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PubMed2006

The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation.

RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division.

Cell Division↗

PubMed1997

Rig2, a RING finger protein that interacts with the Kin28/Ccl1 CTD kinase in yeast.

Kin28/Cell, a cyclin-dependent kinase, is essential for the in vivo phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II in Saccharomyces cerevisiae. In a search for mutations co-lethal with a thermosensitive kin28 mutation, we have identified genes whose products interact functionally with Kin28. In the present work, we have studied a new complementation group of synthetic lethal mutations. The corresponding gene, RIG2, encodes a predicted RING finger protein. Rig2 is likely to be a homolog of MAT1 of higher eukaryotes which forms a ternary complex with MO15(cdk7) and cyclin H. Our genetic data suggest that Rig2 is a component of transcription factor TFIIH. Transcription activity in a rig2-ts mutant is impeded at restrictive temperature. However, none of the rig2-ts mutants obtained was UV sensitive, suggesting that Rig2 is dispensable for nucleotide excision repair.

Amino Acid Sequence↗

PubMed1994

A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase.

Phosphorylation by the CDK-activating kinase (CAK) is a required step in the activation of cyclin-dependent kinases. We have purified CAK from mammalian cells; the enzyme comprises two major polypeptides of 42 and 37 kDa. Protein sequencing indicates that the 42 kDa subunit is the mammalian homolog of MO15, a protein kinase known to be a component of CAK in amphibians and echinoderms. Cloning of a cDNA encoding the 37 kDa subunit identifies it as a novel cyclin (cyclin H). We have reconstituted CAK in vitro with the MO15 catalytic subunit and cyclin H, demonstrating that MO15 is a cyclin-dependent kinase (CDK7). Like other CDKs, MO15/CDK7 contains a conserved threonine required for full activity; mutation of this residue severely reduces CAK activity. The CAK holoenzyme activates complexes of CDK2 and CDC2 with various cyclins and also phosphorylates CDK2, but not CDC2, in the absence of cyclin. Thus, CAK is a CDK-cyclin complex implicated in the control of multiple cell cycle transitions.

Amino Acid Sequence↗

PubMed1996

Protein kinase C inhibits the CAK-CDK2 cyclin-dependent kinase cascade and G1/S cell cycle progression in human diploid fibroblasts.

Serum stimulation of human diploid fibroblast IMR-90 cells leads to phosphorylation of p33CDK2 at Thr160 and activation of CDK2 kinase, a necessary event for G1/S transition. We report that serum stimulation causes a gradual, sustained increase in the activity of CDK-activating kinase (CAK) that phosphorylates CDK2 at Thr160, which starts by 5 h after serum stimulation and reaches the maximal plateau level at around the G1/S boundary. In this cell type addition of phorbol-12, 13-dibutyrate 5 h but not 16 h after serum stimulation completely inhibits CDK2 kinase activation and DNA synthesis. Phorbol ester treatment does not reduce the protein level of p33CDK2, but does inhibit serum-stimulated increases in the CAK activity and CDK2 phosphorylation at Thr160. The suppression of the CAK activity by the phorbol ester is accompanied by decreases in the message levels of both CDK7 and cyclin H, the catalytic and the positive regulatory subunit of CAK, respectively. These results indicate that in IMR-90 cells activation of protein kinase C in the late G1 phase causes cell cycle arrest before the G1/S boundary at least in part through downregulation of CAK and CAK-mediated CDK2 phosphorylation and activation.

Antibodies↗

PubMed2004

Effects of pharmacological cyclin-dependent kinase inhibitors on viral transcription and replication.

Cyclin-dependent kinases (CDKs) are required for replication of adeno-, papilloma- and other viruses that replicate only in dividing cells. Surprisingly, CDKs are also required for replication of HIV-1, HSV-1, and other viruses that can replicate in non-dividing cells. Since two low-molecular weight pharmacological CDK inhibitors (PCIs), flavopiridol (Flavo) and roscovitine (Rosco), appear to be non-toxic in human clinical trials against cancer, these drugs have been proposed as potential antiviral drugs. Rosco preferentially inhibits CDKs involved in cell cycle regulation (CDK1, 2, and 7) or neuronal functions (CDK5), whereas Flavo preferentially inhibits CDKs involved in cell cycle (CDK1, 2, 4, 7) or transcription (CDK7, and 9). As potential antivirals, PCIs display several advantages: (i) they are active against many different viruses, including drug-resistant strains of HIV-1 and HSV-1; (ii) PCI-resistant mutants of HIV-1 or HSV-1 have not been identified; and (iii) the antiviral effects of PCIs and conventional antivirals appear to be additive (as expected from drugs that target independent pathways). Moreover, PCIs target both the etiological agents (i.e., the virus) and the pathogenic mechanisms (i.e., unrestricted cell division) of the many diseases that include both a CDK-requiring virus and unrestricted cell division (e.g., Kaposi's sarcoma, cervical carcinoma, HIV-associated nephropathy-HIVAN). This is nicely illustrated in a recent study which demonstrated the efficacy of Flavo in a mouse model of HIVAN. Herein, we will review the involvement of CDKs in viral replication and the antiviral properties of the most extensively characterized PCIs, with special emphasis on the mechanisms of inhibition of viral transcription.

Animals↗

PubMed2006

3-(Indol-2-yl)indazoles as Chek1 kinase inhibitors: Optimization of potency and selectivity via substitution at C6.

The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC(50)=0.30nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.

Cell Cycle↗

PubMed2004

Selective regulation of vitamin D receptor-responsive genes by TFIIH.

Mutations in the XPD subunit of the transcription/repair factor TFIIH cause the Xeroderma pigmentosum disorder. We show that in some XP-D deficient cells, transactivation by the vitamin D receptor (VDR) is selectively inhibited for a subset of responsive genes, such as CYP24, and that the XPD/R683W mutation prevents VDR recruitment on its promoter. Contrary to other nuclear receptors, VDR, which lacks a functional A/B domain, is not phosphorylated and consequently not regulated by the cdk7 kinase of TFIIH. In fact, we demonstrate that the VDR transactivation defect resides in Ets1, another activator that cannot be phosphorylated by TFIIH in XP-D cells. Indeed, the phosphorylated Ets1 seems to promote the binding of VDR to its responsive element and trigger the subsequent recruitment of coactivators and RNA pol II. We propose a model in which TFIIH regulates the activity of nuclear receptors by phosphorylating either their A/B domain or an additional regulatory DNA binding partner.

Cytochrome P-450 Enzyme System↗

PubMed2005

Cyclin-dependent kinase activating kinase/Cdk7 co-localizes with PKC-iota in human glioma cells.

Cyclin-dependent kinase activating kinase (CAK) is a trimeric complex composed of cdk7, cyclin H and MAT1. CAK/cdk7 functions as a master cell cycle regulator by phosphorylating cyclin-dependent kinases for cell cycle progression. We have previously reported that protein kinase C-iota (PKC-iota) associates with CAK/cdk7. In this investigation, immunofluorescence confocal microscopy was used to provide further evidence for the co-localization of PKC-iota with CAK/cdk7. PKC-iota was labeled with Alexa Fluor 488 (green fluorescent dye) and CAK/cdk7 was labeled with Alexa Fluor 555 (red fluorescent dye). The fusion of the red and green fluorescent colors produced a yellow color, which was used to quantify co-localization of PKC-iota and CAK/cdk7. Confocal microscopy revealed the co-localization of PKC-iota with CAK/cdk7 in both the cytoplasm and nucleus of U-373 MG cells.

Cell Line, Tumor↗

PubMed2003

Two forms of RNA polymerase II holoenzyme display different abundance during the cell cycle.

We analyzed the composition and abundance of two forms of RNA polymerase II (pol II) holoenzyme in synchronized HeLa cells. We did not detect significant changes in pol II holoenzyme composition, but we noticed differences in the abundance of the two complexes at different stages of the cell cycle. Summarized data from several independent experiments demonstrate that pol II holoenzyme, which is purified by GST-TFIIS affinity chromatography, is more abundant during G1/S and S phases. Another form of pol II holoenzyme, which is purified by anti-CDK7 antibodies, shows relatively higher amounts in G2/M and early G1 phases.

Blotting, Western↗

PubMed2000

Molecular structure of human TFIIH.

TFIIH is a multiprotein complex required for both transcription and DNA repair. Single particles of human TFIIH were revealed by electron microscopy and image processing at a resolution of 3.8 nm. TFIIH is 16 x 12.5 x 7.5 nm in size and is organized into a ring-like structure from which a large protein domain protrudes out. A subcomplex assembled from five recombinant core subunits also forms a circular architecture that can be superimposed on the ring found in human TFIIH. Immunolabeling experiments localize several subunits: p44, within the ring structure, forms the base of the protruding protein density which includes the cdk7 kinase, cyclin H, and MAT1. Within the ring structure, p44 was flanked on either side by the XPB and XPD helicases. These observations provide us with a quartenary organizational model of TFIIH.

Antibodies, Monoclonal↗

PubMed2002

Molecular cloning and characterisation of p15(CDK-BP), a novel CDK-binding protein.

The suc1/Cks proteins are well-conserved regulatory components of cyclin-dependent kinases 1 and 2 (CDK1/2). These small molecular mass proteins form a stable complex with CDK1/2 and are essential for normal regulation of CDKs during the cell division cycle and for degradation of p27(kip1). Despite the high degree of homology between the nine known CDKs, only CDK1, CDK2 and, to a lesser extent, CDK3 are able to bind to the suc1/Cks proteins. No additional suc1/Cks-related proteins interacting with other CDKs have been reported. We have purified, from starfish oocytes, a 15 kDa protein, p15(CDK-BP), which cross-reacts with anti-Cks antibodies (L. Azzi, L. Meijer, A.C. Ostvold, J. Lew, J.H. Wang, J. Biol. Chem. 269 (1994)). Following microsequencing of internal peptides and generation of corresponding oligonucleotides we cloned two cDNAs encoding two closely related proteins, p15A and p15B. The predicted protein sequences display distant but distinct homology with the Suc1/Cks proteins, including the genuine starfish Cks homologue protein, p9(CksMg). P15 transcripts are essentially expressed in oocytes. Recombinant p15B or native p15(CDK-BP) bind a 34 kDa protein cross-reacting with anti-PSTAIRE antibodies, a feature characteristic of CDK-related proteins. In addition p15B interacts tightly with CDK4, CDK6, CDK8 and the yeast CDC28-related kinase Pho85, but not with CDK1, CDK2 or CDK7. P15 does not appear to alter the catalytic activity of the bound kinases.

Amino Acid Sequence↗

PubMed2002

Modulation of MDM2/p53 and cyclin-activating kinase during the megakaryocyte differentiation of human erythroleukemia cells.

OBJECTIVE: This study was undertaken to address the involvement of CDK activating kinase (CAK), p53, and MDM2 proteins in the mitotic arrest associated with the acquisition of a polyploid DNA content during megakaryocyte differentiation of human erythroleukemia (HEL) cells. METHODS: To evaluate this mechanism we investigated HEL cells as a model system in which there is a marked increase in DNA content during megakaryocyte differentiation induced by phorbol-diesters. Specific cell-cycle phases were separated by centrifugal elutriation and SDS PAGE and Western analysis were performed to determine the relative abundance of these proteins. Kinase assays were carried out following immunoprecipitation of cellular lysates with the antibodies to the proteins. RESULTS: Polyploid HEL cells show an increase in the abundance of the CAK complex proteins, CDK7 and cyclin H, and a sixfold increase in CAK-specific activity. Increased CAK activity in polyploid HEL cells follows both the downregulation of p53 protein and its decreased association with CAK complex. Consistent with the reduction of p53, polyploid HEL cells undergo a dramatic increase in MDM2 protein abundance that in turn facilitates increased interaction of this protein with p53. CONCLUSION: These observations demonstrate that deregulated expression of MDM2 and p53 during megakaryocyte differentiation allow a relaxation of the control over genomic stability, allowing further replicative rounds of DNA synthesis.

Cell Differentiation↗

PubMed2000

Effect of 9-cis-retinoic acid on oral squamous cell carcinoma cell lines.

Retinoic acid (RA) has been shown to be effective in suppressing premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck. However, the precise mechanisms of these effects are still uncertain. In the present study, we examined the effect of 9-cis-RA on the growth of six oral cancer cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, Ho-1-N-1 and Ho-1-u-1). In addition, the relationship among growth and differentiation of tumor cells, RA responsiveness and the expression of nuclear retinoic acid receptors were also investigated. Among the six cell lines examined, five (HSC-2, HSC-3, HSC-4, Ca9-22 and Ho-1-u-1) displayed growth inhibition after treatment with 1x10(-6) M 9-cis-RA, while Ho-1-N-1 cells were resistant to 9-cis-RA. The expression level of RARbeta in 9-cis-RA resistant Ho-1-N-1 cells was very low in comparison with the sensitive cell lines. On the other hand, all of the six the cell lines expressed RARalpha, RARgamma, and RXRalpha at various levels. 9-cis-RA induced accumulation of cell population in G1 phase in HSC-3 cells on the 6th day of the treatment, followed by a marked reduction in the levels of hyperphosphorylated pRB, whereas p53 level was not altered. Interestingly, 9-cis-RA induced transiently the expression of p21(Waf1/Cip1), p27(Kip1), p300, CBP, BAX, Bak and bcl-2 proteins, respectively. This effect was associated with reduction of cyclin D1, cdk4 and CDK-activating kinase (cyclin H and cdk7) protein in HSC-3 cells. These results suggest that the growth inhibitory effect of 9-cis-RA on oral squamous cell carcinoma may depend on the expression levels of RARs, especially RARbeta proteins and RXRalpha proteins, and that 9-cis-RA may provide a powerful therapeutic agent for head and neck cancers.

Alitretinoin↗

PubMed2000

Transcriptional activity of positive transcription elongation factor b kinase in vivo requires the C-terminal domain of RNA polymerase II.

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.

Binding Sites↗

PubMed2003

Nuclear receptors: integration of multiple signalling pathways through phosphorylation.

Nuclear receptors (NRs) orchestrate the transcription of specific gene networks in response to binding of their cognate ligand. They also act as mediators in a variety of signalling pathways through integrating diverse phosphorylation events. NR phosphorylation concerns all three major domains, the N-terminal activation function (AF-1), the ligand-binding and the DNA binding domains. Often, phosphorylation of NRs by kinases that are associated with general transcription factors (e.g. cdk7 within TFIIH), or activated in response to a variety of signals (MAPKs, Akt, PKA, PKC), facilitates the recruitment of coactivators or of components of the transcription machinery and, therefore, cooperates with the ligand to enhance transcription activation. But phosphorylation can also contribute to the termination of the ligand response through inducing DNA dissociation or NR degradation or through decreasing ligand affinity. These different modes of regulation reveal an unexpected complexity of the dynamics of NR-mediated transcription. In addition, deregulation of NR phosphorylation may impact their action in certain diseases or cancers.

Animals↗

PubMed2002

A CDK-activating kinase network is required in cell cycle control and transcription in fission yeast.

Cyclin-dependent kinases (CDKs) involved in cell cycle control require activation by phosphorylation, but CDK-activating kinase (CAK) has diverged between metazoans and budding yeast. Fission yeast has two CAKs: the essential Mcs6 complex, homologous to the metazoan CDK7 complex implicated in cell cycle control and transcription; and Csk1, a nonessential ortholog of budding yeast Cak1. Both can activate the major CDK, Cdc2, but Csk1 can also activate Mcs6, so it was unclear whether the pathway is a linear cascade or a network. Here, we show that a mutation, mcs6-13, which selectively abrogates CDK activation, blocks both G1/S and G2/M transitions, but only when csk1(+) is absent. In contrast, gradual depletion or rapid inactivation of Mcs6 in csk1(+) cells causes cell separation defects or growth arrest, respectively, accompanied by decreased phosphorylation of RNA polymerase II (RNAP II), but not of Cdc2. Finally, neither cell cycle arrest nor CAK failure is recapitulated by a second mutation in mcs6-13 that prevents Mcs6 activation by Csk1, indicating that Csk1 activates Cdc2 directly in vivo. Thus, Mcs6 acts in concert with Csk1 to activate Cdc2 and independently to support transcription and facilitate cell separation. Csk1 likewise has multiple physiologic targets, including Mcs6 and Cdc2.

CDC2 Protein Kinase↗

PubMed1999

Cdc2 activation in fission yeast depends on Mcs6 and Csk1, two partially redundant Cdk-activating kinases (CAKs).

Cyclin-dependent kinases (Cdks) are fully active only when phosphorylated by a Cdk-activating kinase (CAK) [1]. Metazoan CAK is itself a Cdk, Cdk7, whereas the CAK of Saccharomyces cerevisiae is a distinct enzyme unrelated to Cdks [1]. The Mcs6-Mcs2 complex of Schizosaccharomyces pombe is a putative CAK related to the metazoan enzyme [2] [3]. Although the loss of Mcs6 is lethal, it results in a phenotype that is inconsistent with a failure to activate Cdc2, the major Cdk in S. pombe [3]. We therefore tested the ability of Csk1, a putative regulator of Mcs6 [4], to activate Cdk-cyclin complexes in vitro. Csk1 activated both the monomeric and the Mcs2-bound forms of Mcs6. Surprisingly, Csk1 also activated Cdc2 in complexes with either Cdc13 or Cig2 cyclins. When a double mutant carrying a csk1 deletion and a temperature-sensitive mcs6 allele was incubated at the restrictive temperature, Cdc2 was not activated and the cells underwent a cell division arrest prior to mitosis. Cdc2-cyclin complexes isolated from the arrested cells could be activated in vitro by recombinant CAK, whereas complexes from wild-type cells or either of the single mutants were refractory to activation. Thus, fission yeast contains two partially redundant CAKs: the Mcs6-Mcs2 complex and Csk1. Inactivation of both CAKs is necessary and sufficient to prevent Cdc2 activation and cause a cell-cycle arrest. Mcs6, which is essential, may therefore have required functions other than Cdk activation.

Adenosine Triphosphate↗

PubMed2002

Functional coupling of capping and transcription of mRNA.

In humans, 5' m(7)G cap addition is accomplished cotranscriptionally by the sequential action of the capping enzyme (Hce1) and the cap methyltransferase (Hcm1). We found that guanylylation and methylation occur efficiently during transcription with t(1/2)'s of less than 15 and 70 s, respectively. A two to four order of magnitude increase was found in the rate of guanylylation of RNA in transcription complexes compared to free RNA. This stimulation required only the RNA polymerase II elongation complex and Hce1. Capping activity was weakly associated with elongation but not preinitiation complexes. The CTD was not required for functional coupling but stimulated the rate of capping 4-fold. Inhibition of Cdk7 but not Cdk9 similarly slowed the rate of capping.

Antibodies↗