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Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The incidence of lung cancer has been increasing over recent decades. Previous studies show that polymorphisms of the genes involved in carcinogen-detoxication, DNA repair and cell cycle control compose of the risk factors for lung cancer. Recent observations reveal that the components of CAK: Cdk7, MAT1 and cyclin H, may play important roles in cell cycle control, transcriptional control, and DNA repairing process, all of which are important in carcinogenesis. To test whether the genetic variants of CAK genes modify the risk of lung cancer, we compared the manifestation of 25 single nucleotide polymorphisms (SNPs) and the haplotypes of Cdk7, MAT1 and cyclin H between 500 patients with lung cancer and 517 healthy controls. Our results indicated that the genotype frequency of MAT1 79023A/G (p = 0.042) and MAT1 85693C/T (p = 0.005) of cases significantly differed from those of the controls. Further analyses revealed that cyclin H 11817C/T, MAT1 12199A/G, MAT1 70650A/G, MAT1 79023A/G and MAT1 85693C/T significantly influenced the susceptibility of lung cancer in a dominant genetic model while cyclin H 12128A/T and MAT1 42172A/G did in a recessive model. Strongest association between cyclin H alleles and lung cancer patients was found in the non-smoke subpopulation. The haplotype 'TAC' (p = 0.007) increased and the haplotype 'TTC' (p = 0.043) decreased the risk of lung cancer. The potential gene-gene and gene-environmental interactions on lung cancer risk was evaluated using MDR software. A significant interaction between the three CAK component genes was identified and the combination of smoking status and genetic factors barely increased the accuracy. Our results suggested that genetic variants in CAK genes, Cdk7, cyclin H, MAT1, might modulate the risk of lung cancer in a gene-gene interaction mode, which consist to the biochemical interaction of corresponding proteins.

Little is known about the mechanisms by which retinoic acid receptor alpha (RAR alpha) mediates the effects of retinoic acid (RA) to coordinate granulocytic proliferation/differentiation (P/D) transition. Cyclin-dependent kinase-activating kinase (CAK) complex, whose activity in phosphorylation of RAR alpha is determined by its targeting subunit ménage à trois 1 (MAT1), regulates G(1) exit, a cell cycle stage when cells commonly commit to proliferation or to differentiation. We previously found that in myeloid leukemia cells, the lack of RA-induced RAR alpha-CAK dissociation and MAT1 degradation suppresses cell differentiation by inhibiting CAK-dependent G(1) exit and sustaining CAK hyperphosphorylation of RAR alpha. This contrasts with our recent findings about the P/D transition in normal primitive hematopoietic cells, where MAT1 degradation proceeds intrinsically together with granulocytic development, in accord with dynamic expression of aldehyde dehydrogenases (ALDHs) 1A1 and 1B1, which catalyze RA synthesis. Blocking ALDH activity inhibits MAT1 degradation and granulocytic differentiation, whereas loss of RAR alpha phosphorylation by CAK induces RA-target gene expression and granulocytic differentiation. These studies suggest that the subversion of RAR alpha-CAK signaling during normal granulopoiesis is crucial to myeloid leukemogenesis and challenges the current paradigm that RA induces cell differentiation solely by transactivating target genes. Disclosure of potential conflicts of interest is found at the end of this article.

Cyclin-dependent kinase 7 (Cdk7) is the catalytic subunit of the metazoan Cdk-activating kinase (CAK). Activation of Cdk7 requires its association with a regulatory subunit, Cyclin H. Although the Cdk7/Cyclin H complex has been implicated in the regulation of RNA polymerase in several species, the precise function of their orthologs in zebrafish has not been fully elucidated. In this study, we isolated from zebrafish blastula embryos two cDNAs encoding the orthologs of human Cyclin H and Cdk7, and examined the role of Cdk7/Cyclin H in zebrafish embryogenesis. Sequence analysis showed that the zebrafish Cyclin H and Cdk7 cDNAs encode proteins with 65% and 86% identity to the respective human orthologs. RT-PCR and whole-mount in situ hybridization analyses of their expression in unfertilized eggs, embryos and organs of adult fish suggested that Cyclin H and Cdk7 messages are maternally loaded. Our data also showed that their transcripts were detected throughout development. Distribution of Cyclin H transcripts was found to be ubiquitous during early stages of development and become restricted to the anterior neural tube, brain, eyes, procreate tissues, liver and heart by 5 days post-fertilization. Expression of a dominant-negative form of Cyclin H delayed the onset of zygotic transcription in the early embryo, resulting in apoptosis at 5 hours post-fertilization and leading to sever defects in tissues normally exhibiting high levels of Cyclin H expression. These results implicate Cyclin H in the regulation of the transcriptional machinery during midblastula transition and suggest that it is an essential gene in early zebrafish larval development.

For the full activation of cyclin-dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T-loop is required. This phosphorylation is catalyzed by CDK-activating kinases (CAKs). In Arabidopsis three D-type CDK genes (CDKD;1-CDKD;3) encode vertebrate-type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP-binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK - CDKF;1 - exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T-loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.

Nuclear retinoic acid receptors (RARs) work as ligand-dependent heterodimeric RAR/retinoid X receptor transcription activators, which are targets for phosphorylations. The N-terminal activation function (AF)-1 domain of RARalpha is phosphorylated by the cyclin-dependent kinase (cdk) 7/cyclin H complex of the general transcription factor TFIIH and the C-terminal AF-2 domain by the cAMP-dependent protein kinase A (PKA). Here, we report the identification of a molecular pathway by which phosphorylation by PKA propagates cAMP signaling from the AF-2 domain to the AF-1 domain. The first step is the phosphorylation of S369, located in loop 9-10 of the AF-2 domain. This signal is transferred to the cyclin H binding domain (at the N terminus of helix 9 and loop 8-9), resulting in enhanced cyclin H interaction and, thereby, greater amounts of RARalpha phosphorylated at S77 located in the AF-1 domain by the cdk7/cyclin H complex. This molecular mechanism relies on the integrity of the ligand-binding domain and the cyclin H binding surface. Finally, it results in higher DNA-binding efficiency, providing an explanation for how cAMP synergizes with retinoic acid for transcription.

The transcriptional activity of nuclear retinoic acid receptors (RARs), which act as RAR/retinoid X receptor (RXR) heterodimers, depends on two activation functions, AF-1 and AF-2, which are targets for phosphorylations and synergize for the activation of retinoic acid target genes. The N-terminal AF-1 domain of RARalpha is phosphorylated at S77 by the cyclin-dependent kinase (cdk)-activating kinase (CAK) subcomplex (cdk7/cyclin H/MAT1) of the general transcription factor TFIIH. Here, we show that phosphorylation of S77 governing the transcriptional activity of RARalpha depends on cyclin H binding at a RARalpha region that encompasses loop 8-9 and the N-terminal tip of helix 9 of the AF-2 domain. We propose a model in which the structural constraints of this region control the architecture of the RAR/RXR/TFIIH complex and therefore the efficiency of RARalpha phosphorylation by cdk7. To our knowledge, this study provides the first example of a cooperation between the AF-2 and AF-1 domains of RARs through a kinase complex.

TFIIH is a general transcription and repair factor implicated in RNA polymerase II transcription, nucleotide excision repair, and transcription-coupled repair. Genetic defects in TFIIH lead to three distinct inheritable diseases: xeroderma pigmentosa, Cockayne syndrome, and trichothiodystrophy, with xeroderma pigmentosa patients being highly susceptible to skin cancer. Earlier data revealed that the cyclin H subunit of TFIIH associates with U1 small nuclear RNA, a core-splicing component. In addition to its role in RNA processing U1 small nuclear RNA also regulates diverse stages of transcription by RNA polymerase II both in vivo and in vitro, including abortive initiation and re-initiation. Here we identify structural components of U1 and cyclin H implicated in the direct interaction and show how they affect function. Because of unique features of cyclin H we have developed a new methodology for mapping RNA interaction with the full-length cyclin H polypeptide based on electrospray ionization tandem mass spectrometry. We also demonstrate the importance of U1 stem-loops 1 and 2 for the interaction with cyclin H. Functional assays implicate the identified interaction with U1 in regulation of the activity of the cyclin H associated kinase CDK7.

Cyclin-dependent kinases (CDKs) play essential roles in coordinate control of cell cycle progression. Activation of CDKs requires interaction with specific cyclin partners and phosphorylation of their T-loops by CDK-activating kinases (CAKs). The Arabidopsis thaliana genome encodes four potential CAKs. CAK2At (CDKD;3) and CAK4At (CDKD;2) are closely related to the vertebrate CAK, CDK7/p40MO15; they interact with cyclin H and phosphorylate CDKs, as well as the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. CAK1At (CDKF;1) shows cyclin H-independent CDK-kinase activity and can activate a heterologous CAK, Mcs6, in fission yeast. In Arabidopsis, CAK1At is a subunit of a protein complex of 130 kD, which phosphorylates the T-loop of CAK2At and CAK4At and activates the CTD-kinase activity of CAK4At in vitro and in root protoplasts. These results suggest that CAK1At is a novel CAK-activating kinase that modulates the activity of CAK2At and CAK4At, thereby controlling CDK activities and basal transcription in Arabidopsis.

Increasing evidence has indicated that perturbation of cyclins is one of the major factors leading to cancer. The aim of this study was not only to investigate various cell cycle-related kinase activities in hepatocellular carcinoma (HCC), but also to analyze the difference of cell cycle-related kinase activity levels between hepatitis C virus (HCV)-induced HCC and HCV-induced cirrhosis. The protein levels of cyclins D1, E, A, and H, and of cyclin dependent kinase 1 (Cdk1), Cdk2, Cdk4, Cdk6, and Cdk7 in HCC and in surrounding nontumorous cirrhosis were determined by Western blot. The enzymatic activities of cyclins D1, E, A, Cdk1, Cdk4, Cdk6, Cdk7, and Wee1 were measured using in vitro kinase assays. Protein levels and kinase activities of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 were significantly elevated in HCC compared with surrounding cirrhotic tissues. The enhanced cyclin D1-related kinase activity in HCC was accompanied by the up-regulation of Cdk4 activity, but not Cdk6 activity. The kinase activities of Cdk6, Cdk7, and Cdk1 did not differ between HCC and surrounding cirrhotic tissues. In addition, the protein levels and kinase activities of cyclin D1, Cdk4, and cyclin E were higher in poorly differentiated HCC and advanced HCC. In conclusion, the increases of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 play an important role in the development of HCC from cirrhosis. Cyclin D1, Cdk4, and cyclin E activation may be closely related to the histopathologic grade and progression of HCC.

Cyclin dependent kinases are regulated by phosphorylation and dephosphorylation of the catalytic cdk subunits, by assembly with specific cyclins and by specific inhibitor molecules. Recently, it turned out that cyclins are also phosphoproteins, which means that they are also potential targets for a regulation by phosphorylation and dephosphorylation. Here, we show that cyclin H was phosphorylated by protein kinase CK2. Like most other CK2 substrates cyclin H was much better phosphorylated by the CK2 holoenzyme than by the alpha-subunit alone. By using point mutants derived from the cyclin H sequence we mapped the CK2 phosphorylation site at threonine 315 at the C-terminal end of cyclin H. Phosphorylation at this position had no influence on the assembly of the cyclin H/cdk7/Mat1 complex. However, phosphorylation at amino acid 315 of cyclin H turned out to be critical for a full cyclin H/cdk7/Mat1 kinase activity when the CTD peptide of RNA polymerase II or cdk2 was used as a substrate.

To understand the role of the various components of TFIIH, a DNA repair/transcription factor, a yeast four-hybrid system was designed. When the ternary Cdk-activating kinase (CAK) complex composed of Cdk7, cyclin H, and MAT1 was used as bait, the xeroderma pigmentosum (XP) D helicase of transcription factor IIH (TFIIH), among other proteins, was identified as an interacting partner. Deletion mutant analyses demonstrated that the coiled-coil and the hydrophobic domains of MAT1 interlink the CAK complex directly with the N-terminal domain of XPD. Using immunoprecipitates from cells coinfected with baculoviruses, we further validated the bridging function of XPD, which anchors CAK to the core TFIIH. In addition we show that upon interaction with MAT1, CAK inhibits the helicase activity of XPD. This inhibition is overcome upon binding to p44, a subunit of the core TFIIH. It is not surprising that under these conditions some XPD mutations affect interactions not only with p44, but also with MAT1, thus preventing either the CAK inhibitory function within CAK.XPD and/or the role of CAK within TFIIH and, consequently, explaining the variety of the XP phenotypes.

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.

Cell division requires that cyclin-dependent kinases (Cdks) be activated by phosphorylation. In mitotic cells, this is accomplished by the Cdk-activating-kinase (CAK), which is a complex of cyclin H and Cdk7. There are currently no data on the role of CAK in meiotic cells. Previously, we have shown that cyclin A1 is meiosis-specific and forms an active kinase with Cdk2. Because cyclin A1 is required for meiosis, and its associated kinase must be phosphorylated (activated), we propose that cyclin H/Cdk7 function to activate cyclin A1/Cdk2 in meiotic cells. Here, we show that cyclin H and Cdk7 are present during meiosis. Using reverse transcription-polymerase chain reaction and in situ hybridization, we show that the mRNAs encoding cyclin H and Cdk7 are abundant in spermatocytes. Immunohistochemistry localized cyclin H and Cdk7 to the nucleus of spermatocytes in stages IV to XII of the spermatogenic cycle, overlapping the same stages that express cyclin A1-associated kinases. Finally, immunoprecipitation and histone H1-kinase assays of cyclin H and Cdk7 from testicular extracts show that these proteins interact to form an active kinase. We conclude that cyclin H/Cdk7 complexes are present and during meiosis, form active complexes in testicular cells and are strong candidates for the activating kinase for cyclin A1-associated kinase.

Knowledge about adhesion checkpoints is important to counteract dissemination of cells from solid tumors. Lack of anchorage in adherent cells is associated with growth arrest and inhibition of cyclin-dependent kinases (cdks) required to drive cell cycle progression. Because cyclin-cdk complex activation requires CDK-activating kinase comprising cdk7 and cyclin H, we now investigated their relationship to decreased proliferation by lack of cell spreading. This report shows that either UV irradiation on an adhesive substrate or culture on a nonadhesive substrate produced K1735 melanoma growth arrest. Inhibition of proliferation by UV primarily induced the cdk inhibitor p21WAF1 without a significant effect on cyclin H and cdk7. In contrast, lack of adhesion to substratum decreased cyclin H but not cdk7 with accumulation of a slower migrating, presumably unphosphorylated cdk4 isoform. These results were paralleled by decreased cdk7-mediated phosphorylation of GST-cdk2 and lower activation of a baculovirus-derived cdc2-cyclin B kinase complex. This is the first report showing that cyclin H-mediated down-regulation of cdk-activating kinase activity is involved in growth arrest induced by lack of anchorage.

Cyclin-dependent kinase 7 (Cdk7) forms a trimeric complex with cyclin H and Mat1 to form the mammalian Cdk-activating kinase, CAK, as well as a part of the basal transcription factor TFIIH, where Cdk7 phosphorylates the C-terminal domain (CTD) of the large subunit of RNA polymerase II. Here, we report a novel interaction between Cdk7 and a histidine triad (HIT) family protein, Hint/PKCI-1. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation and subcellular localization studies, where overexpression of Cdk7 leads to partial relocalization of Hint to the nucleus. The physical association is independent of cyclin H binding or Cdk7 kinase activity and is conserved between the related Sacharomyces cerevisiae CTD kinase Kin28 and the HIT protein Hnt1. Furthermore, combination of a disruption of HNT1 and a KIN28 temperature-sensitive allele in S. cerevisiae led to highly elongated cell morphology and reduced colony formation, indicating a genetic interaction between KIN28 and HNT1. The physical and genetic interactions of Hint and Hnt1 with Cdk7 and Kin28 suggest a role for this class of histidine triad proteins in the regulation of Cdk7 and Kin28 functions.

MAT1 (ménage à trois1) functions as an assembly/targeting factor of CAK (cyclin-dependent kinase-activating kinase). In a search for MAT1-interacting proteins using yeast two-hybrid system, MCM7 (minichromosome maintenance 7), a member of a family of DNA licensing factors, was identified. The physical interaction between MAT1 and MCM7 was confirmed in vivo in yeast cells and verified with in vitro protein binding assays. Further studies showed the RING-finger motif of MAT1 is not required for the interaction with MCM7, while the C-terminal domain of MAT1 is indispensable. Immunoprecipitation of MCM7 in human osteosarcoma MG63 cells demonstrated that MCM7 associates with the CAK complex in vivo.

The mammalian cyclin-dependent kinase 8 (cdk8) gene has been linked with a subset of acute lymphoblastic leukaemias, and its corresponding protein has been functionally implicated in regulation of transcription. Mammalian cdk8 and cyclin C, and their respective yeast homologues, Srb10 and Srb11, are components of the RNA polymerase II holoenzyme complex where they function as a protein kinase that phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 7). The yeast SRB10 and SRB11 genes have been implicated in the negative regulation of transcription. The cdk8/cyclin C protein complex is also found in a number of mammalian Mediator-like protein complexes, which repress activated transcription independently of the CTD in vitro. Here we show that cdk8/cyclin C can regulate transcription by targeting the cdk7/cyclin H subunits of the general transcription initiation factor IIH (TFIIH). cdk8 phosphorylates mammalian cyclin H in the vicinity of its functionally unique amino-terminal and carboxy-terminal alpha-helical domains. This phosphorylation represses both the ability of TFIIH to activate transcription and its CTD kinase activity. In addition, mimicking cdk8 phosphorylation of cyclin H in vivo has a dominant-negative effect on cell growth. Our results link the Mediator complex and the basal transcription machinery by a regulatory pathway involving two cyclin-dependent kinases. This pathway appears to be unique to higher organisms.

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.

Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathione S-transferase-C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat-P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.

The transcription/DNA repair factor TFIIH may be resolved into at least two subcomplexes: the core TFIIH and the cdk-activating kinase (CAK) complex. The CAK complex, which is also found free in the cell, is composed of cdk7, cyclin H, and MAT1. In the present work, we found that the C terminus of MAT1 binds to the cdk7 x cyclin H complex and activates the cdk7 kinase activity. The median portion of MAT1, which contains a coiled-coil motif, allows the binding of CAK to the TFIIH core through interactions with both XPD and XPB helicases. Furthermore, using recombinant TFIIH complexes, it is demonstrated that the N-terminal RING finger domain of MAT1 is crucial for transcription activation and participates to the phosphorylation of the C-terminal domain of the largest subunit of the RNA polymerase II.

The androgen receptor (AR), like other steroid receptors, modulates the activity of the general transcription machinery on the core promoter to exert its function as a regulator. Co-immunoprecipitation of prostate cancer LNCaP cell extract using protein A-Sepharose coupled with anti-AR antibody indicates that the AR interacts with the general transcription factor TFIIH in a physiological condition. Co-transfection of cdk activating kinase (CAK), the kinase moiety of TFIIH, enhanced AR-mediated transcription in a ligand-dependent manner in human prostate cancer PC-3 and LNCaP cells, and in a ligand-independent manner in human prostate cancer DU145 cells. Detailed interaction studies further revealed that the AR NH(2)-terminal domain interacting with CAK was essential for the CAK-induced AR transactivation. Together, our data suggest that the AR may interact with TFIIH for efficient communication with the general transcription factors/RNA polymerase II on the core promoter.

Transforming growth factor beta (TGF-beta)-mediated G(1) arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. We report here that TGF-beta-treated human HepG2 hepatocellular carcinoma cells arrest in G(1), but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-beta-treated cells failed to induce p15(INK4b), down-regulate CDC25A, or increase levels of p21(CIP1), p27(KIP1), and p57(KIP2). However, TGF-beta treatment resulted in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr(160) phosphorylation on Cdk2. Whole-cell lysates from TGF-beta-treated cells showed inhibition of Cdk2 Thr(160) Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, was not altered. Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7. Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regulated in response to TGF-beta treatment. Taken together, these observations demonstrate that TGF-beta signaling mediates a G(1) arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.

Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II is important for basal transcriptional processes in vivo and for cell viability. Several kinases, including certain cyclin-dependent kinases, can phosphorylate this substrate in vitro. It has been proposed that differential CTD phosphorylation by different kinases may regulate distinct transcriptional processes. We have found that two of these kinases, cyclin C/CDK8 and cyclin H/CDK7/p36, can specifically phosphorylate distinct residues in recombinant CTD substrates. This difference in specificity may be largely due to their varying ability to phosphorylate lysine-substituted heptapeptide repeats within the CTD, since they phosphorylate the same residue in CTD consensus heptapeptide repeats. Furthermore, this substrate specificity is reflected in vivo where cyclin C/ CDK8 and cyclin H/CDK7/p36 can differentially phosphorylate an endogenous RNA polymerase II substrate. Several small-molecule kinase inhibitors have different specificities for these related kinases, indicating that these enzymes have diverse active-site conformations. These results suggest that cyclin C/CDK8 and cyclin H/CDK7/p36 are physically distinct enzymes that may have unique roles in transcriptional regulation mediated by their phosphorylation of specific sites on RNA polymerase II.

Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK).

Cyclin-dependent kinases (CDKs), which play a key role in cell cycle control, are activated by the CDK-activating kinase (CAK), which activates cyclin-bound CDKs by phosphorylation at the specific threonine residue. Mammalian CAK contains three components: CDK7, cyclin H, and an assembly factor called MAT1. The CDK7-cyclin H-MAT1 complex is tightly associated with a multiprotein complex TFIIH, which plays a dual role in transcription and DNA repair. Here, we have determined chromosomal localizations of the human genes encoding cyclin H (CCNH) and MAT1 (HGMW-approved symbol MNAT1) to chromosome bands 5q13.3-q14 and 14q23, respectively, by using fluorescence in situ hybridization, somatic cell hybrid analyses, and mapping to the human YAC contigs.

The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery.

We describe a three-hybrid system that involves three polypeptides that allow or prevent the formation of the transcriptional activator. Beside the two-hybrid fusion proteins, the third partner is under the control of the Met25 promoter, which is positively regulated in medium lacking methionine. We document a situation where such a third partner promotes interaction between two proteins, one fused to a DNA-binding domain and the other fused to an activator domain. This is demonstrated for cdk7-MAT1 interaction stabilized by the presence of cyclin H; these three polypeptides are found either free or associated with the transcription/DNA repair factor TFIIH. We also document the capacity of our system to conditionally inhibit the interaction between two polypeptides that otherwise elicit a positive two-hybrid response. This is demonstrated for Ras-Raf interaction precluded by an excess of Raf. The presence of a methionine-regulated promoter provides an "on" or "off" switch for the formation of the transcriptional activator, thus also providing an excellent control to evaluate the activation or inhibition properties of the third partner.

TFIIH is a general transcription factor for RNA polymerase II that in addition is involved in DNA excision repair. TFIIH is composed of eight or nine subunits and we show that at least four of them, namely cdk7, cyclin H, MAT1, and p62 are localized in the coiled body, a distinct subnuclear structure that is transcription dependent and highly enriched in small nuclear ribonucleoproteins. Although coiled bodies do not correspond to sites of transcription, in vivo incorporation of bromo-UTP shows that they are surrounded by transcription foci. Immunofluorescence analysis using antibodies directed against the essential repair factors proliferating cell nuclear antigen and XPG did not reveal labeling of the coiled body in either untreated cells or cells irradiated with UV light, arguing that coiled bodies are probably not involved in DNA repair mechanisms. The localization of cyclin H in the coiled body was predominantly detected during the G1 and S-phases of the cell cycle, whereas in G2 coiled bodies were very small or not detected. Finally, both cyclin H and cdk7 did not colocalize with P80 coilin after disruption of the coiled body, indicating that these proteins are specifically targeted to the small nuclear ribonucleoprotein-containing domain.

Inhibitory proteins for Cdks (CKIs) are involved in cell cycle arrest induced by anti-mitotic factors, chemicals, or DNA damage in mammalian cells. High cell density also induces cell cycle arrest with unreplicated genomic DNA even in the presence of mitotic dose of the growth factors, termed contact inhibition. Although rat fibroblast cell line, 3Y1, arrested in quiescence by contact inhibition, Cdk4 bound its regulatory subunit, cyclin D1 or D3. However, these complexes were enzymatically inactive. Phosphorylation of the cyclin D1-bound Cdk4 by Cdk4-activating kinase, composed of cyclin H and MO15 (alias Cdk7), which was reconstituted in Spodoptera frugiperda cells (Sf9) could convert inactive cyclin D1-Cdk4 complex into active form in vitro, suggesting that threonine 172 in the Cdk4, whose phosphorylation is required for its activation, was in part unphosphorylated in the contact-inhibited 3Y1. Although MO15 was active in cell extracts prepared from the contact-inhibited 3Y1, activation of bacterially produced Cdk4 in the cell extracts was inhibited. Removing p27kip1 from the cell extracts allowed MO15 holoenzyme to phosphorylate the Cdk4 and to activate it, indicating that an access of MO15 to Cdk4 was inhibited by p27kip1 in the contact-inhibited 3Y1.

The crystal structure of human cyclin H refined at 2.6 A resolution is compared with that of cyclin A. The core of the molecule consists of two repeats containing five helices each and forming the canonical cyclin fold also observed in TFIIB. One hundred and thirty-two out of the 217 C alpha atoms from the cyclin fold can be superposed with a root-mean-square difference of 1.8 A. The structural homology is even higher for the residues at the interface with the kinase, which is of functional significance, as shown by our observation that cyclin H binds to cyclin-dependent kinase 2 (cdk2) and that cyclin A is able to activate cdk7 in the presence of MAT1. Based on this superposition, a new signature sequence for cyclins was found. The specificity of the cyclin H molecule is provided mainly by two long helices which extend the cyclin fold at its N- and C-termini and pack together against the first repeat on the side opposite to the kinase. Deletion mutants show that the terminal helices are required for a functionally active cyclin H.

A cyclin-dependent kinase (cdk)-activating kinase (CAK) has been shown previously to catalyze T-loop phosphorylation of cdks in most eukaryotic cells. This enzyme exists in either of two forms: the major one contains cdk7, cyclin H and an assembly factor called MAT-1, whilst the minor one lacks MAT-1. Cdk7 is unusual among cdks because it contains not one but two residues (S170 and T176 in Xenopus cdk7) in its T-loop that are phosphorylated in vivo. We have investigated the role of S170 and T176 phosphorylation in the assembly and activity of cyclin H-cdk7 dimers. In the absence of MAT-1, phosphorylation of the T-loop appears to be required for cdk7 to bind cyclin H. Phosphorylation of both residues does not require cyclin H binding in vitro. Phosphorylation of S170 is sufficient for cdk7 to bind cyclin H with low affinity, but high affinity binding requires T176 phosphorylation. By mutational analysis, we demonstrate that in addition to its role in promotion of cyclin H binding, S170 phosphorylation plays a direct role in the control of CAK activity. Finally, we show that dual phosphorylation of S170 and T176, or substitution of both phosphorylatable residues by aspartic residues, is sufficient to generate CAK activity to one-third of its maximal value in vitro, even in the absence of cyclin H and MAT-1, and may thus provide further clues as to how cyclins activate cdk subunits.

Cyclin-dependent kinases (CDKs), which play a key role in cell cycle control, are activated by the CDK activating kinase (CAK), which activates cyclin-bound CDKs by phosphorylation at a specific threonine residue. Vertebrate CAK contains two key components: a kinase subunit with homology to its substrate CDKs and a regulatory subunit with homology to cyclins. We have determined the X-ray crystal structure of the regulatory subunit of CAK, cyclin H, at 2.6 A resolution. Cyclin H contains two alpha-helical core domains with a fold similar to that of cyclin A, a regulatory subunit of CAK substrate CDK2, and of TFIIB, a transcription factor. Outside of the core domains, the N- and C-terminal regions of the three structures are completely different. The conformational differences between cyclin H and A structures may reflect functional differences between the two cyclins.

A number of cyclin/kinase complexes have been identified in mammalian cells that are essential for controlled cell proliferation. Cyclin C was isolated by virtue of its ability to rescue the triple CLN mutation in yeast; however, until now its function has remained unclear. Cyclin C associates with a novel cyclin dependent kinase, CDK8, and we demonstrate that this complex is associated with kinase activity towards the carboxy-terminal domain (CTD) of RNA polymerase II. We have identified at least two distinct cyclin C/CDK8 containing complexes within the cell, a larger complex over 500 kD in size, that also contains the largest subunit of RNA polymerase II, and a smaller 170 kD species. Both of these cyclin C complexes retain potent CTD kinase activity. We further demonstrate that the cyclin C/CDK8 complex associates with the large subunit of RNA polymerase II in vivo, implicating a potential role for cyclin C/CDK8 in regulating its activities.

Cyclin-dependent kinases (Cdks) form complexes with cyclins, and as a consequence they generally express kinase activities. One of these Cdks, Cdk2, is known to bind with cyclins A and E, and plays an important role in the progression of the cell cycle via phosphorylation of target proteins such as the product of the retinoblastoma tumor-suppressor gene (pRB). It has been suggested that Cdk2 bound with cyclin D1 and Cdk2-cyclin-D1 complex show neither H1 histone nor pRB kinase activity. However, it is not clear whether Cdk2-cyclin-D1 has unknown targets and why Cdk2 is not activated by binding with cyclin D1. We investigated these questions using Cdk, cyclin and Cdk-cyclin complexes produced in a baculovirus expression system. Cdk2 formed a complex with cyclin D1 in this system. After extensive purification, Cdk2 was still bound to cyclin D1. The Cdk2-cyclin-D1 complex did not phosphorylate any tested substrates, such as H1 histone, pRB, SV40 large T antigen, p53, E2F-1 or a preparation of nuclear proteins from HeLa cells; in contrast, Cdk2-cyclin-E and Cdk2-cyclin-A phosphorylated these proteins. Moreover, the Cdk2-cyclin-D1 complex was not activated by incubation with Cdk4 or cyclin E. Thus, Cdk2 and cyclin D1 formed a stable complex that was not activated. In order to determine why Cdk2-cyclin-D1 lacks kinase activity, we investigated the phosphorylation of Cdk2. Under-shifted Cdk2, the active form of Cdk2, was not detected in the Cdk2-cyclin-D1 complex in the baculovirus system. In human WI-38 cells, cyclin D1 began to form a complex with Cdk2 as well as with Cdk4 from the mid-G1 phase of the cell cycle. The Cdk2 bound to cyclin D1 in human cells was also the inactive form that was slowly migrated. Moreover, we found that Cdk2 bound to cyclin D1 was not phosphorylated by Cdk7-cyclin-H, while Cdk2 bound to cyclin E, as well as free Cdk2, was was phosphorylated by Cdk7-cyclin-H. Additionally, Cdk2 phosphorylated by Cdk7-cyclin-H did not bind to cyclin D1. These results strongly suggest that Cdk2 forms a stable complex with cyclin D1 but is not activated because the Cdk2 molecule in the complex is not phosphorylated by Cdk7-cyclin-H and the phosphorylated Cdk2, an active form, does not bind to cyclin D1.

MAT1, cyclin H and cdk7 are part of TFIIH, a class II transcription factor which possesses numerous subunits of which several have been shown to be involved in processes other than transcription. Two of them, XPD (ERCC2) and XPB (ERCC3), are helicases involved in nucleotide excision repair (NER), whereas cdk7, cyclin H and MAT1 are thought to participate in cell cycle regulation. MAT1, cyclin H and cdk7 exist as a ternary complex either free or associated with TFIIH from which the latter can be dissociated at high salt concentration. MAT1 is strongly associated with cdk7 and cyclin H. Although not strictly required for the formation and activity of the complex, it stimulates its kinase activity. The kinase activity of TFIIH, which is constant during the cell cycle, is reduced after UV light irradiation.

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.

The cyclin-dependent kinase (CDK)-activating kinase, CAK, from mammals and amphibians consists of MO15/CDK7 and cyclin H, a complex which has been identified also as a RNA polymerase II C-terminal domain (CTD) kinase. While the Schizosaccharomyces pombe cdc2 gene product also requires an activating phosphorylation, the enzyme responsible has not been identified. We have isolated an essential S.pombe gene, mop1, whose product is closely related to MO15 and to Saccharomyces cerevisiae Kin28. The functional similarity of Mop1 and MO15 is reflected in the ability of MO15 to rescue a mop1 null allele. This suggests that Mop1 would be a CDK, and indeed Mop1 associates with a previously characterized cyclin H-related cyclin Mcs2 of S.pombe. Also, Mop1 and Mcs2 can associate with the heterologous partners human cyclin H and MO15, respectively. Moreover, the rescue of a temperature-sensitive mcs2 strain by expression of mop1+ demonstrates a genetic interaction between mop1 and mcs2. In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. The CAK activity of Mop1-Mcs2 distinguishes it from the related CDK-cyclin pair Kin28-Ccl1 from S.cerevisiae, and supports the notion that Mop1-Mcs2 may represent a homolog of MO15-cyclin H in S.pombe with apparent dual roles as a RNA polymerase CTD kinase and as a CAK.

Mammalian CDK7 is a protein kinase identified as the catalytic subunit of cyclin-dependent kinase (CDK)-activating kinase and as an essential component of the transcription factor TFIIH that is involved in transcription initiation and DNA repair. We have identified in human cells a number of CDK7-associated cellular proteins that appear to fall into two classes based on their relative [35S] metabolic labeling intensity. One class of proteins present in CDK7 immunocomplexes as a minor fraction contains components of the TFIIH transcription complex such as p62 and p89ERCC3, whereas the other fraction contains four polypeptides (p35, p37Cyclin H, p75, and p95) that are stoichiometrically associated with CDK7. Whereas the levels of association of p35, p37Cyclin H, and p75 with CDK7 remain unchanged between density-arrested and proliferating Ewing sarcoma EW-1 cells, the association of p95 with CDK7 was significantly decreased as cells reached confluency. Through a large-scale immunopurification of CDK7 complexes and protein microsequencing, we have isolated a cDNA that encodes p35 and have shown that it is the human homologue of Mat1 that is involved in the assembly of CAK. MAT1 contains a highly conserved C3HC4 motif at its NH2 terminus, a characteristic feature shared among RING finger proteins. The human MAT1 gene expresses a single 1.6-kb transcript, the steady-state level of which, like CDK7 and cyclin H, varies significantly in different cell lines and in different terminally differentiated tissues.

It is proposed that the CDK7-cyclin H complex functions in cell cycle progression, basal transcription and DNA repair. Here we report that in vitro reconstitution of an active CDK7-cyclin H complex requires stoichiometric amounts of a novel 36 kDa assembly factor termed MAT1 (ménage à trois 1). Sequencing of MAT1 reveals a putative zinc binding motif (a C3HC4 RING finger) in the N-terminus; however, this domain is not required for ternary complex formation with CDK7-cyclin H. MAT1 is associated with nuclear CDK7-cyclin H at all stages of the cell cycle in vivo. Ternary complexes of CDK7, cyclin H and MAT1 display kinase activity towards substrates mimicking both the T-loop in CDKs and the C-terminal domain of RNA polymerase II, regardless of whether they are immunoprecipitated from HeLa cells or reconstituted in a reticulocyte lysate. MAT1 constitutes the first example of an assembly factor that appears to be essential for the formation of an active CDK-cyclin complex.

The kinase responsible for Thr161-Thr160 phosphorylation and activation of cdc2/cdk2 (CAK:cdk-activating kinase) has been shown previously to comprise at least two subunits, cdk7 and cyclin H. An additional protein co-purified with CAK in starfish oocytes, but its sequencing did not reveal any similarity with any known protein. In the present work, a cDNA encoding this protein is cloned and sequenced in both starfish and Xenopus oocytes. It is shown to encode a new member of the RING finger family of proteins with a characteristic C3HC4 motif located in the N-terminal domain. We demonstrate that the RING finger protein (MAT1: 'menage à trois') is a new subunit of CAK in both vertebrate and invertebrates. However, CAK may also exist in oocytes as heterodimeric complexes between cyclin H and cdk7 only. Stable heterotrimeric CAK complexes were generated in reticulocyte lysates programmed with mRNAs encoding Xenopus cdk7, cyclin H and MAT1. In contrast, no heterodimeric cyclin H-cdk7 complex could be immunoprecipitated from reticulocyte lysates programmed with cdk7 and cyclin H mRNAs only. Stabilization of CAK complexes by MAT1 does not involve phosphorylation of Thr176, as the Thr176-->Ala mutant of Xenopus cdk7 could engage as efficiently as wild-type cdk7 in ternary complexes. Even though starfish MAT1 is almost identical to Xenopus MAT1 in the RING finger domain, the starfish subunit could not replace the Xenopus subunit and stabilize cyclin H-cdk7 in reticulocyte lysate, suggesting that the MAT1 subunit does not (or not only) interact with cyclin H-cdk7 through the RING finger domain.

An array of tandem heptapeptide repeats at the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II constitute a highly conserved structure essential for viability. Studies have established that the CTD is phosphorylated at different stages of the transcription cycle, and that it may be involved in transcriptional regulation. The exact role of the CTD remains elusive, as in vitro reconstituted transcription using the adenovirus major late promoter does not require the CTD. Previous studies showed that transcription from the murine dihydrofolate reductase (DHFR) promoter can be only accomplished by the form of RNA polymerase II that contains the hypophosphorylated CTD (RNAPIIA), but not by the form that lacks it (RNAPIIB). Here we show that the CTD, but not its phosphorylation, is required for initiation of transcription. We also show that transcription requires CTD kinase activity provided by the CDK subunit of TFIIH.

We have cloned a mouse cDNA that encodes p36, a novel subunit of the CDK-activating kinase (CAK). p36 contains a C3HC4 zinc-binding domain or RING factor and is associated both with a TFIIH-bound form of CAK and with a free trimeric form. p36 promotes the assembly of CDK7 and cyclin H in vitro, stabilizing the transient CDK7-cyclin H complex. Stabilization and activation of CAK by p36 is independent of the phosphorylation state of T170, the conserved activating residue of CDK7. Assembly of active CDK7-cyclin H dimers can also occur through an alternative p36-independent pathway that requires phosphorylation of T170 by a CAK-activating kinase, or CAKAK. Thus, CDK7-cyclin H complex formation can be achieved by multiple mechanisms.

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.

Transcription factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). Here we report the identification of the Cdk-activating kinase (Cak) complex (Cdk7 and cyclin H) as a component of TFIIH after extensive purification of TFIIH by chromatography. We find that affinity-purified antibodies directed against cyclin H inhibit TFIIH-dependent transcription and that both cyclin H and Cdk7 antibodies inhibit phosphorylation of the CTD of the largest subunit of the RNAPII in the preinitiation complex. Cak is present in at least two distinct complexes, TFIIH and a smaller complex that is unable to phosphorylate RNAPII in the preinitiation complex. Both Cak complexes, as well as recombinant Cak, phosphorylate a CTD peptide. Finally, TFIIH was shown to phosphorylate both Cdc2 and Cdk2, suggesting that there could be a link between transcription and the cell cycle machinery.

The eukaryotic cell cycle is regulated by the sequential activation of cyclin-dependent kinases (CDKs). CDK activation is dependent on cyclin binding and phosphorylation of a conserved threonine (T161 in Cdc2) mediated by the CDK-activating kinase CAK. A CDK-related kinase, MO15 (ref. 10), has been identified as the catalytic subunit of CAK (refs 11-13). Here we use a yeast two-hybrid screen to show that a new human cyclin (cyclin H) is a MO15-associated protein. Cyclin H is a major MO15 partner in vivo and enhances the kinase activity of MO15 towards Cdk2/cyclin A. These findings demonstrate that a cyclin/kinase complex can function as a regulator of other cyclin/kinase complexes, and suggest that cyclin/kinase cascades may exist.

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.

ObjectiveThe site-specificity of endothelial phenotype is attributable to the local hemodynamic forces. The flow regulation of microRNAs in endothelial cells (ECs) plays a significant role in vascular homeostasis and diseases. The objective of this study was to elucidate the molecular mechanism by which the pulsatile shear flow-induced microRNA-23b (miR-23b) exerts antiproliferative effects on ECs.Approach and resultsWe used a combination of a cell perfusion system and experimental animals to examine the flow regulation of miR-23b in modulating EC proliferation. Our results demonstrated that pulsatile shear flow induces the transcription factor Krüppel-like factor 2 to promote miR-23b biosynthesis; the increase in miR-23b then represses cyclin H to impair the activity and integrity of cyclin-dependent kinase-activating kinase (CAK) complex. The inhibitory effect of miR-23b on CAK exerts dual actions to suppress cell cycle progression, and reduce basal transcription by deactivating RNA polymerase II. Whereas pulsatile shear flow regulates the miR-23b/CAK pathway to exert antiproliferative effects on ECs, oscillatory shear flow has little effect on the miR-23b/CAK pathway and hence does not cause EC growth arrest. Such flow pattern-dependent phenomena are validated with an in vivo model on rat carotid artery: the flow disturbance induced by partial carotid ligation led to a lower expression of miR-23b and a higher EC proliferation in comparison with the pulsatile flow regions of the unligated vessels. Local delivery of miR-23b mitigated the proliferative EC phenotype in partially ligated vessels.ConclusionsOur findings unveil a novel mechanism by which hemodynamic forces modulate EC proliferative phenotype through the miR-23b/CAK pathway.

Eukaryotic cell division is controlled by cyclin-dependent kinases (CDKs), which require phosphorylation by a CDK-activating kinase (CAK) for full activity. Chemical genetics uncovered requirements for the metazoan CAK Cdk7 in determining cyclin specificity and activation order of Cdk2 and Cdk1 during S and G2 phases. It was unknown if Cdk7 also activates Cdk4 and Cdk6 to promote passage of the restriction (R) point, when continued cell-cycle progression becomes mitogen independent, or if CDK-activating phosphorylation regulates G1 progression. Here we show that Cdk7 is a Cdk4- and Cdk6-activating kinase in human cells, required to maintain activity, not just to establish the active state, as is the case for Cdk1 and Cdk2. Activating phosphorylation of Cdk7 rises concurrently with that of Cdk4 as cells exit quiescence and accelerates Cdk4 activation in vitro. Therefore, mitogen signaling drives a CDK-activation cascade during G1 progression, and CAK might be rate-limiting for R point passage.