The Experts below are selected from a list of 78321 Experts worldwide ranked by ideXlab platform

Robert P Fisher - One of the best experts on this subject based on the ideXlab platform.

  • Cdk7: a Kinase at the core of transcription and in the crosshairs of cancer drug discovery.
    Transcription, 2018
    Co-Authors: Robert P Fisher
    Abstract:

    The transcription cycle of RNA polymerase II (Pol II) is regulated by a set of cyclin-dependent Kinases (CDKs). Cdk7, associated with the transcription initiation factor TFIIH, is both an effector CDK that phosphorylates Pol II and other targets within the transcriptional machinery, and a CDK-activating Kinase (CAK) for at least one other essential CDK involved in transcription. Recent studies have illuminated Cdk7 functions that are executed throughout the Pol II transcription cycle, from promoter clearance and promoter-proximal pausing, to co-transcriptional chromatin modification in gene bodies, to mRNA 3´-end formation and termination. Cdk7 has also emerged as a target of small-molecule inhibitors that show promise in the treatment of cancer and inflammation. The challenges now are to identify the relevant targets of Cdk7 at each step of the transcription cycle, and to understand how heightened dependence on an essential CDK emerges in cancer, and might be exploited therapeutically.

  • The CDK-activating Kinase Cdk7: taking yes for an answer.
    Cell cycle (Georgetown Tex.), 2013
    Co-Authors: Miriam Merzel Schachter, Robert P Fisher
    Abstract:

    The cyclin-dependent Kinases (CDKs) that control cell division require activation (T) loop phosphorylation for full activity. In metazoans, the only known CDK-activating Kinase (CAK) is the Cdk7 complex, which was discovered ~20 y ago and subsequently implicated in CDK activation in vivo in flies and worms. Cdk7 has another essential function as part of transcription factor IIH, to phosphorylate RNA polymerase (RNAP) II and other transcription factors. These two seemingly disparate roles, and the existence of divergent, non-cyclin-dependent CAKs in yeast, initially raised doubts about whether Cdk7 is a CAK in vivo, but no other metazoan enzyme capable of activating CDKs has been identified.1 Conclusive evidence that human Cdk7 is a bona fide CAK emerged from chemical genetics—expanding the ATP binding pocket to accommodate bulky adenine analogs that inhibit the resulting analog-sensitive (AS) Cdk7, but not any wild-type Kinase. Selective inhibition of Cdk7, in HCT116 colon cancer cells in which wild-type Cdk7 was replaced with Cdk7as, blocked activation of Cdk1 and Cdk2 and caused arrest at both G1/S and G2/M transitions.2 Cdk7 supports distinct activation pathways for the 2 CDKs, despite their structural similarities; in the case of Cdk1, cyclin binding and T-loop phosphorylation must occur in concert, whereas Cdk2 can be phosphorylated as a monomer.3 This difference helps ensure activation of Cdk2 before Cdk1, and might obviate the need for a separate CAK that prefers monomeric substrates—a feature of CDK networks in yeast. Moreover, different activation mechanisms might explain why previous studies detected effects of Cdk7 impairment on Cdk1 but not Cdk2.1 Still to be identified was a CAK for Cdk4 and Cdk6, which control cell cycle commitment by phosphorylating the retinoblastoma tumor suppressor protein Rb at the restriction point. It had been suggested that Cdk4 was activated by another CAK, based on instances in which Cdk4 T-loop phosphorylation fluctuates while Cdk7 activity appears constant, and on structural differences between the Cdk4 T loop and those of other CDKs.4 We showed, however, that Cdk7 is responsible for activation of Cdk4 and Cdk6 through another distinct pathway.5 Cdk2 and Cdk1 remain phosphorylated for several hours after Cdk7 inactivation, even though their activation de novo is blocked.2,3 In contrast, Cdk4 and Cdk6 lose activity rapidly upon Cdk7 inhibition in human cells.5 Differential susceptibility to T-loop dephosphorylation, due to structural differences between Cdk4 and Cdk2 complexes, might place greater demand on a Cdk4-activating Kinase to overcome antagonism by phosphatases and provide an opportunity for regulation during G1. Consistent with this scenario, activation of Cdk4 (but not Cdk2) in vitro was stimulated by phosphorylation of Cdk7’s own T loop. In vivo, Cdk7 T-loop phosphorylation increased during G1 when quiescent cells were stimulated by mitogens—the first evidence that changes in CAK activity might regulate a key cell cycle transition. Nonetheless, doubts (and doubters) persist. Another study in Cdk7as HCT116 cells showed that Cdk7 inhibition caused rapid inactivation of Cdk4 and Cdk6 (in agreement with our results) but loss of Cdk4 T-loop phosphorylation only in the population bound to the CDK inhibitor p21.6 This was taken as evidence that another CAK works on p21-free Cdk4, even though it cannot support Cdk4 activity or cell cycle progression in Cdk7as/as cells treated with allele-specific inhibitors. Moreover, there was no demonstration that p21-free Cdk4 became phosphorylated when Cdk7 was inactive; it might simply remain so, possibly due to different rates of dephosphorylation in distinct Kinase sub-populations, for which Cdk2 provides a precedent.3 In another study, conditional disruption of Cdk7 in mouse embryonic fibroblasts (MEFs) blocked cell division and activation of Cdk1, Cdk2, Cdk4, and Cdk6. Proliferation and CDK T-loop phosphorylation were maintained in Cdk7mut/mut MEFs, however, when an SV40 large T antigen fragment was expressed to inactivate the pocket proteins Rb, p107 and p130.7 This seems to challenge the “single-CAK theory” and provide evidence for a cryptic, Cdk7-independent CDK activation pathway normally suppressed by Rb. This pathway remains hypothetical, however, until the responsible activity is detected and proven not to be due to the residual Cdk7 complexes in extracts of Cdk7mut/mut MEFs rescued by T antigen (or by “CAK-bypass” variants of Cdk1 or Cdk2).7 Is Cdk7 the major CAK in metazoans? The answer, from recent chemical-genetic and knockout studies, as well as older “classical” genetics, is an unequivocal “yes,” which is corroborated by demonstrations that removal or chemical inhibition of Cdk7 abolishes CAK activity of whole-cell extracts.1,8 Cdk7 is a common activator of cell cycle CDKs, but its CAK function is not restricted to the cell cycle machinery: one of its targets, Cdk9,8 is part of positive transcription elongation factor b, which regulates elongation and maturation of RNAP II transcripts. Whether Cdk7 is the only CAK is likely to remain an open question. In the 2 decades since its discovery, however, there have been several, ultimately unsubstantiated challenges to Cdk7’s position as the major CAK in vivo. Despite differences in interpretation, the 3 recent studies discussed here do not differ on a key point: inactivating Cdk7, either by chemical inhibition5,6 or gene disruption,7 causes general failure of activating phosphorylation affecting G1, S phase, and mitotic CDKs. For now then, Cdk7 is the only CAK we know, and novel insights continue to emerge from studies that focus on how it activates different CDKs at the right time and place.

  • A Cdk7-Cdk4 T-Loop Phosphorylation Cascade Promotes G1 Progression
    Molecular cell, 2013
    Co-Authors: Miriam Merzel Schachter, Stephane Larochelle, Karl A. Merrick, Alexander Hirschi, Chao Zhang, Kevan M. Shokat, Seth M. Rubin, Robert P Fisher
    Abstract:

    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.

  • The CDK-activating Kinase (CAK) Csk1 is required for normal levels of homologous recombination and resistance to DNA damage in fission yeast.
    PloS one, 2008
    Co-Authors: Hilary B. Gerber, Yana Pikman, Robert P Fisher
    Abstract:

    Background Cyclin-dependent Kinases (CDKs) perform essential roles in cell division and gene expression in all eukaryotes. The requirement for an upstream CDK-activating Kinase (CAK) is also universally conserved, but the fission yeast Schizosaccharomyces pombe appears to be unique in having two CAKs with both overlapping and specialized functions that can be dissected genetically. The Mcs6 complex—orthologous to metazoan Cdk7/cyclin H/Mat1—activates the cell-cycle CDK, Cdk1, but its non-redundant essential function appears to be in regulation of gene expression, as part of transcription factor TFIIH. The other CAK is Csk1, an ortholog of budding yeast Cak1, which activates all three essential CDKs in S. pombe—Cdk1, Mcs6 and Cdk9, the catalytic subunit of positive transcription elongation factor b (P-TEFb)—but is not itself essential. Methodology/Principal Findings Cells lacking csk1+ are viable but hypersensitive to agents that damage DNA or block replication. Csk1 is required for normal levels of homologous recombination (HR), and interacts genetically with components of the HR pathway. Tests of damage sensitivity in csk1, mcs6 and cdk9 mutants indicate that Csk1 acts pleiotropically, through Cdk9 and at least one other target (but not through Mcs6) to preserve genomic integrity. Conclusions/Significance The two CAKs in fission yeast, which differ with respect to their substrate range and preferences for monomeric CDKs versus CDK/cyclin complexes as substrates, also support different functions of the CDK network in vivo. Csk1 plays a non-redundant role in safeguarding genomic integrity. We propose that specialized activation pathways dependent on different CAKs might insulate CDK functions important in DNA damage responses from those capable of triggering mitosis.

  • requirements for cdk7 in the assembly of cdk1 cyclin b and activation of cdk2 revealed by chemical genetics in human cells
    Molecular Cell, 2007
    Co-Authors: Stephane Larochelle, Karl A. Merrick, Chao Zhang, Kevan M. Shokat, Lara Wohlbold, Nora M Barboza, Marieemilie Terret, Prasad V Jallepalli, Robert P Fisher
    Abstract:

    Cell division is controlled by cyclin-dependent Kinases (CDKs). In metazoans, S phase onset coincides with activation of Cdk2, whereas Cdk1 triggers mitosis. Both Cdk1 and -2 require cyclin binding and T loop phosphorylation for full activity. The only known CDK-activating Kinase (CAK) in metazoans is Cdk7, which is also part of the transcription machinery. To test the requirements for Cdk7 in vivo, we replaced wild-type Cdk7 with a version sensitive to bulky ATP analogs in human cancer cells. Selective inhibition of Cdk7 in G1 prevents activation (but not formation) of Cdk2/cyclin complexes and delays S phase. Inhibiting Cdk7 in G2 blocks entry to mitosis and disrupts Cdk1/cyclin B complex assembly, indicating that the two steps of Cdk1 activation-cyclin binding and T loop phosphorylation-are mutually dependent. Therefore, by combining chemical genetics and homologous gene replacement in somatic cells, we reveal different modes of CDK activation by Cdk7 at two distinct execution points in the cell cycle.

Jean Marc Egly - One of the best experts on this subject based on the ideXlab platform.

  • In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair.
    Nature Communications, 2020
    Co-Authors: Stefan Peissert, Arnaud Poterszman, Jean Marc Egly, Jochen Kuper, Florian Sauer, Daniel Grabarczyk, Cathy Braun, Gudrun Sander, Caroline Kisker
    Abstract:

    The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating Kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction. More importantly, mutagenesis of the Arch domain revealed that these residues are essential for the regulation of (i) NER activity by either impairing XPD helicase activity or the interaction of XPD with XPG; (ii) the phosphorylation of the RNA polymerase II and RNA synthesis. Our results reveal how MAT1 shields these functionally important residues thereby providing insights into how XPD is regulated by MAT1 and defining the Arch domain as a major mechanistic player within the XPD scaffold.

  • A history of TFIIH: Two decades of molecular biology on a pivotal transcription/repair factor
    DNA repair, 2011
    Co-Authors: Jean Marc Egly, Frédéric Coin
    Abstract:

    The TFIIH multiprotein complex is organized into a 7-subunit core associated with a 3-subunit CDK-activating Kinase module (CAK). Three enzymatic subunits are present in TFIIH, two ATP-dependent DNA helicases: XPB and XPD, and the Kinase Cdk7. Mutations in three of the subunits, XPB, XPD and TTDA, lead to three distinct genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD) predisposing patients not only to cancer and ageing but also to developmental and neurological defects. These heterogeneous phenotypes originate from the dual role of TFIIH in transcription and DNA repair. For twenty years, many molecular studies have been conducted with the aim to unveil the role of TFIIH in DNA repair and transcription as well as the origin of the phenotypes of patients. This review intends to give a non-exhaustive survey of the most prominent discoveries on the molecular functioning of TFIIH.

  • Nucleotide excision repair driven by the dissociation of CAK from TFIIH.
    Molecular Cell, 2008
    Co-Authors: Frédéric Coin, Valentyn Oksenych, Vincent Mocquet, Stefanie Groh, Christine Blattner, Jean Marc Egly
    Abstract:

    The transcription/DNA repair factor TFIIH is organized into a core that associates with the CDK-activating Kinase (CAK) complex. Using chromatin immunoprecipitation, we have followed the composition of TFIIH over time after UV irradiation of repair-proficient or -deficient human cells. We show that TFIIH changes subunit composition in response to DNA damage. The CAK is released from the core during nucleotide excision repair (NER). Using reconstituted in vitro NER assay, we show that XPA catalyzes the detachment of the CAK from the core, together with the arrival of the other NER-specific factors. The release of the CAK from the core TFIIH promotes the incision/excision of the damaged oligonucleotide and thereby the repair of the DNA. Following repair, the CAK reappears with the core TFIIH on the chromatin, together with the resumption of transcription. Our findings demonstrate that the composition of TFIIH is dynamic to adapt its engagement in distinct cellular processes.

  • A yeast four-hybrid system identifies CDK-activating Kinase as a regulator of the XPD helicase, a subunit of transcription factor IIH.
    The Journal of biological chemistry, 2001
    Co-Authors: Björn Sandrock, Jean Marc Egly
    Abstract:

    Abstract 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.

  • Distinct Regions of MAT1 Regulate cdk7 Kinase and TFIIH Transcription Activities
    Journal of Biological Chemistry, 2000
    Co-Authors: Didier Busso, Arnaud Poterszman, Anne Keriel, Björn Sandrock, Opher Gileadi, Jean Marc Egly
    Abstract:

    Abstract 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·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.

Alain Devault - One of the best experts on this subject based on the ideXlab platform.

  • Is Cdk7/cyclin H/MAT1 the genuine cdk activating Kinase in cycling xenopus egg extracts?
    Oncogene, 1997
    Co-Authors: Didier Fesquet, Marcel Dorée, Nathalie Morin, Alain Devault
    Abstract:

    : 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 (Menage a 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.

  • is cdk7 cyclin h mat1 the genuine cdk activating Kinase in cycling xenopus egg extracts
    Oncogene, 1997
    Co-Authors: Didier Fesquet, Marcel Dorée, Nathalie Morin, Alain Devault
    Abstract:

    : 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 (Menage a 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.

  • p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate CDK-activating Kinase activity in Xenopus oocytes.
    The EMBO journal, 1994
    Co-Authors: Jean-claude Labbé, Alain Devault, Didier Fesquet, A.m. Martinez, Jean-paul Capony, J.-m. Darbon, Jean Derancourt, N. Morin, Jean-claude Cavadore, Marcel Dorée
    Abstract:

    p40MO15, a cdc2-related protein, is the catalytic subunit of the Kinase (CAK, CDK-activating Kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified Kinase) as features of p40MO15 that are required for the generation of active CAK.

  • Cloning, expression and subcellular localization of the human homolog of p40MO15 catalytic subunit of CDK-activating Kinase.
    Oncogene, 1994
    Co-Authors: J.-m. Darbon, Alain Devault, Didier Fesquet, A.m. Martinez, Jean-claude Cavadore, Taviaux S, Simon Galas, Marcel Dorée
    Abstract:

    Transitions of the cell cycle are controlled by cyclin-dependent protein Kinases (cdks) whose phosphorylation on the Thr residue included in the conserved sequence YTHEVV dramatically increases the activity. A Kinase responsible for this specific phosphorylation, called CAK for CDK-activating Kinase, has been recently purified from starfish and Xenopus oocytes and shown to contain the MO15 gene product as a catalytic subunit. In the present paper, we have cloned the human homolog of Xenopus p40MO15 by probing a HeLa cell cDNA library with degenerate oligonucleotides deduced from Xenopus and starfish MO15 sequences. Human and Xenopus MO15 displayed a strong homology showing 86% identity with regard to amino acid sequences. Northern blot analysis of RNA extracts from a series of human tissues as well as from cultured rodent fibroblasts revealed a unique 1.4 kb MO15 mRNA. No variation in the amount of MO15 transcript or protein was found along the entire course of the fibroblast cell cycle. Fluorescence in situ hybridization on human lymphocyte metaphases showed two distinct chromosomal locations of human MO15 gene at 5q12-q13 and 2q22-q24. By using gene tagging and mammalian cell transfection, we demonstrate that the KRKR motif located at the carboxy terminal end of MO15 is required for nuclear targeting of the protein. Mutation of KRKR to NGER retains MO15 in the cytoplasmic compartment, whilst the wild-type protein is detected exclusively in the nucleus. Interestingly, we demonstrate that the nuclear targeting of MO15 is necessary to confer the protein its CAK activity. In contrast to the wild-type, the NLS-mutated MO15 expressed in Xenopus oocytes is unable to generate CAK as long as the nuclear envelope is not broken. The nuclear localization of both the MO15 gene product and CAK activity may imply that cdks activation primarily occurs in the cell nucleus.

David O Morgan - One of the best experts on this subject based on the ideXlab platform.

  • The HIV transactivator TAT binds to the CDK-activating Kinase and activates the phosphorylation of the carboxy-terminal domain of RNA polymerase II
    Genes & development, 1997
    Co-Authors: Thomas P. Cujec, Holly M Chamberlin, David O Morgan, Okamoto H, Koh Fujinaga, J. Meyer, B M Peterlin
    Abstract:

    The human immunodeficiency virus encodes the transcriptional transactivator Tat, which binds to the transactivation response (TAR) RNA stem–loop in the viral long terminal repeat (LTR) and increases rates of elongation rather than initiation of transcription by RNA polymerase II (Pol II). In this study, we demonstrate that Tat binds directly to the cyclin-dependent Kinase 7 (CDK7), which leads to productive interactions between Tat and the CDK-activating Kinase (CAK) complex and between Tat and TFIIH. Tat activates the phosphorylation of the carboxy-terminal domain (CTD) of Pol II by CAK in vitro. The ability of CAK to phosphorylate the CTD can be inhibited specifically by a CDK7 pseudosubstrate peptide that also inhibits transcriptional activation by Tat in vitro and in vivo. We conclude that the phosphorylation of the CTD by CAK is essential for Tat transactivation. Our data identify a cellular protein that interacts with the activation domain of Tat, demonstrate that this interaction is critical for the function of Tat, and provide a mechanism by which Tat increases the processivity of Pol II.

  • three dimensional structure of human cyclin h a positive regulator of the cdk activating Kinase
    Nature Structural & Molecular Biology, 1996
    Co-Authors: Holly M Chamberlin, David O Morgan
    Abstract:

    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 α-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 cyclin-dependent Kinase-activating Kinase (CAK) in budding yeast unrelated to vertebrate CAK.
    Science, 1996
    Co-Authors: F. Hernan Espinoza, Alison Farrell, Hediye Erdjument-bromage, Paul Tempst, David O Morgan
    Abstract:

    Progress through the cell cycle is governed by the cyclin-dependent Kinases (CDKs), the activation of which requires phosphorylation by the CDK-activating Kinase (CAK). In vertebrates, CAK is a trimeric enzyme containing CDK7, cyclin H, and MAT1. CAK from the budding yeast Saccharomyces cerevisiae was identified as an unusual 44-kilodalton protein Kinase, Cak1, that is only distantly related to CDKs. Cak1 accounted for most CAK activity in yeast cell lysates, and its activity was constant throughout the cell cycle. The CAK1 gene was essential for cell viability. Thus, the major CAK in S. cerevisiae is distinct from the vertebrate enzyme, suggesting that budding yeast and vertebrates may have evolved different mechanisms of CDK activation.

  • Alternative mechanisms of CAK assembly require an assembly factor or an Activating Kinase
    Cell, 1995
    Co-Authors: Robert P Fisher, Holly M Chamberlin, David O Morgan
    Abstract:

    Abstract We have cloned a mouse cDNA that encodes p36, a novel subunit of the CDK-activating Kinase (CAK). p36 contains a C 3 HC 4 zinc-binding domain or RING finger 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.

  • CDK-activating Kinase complex is a component of human transcription factor TFIIH
    Nature, 1995
    Co-Authors: Ramin Shiekhattar, David O Morgan, Robert P Fisher, Fred Mermelstein, Brian David Dynlacht, Holly C. Wessling, Ronny Drapkin, Danny Reinberg
    Abstract:

    TRANSCRIPTION factor IIH (TFIIH) contains a Kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII)1–3. 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.

Marcel Dorée - One of the best experts on this subject based on the ideXlab platform.

  • Is Cdk7/cyclin H/MAT1 the genuine cdk activating Kinase in cycling xenopus egg extracts?
    Oncogene, 1997
    Co-Authors: Didier Fesquet, Marcel Dorée, Nathalie Morin, Alain Devault
    Abstract:

    : 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 (Menage a 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.

  • is cdk7 cyclin h mat1 the genuine cdk activating Kinase in cycling xenopus egg extracts
    Oncogene, 1997
    Co-Authors: Didier Fesquet, Marcel Dorée, Nathalie Morin, Alain Devault
    Abstract:

    : 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 (Menage a 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.

  • p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate CDK-activating Kinase activity in Xenopus oocytes.
    The EMBO journal, 1994
    Co-Authors: Jean-claude Labbé, Alain Devault, Didier Fesquet, A.m. Martinez, Jean-paul Capony, J.-m. Darbon, Jean Derancourt, N. Morin, Jean-claude Cavadore, Marcel Dorée
    Abstract:

    p40MO15, a cdc2-related protein, is the catalytic subunit of the Kinase (CAK, CDK-activating Kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified Kinase) as features of p40MO15 that are required for the generation of active CAK.

  • Cloning, expression and subcellular localization of the human homolog of p40MO15 catalytic subunit of CDK-activating Kinase.
    Oncogene, 1994
    Co-Authors: J.-m. Darbon, Alain Devault, Didier Fesquet, A.m. Martinez, Jean-claude Cavadore, Taviaux S, Simon Galas, Marcel Dorée
    Abstract:

    Transitions of the cell cycle are controlled by cyclin-dependent protein Kinases (cdks) whose phosphorylation on the Thr residue included in the conserved sequence YTHEVV dramatically increases the activity. A Kinase responsible for this specific phosphorylation, called CAK for CDK-activating Kinase, has been recently purified from starfish and Xenopus oocytes and shown to contain the MO15 gene product as a catalytic subunit. In the present paper, we have cloned the human homolog of Xenopus p40MO15 by probing a HeLa cell cDNA library with degenerate oligonucleotides deduced from Xenopus and starfish MO15 sequences. Human and Xenopus MO15 displayed a strong homology showing 86% identity with regard to amino acid sequences. Northern blot analysis of RNA extracts from a series of human tissues as well as from cultured rodent fibroblasts revealed a unique 1.4 kb MO15 mRNA. No variation in the amount of MO15 transcript or protein was found along the entire course of the fibroblast cell cycle. Fluorescence in situ hybridization on human lymphocyte metaphases showed two distinct chromosomal locations of human MO15 gene at 5q12-q13 and 2q22-q24. By using gene tagging and mammalian cell transfection, we demonstrate that the KRKR motif located at the carboxy terminal end of MO15 is required for nuclear targeting of the protein. Mutation of KRKR to NGER retains MO15 in the cytoplasmic compartment, whilst the wild-type protein is detected exclusively in the nucleus. Interestingly, we demonstrate that the nuclear targeting of MO15 is necessary to confer the protein its CAK activity. In contrast to the wild-type, the NLS-mutated MO15 expressed in Xenopus oocytes is unable to generate CAK as long as the nuclear envelope is not broken. The nuclear localization of both the MO15 gene product and CAK activity may imply that cdks activation primarily occurs in the cell nucleus.