Cyclin-dependent kinase 7


Yeast CAK plays a role in cell cycle but is not a component of the basal transcripition factor

Activation of the cyclin-dependent kinases to promote cell cycle progression requires their association with cyclins as well as phosphorylation of a threonine residue. This phosphorylation is carried out by the Cdk-activating kinase (CAK). Purification of CAK from mammals, starfish, and Xenopus has identified it as a heterotrimeric complex composed of a catalytic subunit (p40MO15/cdk7), a regulatory subunit (cyclin H), and an assembly factor (MAT1). CAK phosphorylates not only c34cdc2 but also other Cdks, including p33cdk2 and cdk4, which function earlier in the cell cycle. The CAK subunits are components of TFIIH, a basal transcription factor involved in the initiation of transcription, phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II and DNA repair. The cloning of the CAK from S. cerevisiae raises the possibility that the predominant CAK in vertebrate cell extracts may not function as a physiological CAK. S. cerevisiae CAK is active as a monomer and is not a component of the basal transcription factor (Kaldis, 1996 and references).

In Saccharomyces cerevisiae, entry into S phase requires the activation of the protein kinase Cdc28p through binding with cyclin Clb5p or Clb6p, as well as the destruction of the cyclin-dependent kinase inhibitor Sic1p. Mutants that are defective in this activation event arrest after START, with unreplicated DNA and multiple, elongated buds. These mutants include cells defective in CDC4, CDC34 or CDC53, as well as cells that have lost all CLB function. This paper describes mutations in another gene, CAK1, that lead to a similar arrest. Cells that are defective in CAK1 are inviable and arrest with a single nucleus and multiple, elongated buds. CAK1 encodes a protein kinase most closely related to the Cdc2p family of protein kinases. Mutations that lead to the production of an inactive kinase that can neither autophosphorylate, nor phosphorylate Cdc28p in vitro are also incapable of rescuing a cell with a deletion of CAK1. These results underscore the importance of the Cak1p protein kinase activity in cell cycle progression (Chun, 1997).

The CAK1 gene encodes the major CDK-activating kinase (CAK) in budding yeast and is required for activation of Cdc28p for cell cycle progression from G2 to M phase. A mutant allele of CAK1 was isolated in a synthetic lethal screen with the Sit4 protein phosphatase. Analysis of several different cak1 mutants shows that although the G2 to M transition appears most sensitive to loss of Cak1p function, Cak1p is also required for activation of Cdc28p for progression from G1 into S phase. Further characterization of these mutants suggests that, unlike the CAK identified from higher eukaryotes, Cak1p of budding yeast may not play a role in general transcription. Finally, although Cak1 protein levels and in vitro protein kinase activity do not fluctuate during the cell cycle, at least a fraction of Cak1p associates with higher molecular weight proteins, which may be important for its in vivo function (Sutton, 1997).

Cdc7p is a protein kinase that is required for G1/S transition and initiation of DNA replication in Saccharomyces cerevisiae. The mechanisms whereby Cdc7p and its substrates exerts their effects are unknown. The characterization in S. cerevisiae of a recessive mutation is reported in a member of the MCM family, MCM5/CDC46, that bypasses the requirement for Cdc7p and its interacting factor Dbf4p. Because the MCM family of evolutionarily conserved proteins have been implicated in restricting DNA replication to once per cell cycle, these studies suggest that Cdc7p is required late in G1 because in its absence the Mcm5p/Cdc46p blocks the initiation of DNA replication. Moreover, Mcm5p/Cdc46p may have both positive and negative effects on the ability of cell to initiate replication (Hardy, 1997).

Interaction of Cdk7 and DNA replication

The Cdc7-Dbf4 kinase is essential for regulating initiation of DNA replication in Saccharomyces cerevisiae. A human Cdc7 homolog, HsCdc7 has been identified as has a human Dbf4 homolog, HsDbf4. HsDbf4 binds to HsCdc7 and activates HsCdc7 kinase activity when HsDbf4 and HsCdc7 are coexpressed in insect and mammalian cells. HsDbf4 protein levels are regulated during the cell cycle with a pattern that matches that of HsCdc7 protein kinase activity. They are low in G1, increase during G1-S, and remain high during S and G2-M. Purified baculovirus-expressed HsCdc7-HsDbf4 selectively phosphorylates the MCM2 subunit of the minichromosome maintenance (MCM) protein complex isolated by immunoprecipitation with MCM7 antibodies in vitro. Two-dimensional tryptic phosphopeptide-mapping analysis of in vivo 32P-labeled MCM2 from HeLa cells reveals that several major tryptic phosphopeptides of MCM2 comigrate with those of MCM2 phosphorylated by HsCdc7-HsDbf4 in vitro, suggesting that MCM2 is a physiological HsCdc7-HsDbf4 substrate. Immunoneutralization of HsCdc7-HsDbf4 activity by microinjection of anti-HsCdc7 antibodies into HeLa cells blocks initiation of DNA replication. These results indicate that the HsCdc7-HsDbf4 kinase is directly involved in regulating the initiation of DNA replication by targeting MCM2 protein in mammalian cells (Jiang, 1999).

CDK7 in C. elegans

CDK7 is a cyclin-dependent kinase proposed to function in two essential cellular processes: transcription and cell cycle regulation. CDK7 is the kinase subunit of the general transcription factor TFIIH that phosphorylates the C-terminal domain (CTD) of RNA polymerase II, and has been shown to be broadly required for transcription in Saccharomyces cerevisiae. CDK7 can also phosphorylate CDKs that promote cell cycle progression, and has been shown to function as a CDK-activating kinase (CAK) in Schizosaccharomyces pombe and Drosophila. That CDK7 performs both functions in metazoans has been difficult to prove because transcription is essential for cell cycle progression in most cells. A temperature-sensitive mutation has been isolated in C. elegans cdk-7 and it has been used to analyze the role of cdk-7 in embryonic blastomeres, where cell cycle progression is independent of transcription. Partial loss of cdk-7 activity leads to a general decrease in CTD phosphorylation and embryonic transcription, and severe loss of cdk-7 activity blocks all cell divisions. These results support a dual role for metazoan CDK7 as a broadly required CTD kinase, and as a CAK essential for cell cycle progression (Wallenfang, 2002).

Signaling upstream of Cdk7

An analysis was performed of how single-strand DNA gaps affect DNA replication in Xenopus egg extracts. DNA lesions generated by etoposide, a DNA topoisomerase II inhibitor, or by exonuclease treatment activate a DNA damage checkpoint that blocks initiation of plasmid and chromosomal DNA replication. The checkpoint is abrogated by caffeine and requires ATR, but not ATM, protein kinase (see Drosophila mei-41). The block to DNA synthesis is due to inhibition of Cdc7/Dbf4 protein kinase activity and the subsequent failure of Cdc45 to bind to chromatin. The checkpoint does not require pre-RC assembly but requires loading of the single-strand binding protein, RPA, on chromatin. This is the biochemical demonstration of a DNA damage checkpoint that targets Cdc7/Dbf4 protein kinase (Costanzo, 2003).

Interaction of Cdk7 and cyclin H

The formation of cdk-cyclin complexes has been investigated at the molecular level and quantified using spectroscopic approaches. In the absence of phosphorylation, cdk2, cdc2, and cdk7 form highly stable complexes with their "natural" cyclin partners, with dissociation constants in the nanomolar range. In contrast, nonphosphorylated cdc2-cyclin H, cdk2-cyclin H, and cdk7-cyclin A complexes present a 25-fold lower stability. On the basis of both the structure of the cdk2-cyclin A complex and on kinetic results, it is suggested that interaction of any cyclin with any cdk involves the same hydrophobic contacts and induces a marked conformational change in the catalytic cleft of the cdks. Although cdks bind ATP strongly, they remain in a catalytically inactive conformation. In contrast, binding of the cyclin induces structural rearrangements that result in the selective reorientation of ATP, a concomitant 3-fold increase in its affinity, and a 5-fold decrease of its release from the active site of cdks (Heitz, 1997).

Previously, a cyclin-dependent kinase (cdk)-activating kinase (CAK) has been shown 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 MAT1, while the minor one lacks MAT1. 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. The role of S170 and T176 phosphorylation has been investigated in the assembly and activity of cyclin H-cdk7 dimers. In the absence of MAT1, 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, it has been demonstrated that in addition to its role in promotion of cyclin H binding, S170 phosphorylation plays a direct role in the control of CAK activity. 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 MAT1, and may thus provide further clues as to how cyclins activate cdk subunits (Martinez, 1997).

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 the 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 has been found. The specificity of the cyclin H molecule is provided mainly by two long helices that extend the cyclin fold at its N- and C-termini and pack together against the first repeat on the side opposite the kinase. Deletion mutants show that the terminal helices are required for a functionally active cyclin H (Andersen, 1998).

Cdk7 is part of a ternary complex in higher eukaryotes

The cyclin-dependent kinase (CDK)-activating kinase CAK has been proposed to function in the control of cell cycle progression, DNA repair and RNA polymerase II (pol II) transcription. Most CAK exists as complexes of three subunits: CDK7, cyclin H (CycH) and MAT1. This tripartite CAK occurs in a free form and in association with 'core' TFIIH, which functions in both pol II transcription and DNA repair. The substrate specificities of different forms of CAK have been investigated. Addition of the MAT1 subunit to recombinant bipartite CDK7-CycH switchs its substrate preference to favour the pol II large subunit C-terminal domain (CTD) over CDK2. It is suggested that the MAT1 protein, previously shown to function as an assembly factor for CDK7-CycH, also acts to modulate CAK substrate specificity. The substrate specificities of natural TFIIH and free CAK were also compared. TFIIH has a strong preference for the CTD over CDK2 relative to free CAK. TFIIH, but not free CAK, can efficiently hyperphosphorylate the CTD. In the context of TFIIH, the kinase also acquires specificity for the general transcription factors TFIIE and TFIIF, which are not recognized by free CAK. It is concluded that the substrate preference of the CDK7-CycH kinase is governed by association with both MAT1 and 'core' TFIIH (Yankulov, 1997).

MAT1, cyclin H and cdk7 are part of TFIIH, a class II transcription factor that possesses numerous subunits, several of which have been shown to be involved in processes other than transcription. Two of these subunits, 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, MAT1 stimulates the complex's kinase activity. The kinase activity of TFIIH, which is constant during the cell cycle, is reduced after UV light irradiation (Adamczewski, 1996).

TFIIH is a general transcription factor for RNA polymerase II; in addition, it is involved in DNA excision repair. TFIIH is composed of eight or nine subunits and 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 is predominantly detected during the G1 and S-phases of the cell cycle, whereas in G2 coiled bodies are very small or not detected. Neither cyclin H nor cdk7 colocalize with P80 coilin after disruption of the coiled body, indicating that these proteins are specifically targeted to the small nuclear ribonucleoprotein-containing domain (Jordan, 1997).

Cdk7 as an integral member of TFIIH and Cdk7's role in targeting the CTD of RNA Pol II

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. The transcription/DNA repair factor TFIIH consists of nine subunits, several exhibiting known functions: a helicase/ATPase, a kinase activity and DNA binding activity. Three subunits of TFIIH, cdk7, cyclin H and MAT1, form a ternary complex, cdk-activating kinase (CAK), found either on its own or as part of TFIIH. Purified human CAK complex (free CAK) and recombinant CAK (rCAK) produced in insect cells exhibits a strong preference for the cyclin-dependent kinase 2 (cdk2) over a C-terminal domain (ctd) oligopeptide substrate (which mimics the ctd of the RNA polymerase II). In contrast, TFIIH preferentially phosphorylates the ctd as well as TFIIE alpha, but not cdk2. TFIIH was resolved into four subcomplexes: the kinase complex composed of cdk7, cyclin H and MAT1; the core TFIIH, which contains XPB, p62, p52, p44 and p34; and two other subcomplexes in which XPD is found associated with either the kinase complex or with the core TFIIH. Using these fractions, it was demonstrated that TFIIH lacking the CAK subcomplex completely recovers its transcriptional activity in the presence of free CAK. Studies examining the interactions between TFIIH subunits provide evidence that CAK is integrated within TFIIH via XPB and XPD. XPB and XPD are helicase subunits of TFIIH associated with xeroderma pigmentosum (Rossignol, 1997).

A highly purified TFIIH from rat liver has been described; this, like human and yeast TFIIH, contains associated CTD kinase and helicase activities. 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 (Serizawa, 1995).

Transcription factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). The Cdk-activating kinase (Cak) complex (Cdk7 and cyclin H) is identified as a component of TFIIH after extensive purification of TFIIH by chromatography. Affinity-purified antibodies directed against cyclin H inhibit TFIIH-dependent transcription; 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. TFIIH has been shown to phosphorylate both Cdc2 and Cdk2, suggesting that there could be a link between transcription and the cell cycle machinery (Shiekhattar, 1995).

The cell cycle is regulated by various protein kinases, including cyclin-dependent kinases (CDKs). D-type CDKs (CDK4, and CDK6) phosphorylate retinoblastoma protein and are believed to regulate through the G1 phase of the cell cycle. CDK inhibitor p16INK4A has been characterized as binding CDK4 and CDK6 and as inhibiting phosphorylation of retinoblastoma protein by these CDKs. Thus p16INK4A is implicated in regulating the cell cycle at the G1 phase. The largest subunit of RNA polymerase II (pol II) contains an essential C-terminal domain (CTD). General transcription factor TFIIH, which contains CDK7, phosphorylates the CTD in vitro. The CTD phosphorylation is shown to be involved in transcriptional regulation in vivo and in vitro. Phosphorylation of RNA pol II CTD by TFIIH is thought to play an important role in transcriptional regulation. p16INK4A associates with RNA pol II CTD and TFIIH. p16(INK4A) inhibits the CTD phosphorylation by TFIIH. These findings suggest that p16INK4A may regulate transcription via CTD phosphorylation in the cell cycle (Serizawa, 1998).

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, the hypothesis was tested that the kinases differ in their ability to phosphorylate CTD heptad arrays. The data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO4. This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs (Ramanatyhan, 2001).

Mitotic cyclinB/Cdk targets the transcriptional apparatus

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. These results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis (Long, 1998).

Cdk7 targets p53 and other transcription factors

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, an investigation was carried out as to whether p53 can act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, it has been shown 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. A major site of phosphorylation by CAK has been mapped to Ser-33 of p53, and p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, it is 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 (Ko, 1997).

Phosphorylation is believed to be one of the mechanisms by which p53 becomes activated or stabilized in response to cellular stress. Previously, p53 was shown to interact with three components of transcription factor IIH (TFIIH): excision repair cross-complementing types 2 and 3 (ERCC2 and ERCC3) and p62. p53 is phosphorylated by the TFIIH-associated kinase in vitro. The phosphorylation was found to be catalyzed by the highly purified kinase components of TFIIH, in other words, by the CDK7-cycH-p36 trimeric complex. The phosphorylation sites were mapped to the C-terminal amino acids located between residues 311 and 393. Serines 371, 376, 378, and 392 may be the potential sites for this kinase. Phosphorylation of p53 by this kinase complex enhances the ability of p53 to bind to the sequence-specific p53-responsive DNA element as shown by gel mobility shift assays. These results suggest that the CDK7-cycH-p36 trimeric complex of TFIIH may play a role in regulating p53 functions in cells (Lu, 1997).

Octamer binding transcription factors (Oct factors) play important roles in the activation of transcription of various genes, however, certain genes require cofactors that interact with the DNA binding (POU) domain. In the present study, a yeast two-hybrid screen with the Oct-1 POU domain as a bait identified MAT1 as a POU domain-binding protein. MAT1 is known to be required for the assembly of cyclin-dependent kinase (CDK)-activating kinase (CAK), which is functionally associated with the general transcription factor IIH (TFIIH). Further analyses show that MAT1 interacts with POU domains of Oct-1, Oct-2, and Oct-3 in vitro in a DNA-independent manner. MAT1-containing TFIIH also interacts with POU domains of Oct-1 and Oct-2. MAT1 has been shown to enhance the ability of a recombinant CDK7-cyclin H complex (bipartite CAK) to phosphorylate isolated POU domains, intact Oct-1, and the C-terminal domain of RNA polymerase II, but not the originally defined substrate, CDK2. Phosphopeptide mapping indicates that the site (Ser385) of a mitosis-specific phosphorylation that inhibits Oct-1 binding to DNA is not phosphorylated by CAK. However, one CAK-phosphorylated phosphopeptide comigrates with a Cdc2-phosphorylated phosphopeptide previously shown to be mitosis-specific; this suggests that in vitro, CAK is able to phosphorylate at least one site that is also phosphorylated in vivo. These results suggest (1) that interactions between POU domains and MAT1 can target CAK to Oct factors and result in their phosphorylation, (2) that MAT1 not only functions as a CAK assembly factor but also acts to alter the spectrum of CAK substrates, and (3) that a POU-MAT1 interaction may play a role in the recruitment of TFIIH to the preinitiation complex or in subsequent initiation and elongation reactions (Inamoto, 1997).

RARalpha has two transcriptional activation functions. One of these, known as AF-2, is ligand-dependent, while another, in the amino-terminal region, contains a ligand-independent function. The activity of the N-terminal activation function AF-1 of RAR alpha1 is abrogated upon mutation of a phosphorylatable serine residue (Ser-77). Recombinant RAR alpha is phosphorylated by a variety of proline-directed protein kinases in vitro. However, only the coexpression of cdk7 stimulates Ser-77 phosphorylation in vivo and enhances transactivation by RAR alpha, but not by a S77A RAR mutant. Both free CAK (cdk7, cyclin H, MAT1) and the CAK-containing general transcription factor TFIIH phosphorylate Ser-77 in vitro. RAR alpha binds free CAK and purified TFIIH in vitro, and RAR alpha-TFIIH complexes can be isolated from HeLa nuclear extracts. These findings represent the first example of activation of a transactivator through binding to and phosphorylation by a general transcription factor (Rochette-Egly, 1997).

Cdc7-related kinases play essential roles in the initiation of yeast DNA replication. Mice lacking murine homologs of Cdc7 (muCdc7) genes die between E3.5 and E6.5. A mutant embryonic stem (ES) cell line lacking the muCdc7 genes has been established in the presence of a loxP-flanked transgene expressing muCdc7 cDNA. Upon removal of the transgene by Cre recombinase, mutant ES cells cease DNA synthesis, arresting growth with S-phase DNA content, and generate nuclear Rad51 foci, followed by cell death with concomitant increase in p53 protein levels. Inhibition of p53 leads to partial rescue of muCdc7-/- ES cells from cell death. muCdc7-/-p53-/- embryos survive up to E8.5, and their blastocysts generate inner cell mass of a significant size in vitro, whereas those of the muCdc7-/-p53+/- embryos undergo complete degeneration. These results demonstrate that, in contrast to cell cycle arrest at the G1/S boundary observed in yeasts, loss of Cdc7 in ES cells results in rapid cessation of DNA synthesis within S phase, triggering checkpoint responses leading to recombinational repair and p53-dependent cell death (Kim, 2002).

Cdk7 targets cyclin dependent kinases

Activation of cyclin dependent kinases (see Drososophila Cdc2) 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 Menage a trois (MAT1), 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. It is demonstrated that immunodepletion of cdk7 suppresses CAK activity from cycling Xenopus egg extracts, and arrests them before M-phase. It is also shown 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 (Fesquet, 1997).

When full-grown oocytes of the newt Cynops pyrrhogaster are treated with progesterone in O-R2 solution containing antibiotics, approximately 85% of the oocytes complete meiosis synchronously. Maturation-promoting factor (MPF) activity appears just before germinal vesicle breakdown (GVBD) and the oocytes maintain high MPF activity throughout metaphase I and metaphase II of meiosis. A slight decrease of MPF activity is observed at the first polar body emission. The distribution of cyclin B1 was investigated with anti-cyclin B1 antibody. No cyclin B1 is found in the oocytes before progesterone treatment. Cyclin B1 appears in the cortex of animal hemispheres, especially around and inside the germinal vesicle just before GVBD. A large amount of cyclin B1 accumulates at metaphase I; approximately half disappears at the first polar body emission, and then cyclin B1 accumulates again at metaphase II. An inactive form of cdc2 kinase is observed in both the germinal vesicles and the oocyte cytoplasm, while an active form appears at the M phase. No MPF is observed in the oocytes from which the germinal vesicle has been removed. A cdk7-like molecule is localized in the germinal vesicle, but not in oocyte cytoplasm, indicating that inactive cdc2 kinase associated with cyclin B1 derived from cytoplasm is activated by phosphorylation in the germinal vesicle. The changes in the amount of cyclin B1 are synchronous with the first cell cycle after fertilization. Cyclin B1 is primarily localized in the cortex of the animal hemisphere. A shift in band mobility upon electrophoresis of cyclin B1 is observed from samples taken during the cell cycle; this shift is probably due to the protein's phosphorylation state (Sakamoto, 1998).

Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication

Cdc7, a protein kinase required for the initiation of eukaryotic DNA replication, is activated by a regulatory subunit, Dbf4. A second activator of Cdc7 called Drf1 exists in vertebrates, but its function is unknown. In Xenopus egg extracts, Cdc7-Drf1 is far more abundant than Cdc7-Dbf4, and removal of Drf1 but not Dbf4 severely inhibits phosphorylation of Mcm4 and DNA replication. After gastrulation, when the cell cycle acquires somatic characteristics, Drf1 levels decline sharply and Cdc7-Dbf4 becomes the more abundant kinase. These results identify Drf1 as a developmentally regulated, essential activator of Cdc7 in Xenopus (Takahishi, 2005).

The data indicate that Cdc7-Drf1 plays an essential role in DNA replication in the early embryonic cell cycles. Given the ability of Drf1 to support DNA replication, it is proposed that DDK stand for 'Dbf4- and Drf1-dependent protein kinase.' The regulatory subunits of Cdc7 appear to provide at least two functions: activation of Cdc7 kinase and localization of Cdc7 to the pre-RC, where the substrate of Cdc7 presumably resides. The results identify Mcm4 as an excellent potential substrate for Cdc7-Drf1 during replication in Xenopus egg extracts, since Mcm4 hyperphosphorylation is Cdc7-Drf1 dependent, and it coincides with the chromatin loading of Cdc7-Drf1. Moreover, Mcm4 hyperphosphorylation immediately precedes Cdc45 loading. Thus, phosphorylation of Mcm4 by Cdc7-Drf1 could explain the requirement for Cdc7-Drf1 in Cdc45 loading. A rigorous test of this model must await identification and mutation of Cdc7-Drf1 phosphorylation sites within Mcm4 (Takahishi, 2005).

These data raise the question of which DDK complex is required for DNA replication in somatic cells. The rise in Cdc7-Dbf4 abundance after gastrulation suggests that this kinase might predominate in somatic cells. However, no quantitative comparisons of Dbf4 and Drf1 expression levels have been reported in specific primary tissues, and in the absence of genetic experiments, it is unclear which DDK stimulates DNA replication after early development. In transformed human tissue culture cells, Dbf4 and Drf1 are much more highly expressed than in most primary tissues, and in HeLa cells, antibody injection experiments indicate that Dbf4 is essential for DNA replication. When Drf1 expression is knocked down by RNAi in U2OS cells, there is a detectable slowing of progression through S phase, consistent with data that Drf1 can function in DNA replication. Although the lack of a more dramatic effect may be due to the relatively inefficient Drf1 knockdown, another interpretation is that Dbf4 is the more important regulatory subunit in transformed cells. Clearly, further work is needed to assess the relative roles of the two DDKs in primary and transformed somatic cells (Takahishi, 2005).

This work suggests that Cdc7-Drf1 and Cdc7-Dbf4 share key characteristics: Both complexes can stimulate DNA replication, both are recruited to pre-RCs via their regulatory subunits, and both are able to phosphorylate Mcm2 in vitro. Moreover, the fact that rCdc7-Dbf4 completely rescues replication in a Drf1-depleted extract suggests that Cdc7-Drf1 is not uniquely capable of driving a rapid embryonic S phase. Why then did Drf1 evolve? One possible difference between the two regulators might involve their response to checkpoints. Etoposide, a topoisomerase II inhibitor, arrests replication initiation and induces dissociation of Dbf4, but not Drf1, from Cdc7. These data suggest that Cdc7-Drf1 is resistant to inhibition by certain cell cycle checkpoints, a property that might be important to establish rapid embryonic cell cycles that are refractory to arrest. An alternative hypothesis is that the two DDKs have evolved to deal with different forms of chromatin structure. Cdc7-Drf1 might be designed to facilitate efficient initiation from the open, nontranscribed chromatin present in the embryo, while Cdc7-Dbf4 may be better suited for the highly compacted chromatin seen in somatic cells (Takahishi, 2005).

Incorporation into the prereplicative complex activates the Mcm2-7 helicase for Cdc7-Dbf4 phosphorylation

The essential S-phase kinase Cdc7-Dbf4 acts at eukaryotic origins of replication to trigger a cascade of protein associations that activate the Mcm2-7 replicative helicase. Also known as Dbf4-dependent kinase (DDK), this kinase preferentially targets chromatin-associated Mcm2-7 complexes that are assembled on the DNA during prereplicative complex (pre-RC) formation. This study addresses the mechanisms that control the specificity of DDK action. Incorporation of Mcm2-7 into the pre-RC increased the level and changes the specificity of DDK phosphorylation of this complex. In the context of the pre-RC, DDK preferentially targets a conformationally distinct subpopulation of Mcm2-7 complexes that is tightly linked to the origin DNA. This targeting requires DDK to tightly associate with Mcm2-7 complexes in a Dbf4-dependent manner. Importantly, it was found that DDK association with and phosphorylation of origin-linked Mcm2-7 complexes require prior phosphorylation of the pre-RC. These findings provide insights into the mechanisms that ensure that DDK action is spatially and temporally restricted to the origin-bound Mcm2-7 complexes that will drive replication fork movement during S phase and suggest new mechanisms to regulate origin activity (Francis, 2009).

Rec8 phosphorylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates cohesin cleavage by separase during meiosis

During meiosis, two rounds of chromosome segregation after a single round of DNA replication produce haploid gametes from diploid precursors. At meiosis I, maternal and paternal kinetochores are pulled toward opposite poles, and chiasmata holding bivalent chromosomes together are resolved by cleavage of cohesin's alpha-kleisin subunit (Rec8) along chromosome arms. This creates dyad chromosomes containing a pair of chromatids joined solely by cohesin at centromeres that had resisted cleavage. The discovery that centromeric Rec8 is protected from separase during meiosis I by shugoshin/MEI-S332 proteins that bind PP2A phosphatase suggests that phosphorylation either of separase or cohesin may be necessary for Rec8 cleavage. This study shows that multiple phosphorylation sites within Rec8 as well as two different kinases, casein kinase 1delta/epsilon (CK1delta/epsilon) and Dbf4-dependent Cdc7 kinase (DDK), are required for Rec8 cleavage and meiosis I nuclear division. Rec8 with phosphomimetic mutations is no longer protected from separase at centromeres and is cleaved even when the two kinases are inhibited. These data suggest that PP2A protects centromeric cohesion by opposing CK1delta/epsilon- and DDK-dependent phosphorylation of Rec8 (Katis, 2010).

A vertebrate homolog of the cell cycle regulator Dbf4 is an inhibitor of Wnt signaling required for heart development

Early stages of vertebrate heart development have been linked to Wnt signaling. XDbf4, a known regulator of Cdc7 kinase, is an inhibitor of the canonical Wnt signaling pathway. Depletion of endogenous XDbf4 protein does not disturb gastrulation movements or early organizer genes but results in embryos with morphologically defective heart and eyes and suppressed cardiac markers. These markers are restored by overexpressed XDbf4, or an XDbf4 mutant that inhibits Wnt signaling but lacks the ability to regulate Cdc7. This indicates that the function of XDbf4 in heart development is independent of its role in the cell cycle. Moreover, the data suggest that XDbf4 acts through the physical and functional interaction with Frodo, a context-dependent regulator of Wnt signaling. These findings establish an unexpected function for a vertebrate Dbf4 homolog and demonstrate the requirement for Wnt inhibition in early cardiac specification (Brott, 2005).

XDbf4 has been shown to bind and activate Cdc7, a kinase required for progression of cells through S phase of the cell cycle. Do observed heart defects in XDbf4-depleted embryos result from the effect of XDbf4 on Wnt signaling or the cell cycle? The XDbf4-ΔMC mutant lacking the domains required for activation of Cdc7 was able to rescue heart marker expression in XDbf4-depleted DMZ explants. Since XDbf4-ΔMC inhibits β-catenin signaling, XDbf4 function in heart development is likely to involve Wnt inhibition and does not require its interaction with Cdc7 (Brott, 2005).

No change in cell size was detected in XDbf4-depleted embryos, indicating no inhibition of cell division. The lack of cell cycle defects could be explained by the presence of Drf1, a second family member that may have a redundant function. Lacking direct evidence, the possibility remains that XDbf4 is required for progenitor cell proliferation, which may be critical for cardiogenesis. For instance, XDbf4 might have a dual function in heart development, repressing canonical Wnt signaling while assisting cardiac precursor division. One precedent is the chromatin-associated helicase Reptin. A mutation of zebrafish Reptin has enhanced Wnt inhibitory activity and leads to overproliferation of ventricular cardiomyocytes. Future studies will explore the connection between the role of XDbf4 in Wnt signaling and cell cycle regulation (Brott, 2005).

Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation

The RNA polymerase II (Pol II) C-terminal domain (CTD) serves as a docking site for numerous proteins, bridging various nuclear processes to transcription. The recruitment of these proteins is mediated by CTD phospho-epitopes generated during transcription. The mechanisms regulating the kinases that establish these phosphorylation patterns on the CTD are not known. This study reports that three CTD kinases, CDK7, CDK9, and BRD4, engage in cross-talk, modulating their subsequent CTD phosphorylation. BRD4 phosphorylates PTEFb/CDK9 at either Thr-29 or Thr-186, depending on its relative abundance, which represses or activates CDK9 CTD kinase activity, respectively. Conversely, CDK9 phosphorylates BRD4 enhancing its CTD kinase activity. The CTD Ser-5 kinase CDK7 also interacts with and phosphorylates BRD4, potently inhibiting BRD4 kinase activity. Additionally, the three kinases regulate each other indirectly through the general transcription factor TAF7. An inhibitor of CDK9 and CDK7 CTD kinase activities, TAF7 also binds to BRD4 and inhibits its kinase activity. Each of these kinases phosphorylates TAF7, affecting its subsequent ability to inhibit the other two. Thus, a complex regulatory network governs Pol II CTD kinases (Devaiah, 2012).

Cyclin-dependent kinase 7: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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