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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.

The protein kinase CK2 holoenzyme is composed of two regulatory beta- and two catalytic alpha- or alpha(')-subunits. There is ample evidence for the binding of individual subunits of CK2 to various cellular proteins and, moreover, for functions of the individual subunits, which are different from their roles in the holoenzyme. Here, we report that the regulatory cyclin H subunit of the cyclin H/cdk7/Mat1 complex was associated with a protein kinase activity, which shows some similarity with protein kinase CK2. Coimmunoprecipitation experiments supported the existence of complexes of cyclin H and CK2 in mammalian cells. Far Western blot experiments revealed that cyclin H bound to the alpha-subunit but not the alpha(')- and beta-subunits of CK2. Immunofluorescence analysis showed that cyclin H and CK2alpha were colocated in the nucleus. Although cyclin H functions as the regulatory subunit for the cyclin H/cdk7/Mat1 complex, it could not substitute the regulatory beta-subunit of CK2 in its regulatory function of the CK2 activity.

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.

The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells., (Copyright 2001 Academic Press.)

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.

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 eukaryotic cell cycle is regulated by cyclin-dependent kinases (CDKs). CDK4 and CDK6, which are activated by D-type cyclins during the G(1) phase of the cell cycle, are thought to be responsible for phosphorylation of the retinoblastoma gene product (pRb). The tumor suppressor p16(INK4A) inhibits phosphorylation of pRb by CDK4 and CDK6 and can thereby block cell cycle progression at the G(1)/S boundary. Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II by general transcription factor TFIIH is believed to be an important regulatory event in transcription. TFIIH contains a CDK7 kinase subunit and phosphorylates the CTD. We have previously shown that p16(INK4A) inhibits phosphorylation of the CTD by TFIIH. Here we report that the ability of p16(INK4A) to inhibit CDK7-CTD kinase contributes to the capacity to induce cell cycle arrest. These results suggest that p16(INK4A) may regulate cell cycle progression by inhibiting not only CDK4-pRb kinase activity but also by modulating CDK7-CTD kinase activity. Regulation of CDK7-CTD kinase activity by p16(INK4A) thus may represent an alternative pathway for controlling cell cycle progression.

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.

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.

The eukaryotic cell cycle is regulated by a group of highly conserved cyclin dependent protein kinases (CDKs). Several CDKs have been identified in Plasmodium falciparum, however, their regulatory mechanisms as well as their role in parasite growth and differentiation are not understood fully. To further our understanding of Plasmodium CDK regulation, we have characterized Pfmrk kinase activity. Pfmrk was expressed and purified as a 6xHis tagged recombinant protein from Escherichia coli and assayed for histone H1 kinase activity. Pfmrk has significant histone H1 kinase activity and is autophosphorylated in vitro. Human cyclin H forms a stable complex with Pfmrk and stimulates kinase activity. This is the first indication that Plasmodial CDKs are partially regulated by cyclin subunits, as are human CDKs. CDKs are attractive drug targets due to their role in cellular proliferation. Specific CDK inhibitors were selected to evaluate Pfmrk as a potential drug target. Olomoucine and roscovitine failed to inhibit Pfmrk kinase activity which places Pfmrk with a class of CDKs that are insensitive to these compounds. A molecular model of Pfmrk provides a structural explanation for the failure of these compounds to inhibit Pfmrk.

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.

Background: Cyclin-dependent kinases (CDKs) have a central role in cell-cycle control and are activated by complex formation with positive regulatory proteins called cyclins and by phosphorylation. The overexpression and mutation of cyclins and CDKs has been associated with tumorigenesis and oncogenesis. A virus-encoded cyclin (v-cyclin) from herpesvirus saimiri has been shown to exhibit highest sequence homology to type D cyclins and specifically activates CDK6 of host cells to a very high degree., Results: We have determined the first X-ray structure of a v-cyclin to 3.0 A resolution. The structure of the core domains is very similar to those of cyclin A and cyclin H from human cells. To understand the structural basis for the v-cyclin specificity for CDK6 and the insensitivity of the complex to inhibitors of the p21 and INK4 families, a v-cyclin-CDK2 model was built on the basis of the known structures of human cyclin A in complex with CDK2 and the CDK inhibitor p27(Kip1)., Conclusions: Although many critical interactions between cyclin A and CDK2 would be conserved in a v-cyclin-CDK2 complex, some appear sterically or electrostatically unfavorable due to shifts in the backbone conformation or sidechain differences and may contribute to v-cyclin selectivity for CDK6. The insensitivity of v-cyclin-CDK6 complexes to inhibitors of the p21 family is probably due to structural changes in v-cyclin that lead to a flatter surface area offering fewer potential contacts with the protein inhibitor. In addition, sequence changes in v-cyclin eliminate hydrogen-bonding partners for atoms of the p27(Kip1) inhibitor. This structure provides the first model for interactions between v-cyclins and host cell-cycle proteins; these interactions may be important for virus survival as well as oncogenic transformation of host cells.

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).

The growth suppressor p53 is an important key element which controls cell cycle progression in response to cellular stress like DNA damage. Its ability to act as transcriptional activator or repressor links transcription and cell cycle control. Several target genes selectively transactivated by p53 are implicated in growth control, apoptosis and DNA repair. Here we report the interaction of p53 with another important dual player of cell cycle control and transcription, the protein kinase complex CDK7/cyclin H/Mat1 (CDK activating kinase, CAK kinase). This is implicated in the activating phosphorylation of CDK2/cyclin A kinase required to allow cells to proceed through the G1/S transition, and on the other hand, as a component of the basal transcription factor TFIIH found to be necessary for CTD phosphorylation of RNA polymerase II in order to allow elongation of transcription. Based on previous binding studies of p53 with other C-terminal interaction partners of p53 we demonstrate a direct physical interaction of p53 with cyclin H in vitro and in vivo. As a consequence of this interaction we tested the influence of p53 on the kinase activity of CAK kinase for CTD and CDK2 phosphorylation. The addition of wild type p53 to the kinase reactions resulted in a significant downregulation of CDK2 phosphorylation and CTD phosphorylation by the CDK activating kinase. On the other hand addition of a mutant p53His175 failed to downregulate CDK2 and CTD phosphorylation by the CDK activating kinase. In an attempt to support our findings in vivo we measured CAK kinase activity in p21-/- and p53-/- mice embryonal fibroblasts under conditions when p53 gets activated by irradiation. In the case of p21-/- cells this led to a significant reduction of CTD phosphorylation activity of the CDK activating kinase by irradiation of the cells. On the other hand in p53 cells no downregulation of CTD phosphorylation activity of CAK kinase was observed indicating that this kind of negative regulation of CAK kinase activity is exclusively due to a functional p53. These findings imply a direct involvement of p53 in triggering growth arrest by its interaction with the CDK activating kinase complex without the need of cyclin-dependent kinase inhibitors (CKIs) and potentially suggest a new mechanism for p53-dependent apoptosis.

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.

Formation of active cdk (cyclin dependent kinase)/ cyclin kinases involves phosphorylation of a conserved threonine residue in the T loop of the cdk catalytic-subunit by CAK (Cdk Activating Kinase). CAK was first purified biochemically from higher eukaryotes and identified as a trimeric complex containing a cdk7 catalytic subunit, cyclin H and MAT1 (Ménage à trois), a member of the RING finger family. The same trimeric complex is also part of basal transcription factor TFIIH. In budding yeast, the closest homologs of cdk7 and cyclin H, KIN28 and CCL1, respectively, also associate with TFIIH. However, the KIN28/CCL1 complex does not display CAK activity and a distinct protein kinase able to phosphorylate monomeric CDC28 and GST-cdk2 was recently identified, challenging the identification of cdk7 as the physiological CAK in higher eukaryotes. Here we demonstrate that immunodepletion of cdk7 suppresses CAK activity from cycling Xenopus egg extracts, and arrest them before M-phase. We also show that specific translation of mRNAs encoding Xenopus cdk7 and its associated subunits restores CAK activity in cdk7-immunodepleted Xenopus egg extracts. Hence, the cdk7 complex is necessary and sufficient for activation of cdk-cyclin complexes in cycling Xenopus egg extracts.

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.

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.

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 RNA polymerase II large subunit contains an essential carboxy-terminal domain (CTD) believed to be involved in the response to regulators during transcription initiation. The CTD is phosphorylated on a portion of RNA polymerase II molecules in vivo and it can be phosphorylated by the general transcription factor TFIIH in vitro. A highly purified TFIIH from rat liver has been described; this, like human and yeast TFIIH, contains associated CTD kinase and helicase activities. We report here that two polypeptides of the purified mammalian TFIIH are the MO15/Cdk7 kinase and cyclin H subunits of the Cdk-activating kinase Cak, previously identified as a positive regulator of Cdc2 and Cdk2. TFIIH and Cak preparations are each capable of phosphorylating recombinant CTD and recombinant Cdk2 proteins. The presence of Cak in TFIIH indicates that Cak may have roles in transcriptional regulation and in cell-cycle control.

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.