (PKC) inhibitor, but its major antitumor effects appear to be related to CDK inhibition or "inappropriate" activation of cdc2/CDK1 abrogating the G2 and S checkpoints, inhibition of PDK1/Akt, and induction of apoptosis through a PKC-independent mechanism. Significantly, combining these CDK inhibitors with either conventional cytotoxic drugs or novel agents targeting signal transduction pathways can markedly enhance antitumor activity, particularly induction of apoptosis, in various preclinical models. Such findings may serve as a basis for the introduction of novel combination regimens into clinical trials.
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PMID:Small molecule inhibitors targeting cyclin-dependent kinases as anticancer agents. 1475 Oct 90

Human immunodeficiency virus type 1 (HIV-1) can infect quiescent cells; however, viral production is restricted to actively proliferating cells. Recent evidence has indicated that HIV-1 viral proteins, Vpr and Tat, perturb the cell cycle to optimize HIV-1 replication. Vpr arrests the cell cycle at G2 by inactivating the cyclin B/cdk1 complex. Tat regulates the cell cycle by altering factors involved in proliferation and differentiation (i.e. the cdk inhibitor p21/waf1) and associating with cyclin/cdk complexes (i.e. cyclin E/cdk2, cyclin H/cdk7, and cyclin T/cdk9). These studies indicate the importance of host cellular factors, such as cyclin/cdk complexes, in regulating HIV-1 replication and therefore represent novel targets for antiviral therapeutics. Recently, the efficacy of pharmalogical cdk inhibitors (PCIs) in abrogating viral replication has been under development. To date there are 25-30 PCIs that have been synthesized against known cdks, several of which have been shown to inhibit HIV-1 and other AIDS-associated viruses in vitro and in vivo. Targeting these critical cyclin/cdk complexes needed for viral propagation may solve the problems inherent in current HAART therapy, including the emergence of drug-resistant viruses. Thus, PCIs have the potential to become novel therapeutic antiviral drugs that can inhibit HIV-1 transcription and opens the possibility of new avenues of treatment.
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PMID:Pharmacological cyclin-dependent kinase inhibitors as HIV-1 antiviral therapeutics. 1504 99

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of AIDS. Following entry into the host cell, the viral RNA is reverse transcribed into DNA and subsequently integrated into the host genome as a chromatin template. Chromatin structure may be responsible for silencing retroviral gene expression. Transcriptional activation occurs after ATP-dependent chromatin remodeling complexes alter chromatin structure and positioning of nucleosomes. Histone acetyltransferases (HATs), histone deacetylases (HDACs), kinases, and methyltransferases (HMTs), covalently modify nucleosomes by adding or removing chemical moieties in the N-terminal tails of histones. Recent advances have indicated that HIV-1 encoded proteins interact with chromatin remodeling complexes and histone modifying enzymes, implying that chromatin remodeling plays an important role in the HIV-1 life cycle. Nucleosomes are positioned on the HIV-1 LTR and are barriers to transcription. Following cellular activation, these nucleosomes are modified and repositioned allowing for activation of viral gene expression. Tat recruits various HATs to the HIV-1 promoter region and can also be acetylated by some of these enzymes. Unmodified Tat is involved in binding to the CBP/p300 and cdk9/cyclin T complexes and facilitates transcription initiation. Acetylated Tat dissociates from the TAR RNA structure and recruits bromodomain-containing chromatin modifying complexes such as p/CAF and SWI/SNF to facilitate transcription elongation. This review summarizes our current knowledge and understanding of chromatin remodeling complexes and their regulation of HIV-1 replication, and highlights the important contributions HIV-1 research has made to further our understanding of the transcription process.
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PMID:Chromatin remodeling and modification during HIV-1 Tat-activated transcription. 1504 58

We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His(6)-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni(2+)-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004-14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs.
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PMID:A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. 1532 39

Promoter clearance and transcriptional processivity in eukaryotic cells are fundamentally regulated by the phosphorylation of the carboxy-terminal domain of RNA polymerase II (RNAPII). One of the kinases that essentially performs this function is P-TEFb (positive transcription elongation factor b), which is composed of cyclin-dependent kinase 9 (CDK9) associated with members of the cyclin T family. Here we show that cellular GCN5 and P/CAF, members of the GCN5-related N-acetyltransferase family of histone acetyltransferases, regulate CDK9 function by specifically acetylating the catalytic core of the enzyme and, in particular, a lysine that is essential for ATP coordination and the phosphotransfer reaction. Acetylation markedly reduces both the kinase function and transcriptional activity of P-TEFb. In contrast to unmodified CDK9, the acetylated fraction of the enzyme is specifically found in the insoluble nuclear matrix compartment. Acetylated CDK9 associates with the transcriptionally silent human immunodeficiency virus type 1 provirus; upon transcriptional activation, it is replaced by the unmodified form, which is involved in the elongating phase of transcription marked by Ser2-phosphorylated RNAPII. Given the conservation of the CDK9 acetylated residues in the catalytic task of virtually all CDK proteins, we anticipate that this mechanism of regulation might play a broader role in controlling the function of other members of this kinase family.
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PMID:Acetylation of conserved lysines in the catalytic core of cyclin-dependent kinase 9 inhibits kinase activity and regulates transcription. 1825 Jan 57

The positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26 degrees of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis.
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PMID:The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation. 1856 85

ICP22 is a multifunctional herpes simplex virus 1 (HSV-1) regulatory protein that regulates the accumulation of a subset of late (gamma(2)) proteins exemplified by U(L)38, U(L)41, and U(S)11. ICP22 binds the cyclin-dependent kinase 9 (cdk9) but not cdk7, and this complex in conjunction with viral protein kinases phosphorylates the carboxyl terminus of RNA polymerase II (Pol II) in vitro. The primary function of cdk9 and its partners, the cyclin T variants, is in the elongation of RNA transcripts, although functions related to the initiation and processing of transcripts have also been reported. We report two series of experiments designed to probe the role of cdk9 in infected cells. In the first, infected cells were treated with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), a specific inhibitor of cdk9. In cells treated with DRB, the major effect was in the accumulation of viral RNAs and proteins regulated by ICP22. The accumulation of alpha, beta, or gamma proteins not regulated by ICP22 was not affected by the drug. The results obtained with DRB were duplicated in cells transfected with small interfering RNA (siRNA) targeting cdk9 mRNAs. Interestingly, DRB and siRNA reduced the levels of ICP22 but not those of other alpha gene products. In addition, cdk9 and ICP22 appeared to colocalize with RNA Pol II in wild-type-virus-infected cells but not in DeltaU(L)13-infected cells. We conclude that cdk9 plays a critical role in the optimization of expression of genes regulated by ICP22 and that one function of cdk9 in HSV-1-infected cells may be to bring ICP22 into the RNA Pol II transcriptional complex.
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PMID:Role of cdk9 in the optimization of expression of the genes regulated by ICP22 of herpes simplex virus 1. 1875 2


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