Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics

Larochelle, Stéphane; Batliner, Jasmin; Gamble, Matthew J.; Barboza, Nora M.; Kraybill, Brian; Blethrow, Justin D.; Shokat, Kevan M.; Fisher, Robert P

doi:10.1038/nsmb1028

Cited by 94 publications

(128 citation statements)

References 54 publications

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“…It remains to be investigated if this protein is a transcription factor and whether Pfmrk plays a role in the regulation of gene expression during the P. falciparum life cycle. It is not surprising that Pfmrk may have a role in gene expression as CDK7 and orthologs regulate gene expression in association with the basal transcription RNA Pol II TFIIH complex [16,47]. Additionally, we identified an association of Pfmrk with regulators of DNA replication, PfRFC-5 and PfMCM6.…”

Section: Discussionmentioning

confidence: 78%

The malarial CDK Pfmrk and its effector PfMAT1 phosphorylate DNA replication proteins and co-localize in the nucleus

Jirage

Chen

Caridha

et al. 2010

Molecular and Biochemical Parasitology

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a b s t r a c tCyclin-dependent kinases (CDKs) have an established role in metazoans and yeast in DNA replication, transcription and cell cycle regulation. Several CDKs and their effectors have been identified in the malaria parasite Plasmodium falciparum and their biological functions are beginning to be investigated. Here we report results from the functional characterization of Pfmrk and its effector PfMAT1. We validated the interactions between Pfmrk and PfMAT1 and pinpointed their intracellular location. Co-immunoprecipitation studies demonstrated physical interaction between the two proteins and identified the C-terminal domain of PfMAT1 as the Pfmrk activator domain. Immunofluorescence analyses using GFP and RFP-tagged versions of Pfmrk and PfMAT1, respectively, demonstrated the co-localization of these two proteins to the parasite nucleus. Bacterial two-hybrid screen of a P. falciparum cDNA library using Pfmrk as the bait identified two plasmodial DNA replication proteins, PfRFC-5 and PfMCM6, as interactors with Pfmrk. We demonstrate that that these two proteins are substrates of Pfmrk-mediated phosphorylation and that PfMAT1 confers substrate specificity to the Pfmrk kinase complex. Collectively, these data suggest a role for Pfmrk in the nucleus of the parasite presumably in regulation of the DNA replication machinery.Published by Elsevier B.V.

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Section: Discussionmentioning

confidence: 78%

The malarial CDK Pfmrk and its effector PfMAT1 phosphorylate DNA replication proteins and co-localize in the nucleus

Jirage

Chen

Caridha

et al. 2010

Molecular and Biochemical Parasitology

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“…Therefore, we characterized the ability of forms of PDK1 with mutations in the ATP binding pocket to be inhibited by purine based inhibitors containing bulky groups. This chemical genetic approach has been used for several kinases to identify substrates, for example with JNK [36], ERK2 [37] and Cdk7 [38].…”

Section: Discussionmentioning

confidence: 99%

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

Tamgüney

Zhang

Fiedler

et al. 2008

Experimental Cell Research

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Abstract3-Phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several kinases in the cAMP-dependent, cGMP-dependent and protein kinase C(AGC) family. Many putative PDK1 substrates have been identified, but have not been analyzed following transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the ATP binding pocket of wild type (WT) kinases. When expressed in PDK1 −/− ES cells, PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 optimally inhibited the phosphorylation of PDK1 targets in PDK1 −/− ES cells expressing PDK1 L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, but revealed distinct dephosphorylation kinetics. While PDK1 inhibition had little effect on cell growth, it sensitized cells to apoptotic stimuli. Furthermore, PDK1 loss abolished growth of allograft tumors. Taken together we describe a model system that allows for acute and reversible inhibition of PDK1 in cells, to probe biochemical and biological consequences.

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“…However, multiple so-called transcriptional CDKs (CDK7, -8, -9, and -11) (to which CDK10 may belong; Fig. S11) have been shown to phosphorylate a variety of motifs in a non-proline-directed fashion, especially in the context of molecular docking with the substrate (20). Here, it can be hypothesized that the high-affinity interaction between CDK10 and the Pointed domain of ETS2 (6,9) (Fig.…”

Section: Discussionmentioning

confidence: 99%

CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome

Guen

Gamble

Flajolet

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

104

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Cyclin-dependent kinases (CDKs) regulate a variety of fundamental cellular processes. CDK10 stands out as one of the last orphan CDKs for which no activating cyclin has been identified and no kinase activity revealed. Previous work has shown that CDK10 silencing increases ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2)-driven activation of the MAPK pathway, which confers tamoxifen resistance to breast cancer cells. The precise mechanisms by which CDK10 modulates ETS2 activity, and more generally the functions of CDK10, remain elusive. Here we demonstrate that CDK10 is a cyclin-dependent kinase by identifying cyclin M as an activating cyclin. Cyclin M, an orphan cyclin, is the product of FAM58A, whose mutations cause STAR syndrome, a human developmental anomaly whose features include toe syndactyly, telecanthus, and anogenital and renal malformations. We show that STAR syndrome-associated cyclin M mutants are unable to interact with CDK10. Cyclin M silencing phenocopies CDK10 silencing in increasing c-Raf and in conferring tamoxifen resistance to breast cancer cells. CDK10/cyclin M phosphorylates ETS2 in vitro, and in cells it positively controls ETS2 degradation by the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased cyclin M levels. Altogether, our results reveal an additional regulatory mechanism for ETS2, which plays key roles in cancer and development. They also shed light on the molecular mechanisms underlying STAR syndrome.C yclin-dependent kinases (CDKs) play a pivotal role in the control of a number of fundamental cellular processes (1). The human genome contains 21 genes encoding proteins that can be considered as members of the CDK family owing to their sequence similarity with bona fide CDKs, those known to be activated by cyclins (2). Although discovered almost 20 y ago (3, 4), CDK10 remains one of the two CDKs without an identified cyclin partner. This knowledge gap has largely impeded the exploration of its biological functions. CDK10 can act as a positive cell cycle regulator in some cells (5, 6) or as a tumor suppressor in others (7,8). CDK10 interacts with the ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2) transcription factor and inhibits its transcriptional activity through an unknown mechanism (9). CDK10 knockdown derepresses ETS2, which increases the expression of the c-Raf protein kinase, activates the MAPK pathway, and induces resistance of MCF7 cells to tamoxifen (6).Here, we deorphanize CDK10 by identifying cyclin M, the product of FAM58A, as a binding partner. Mutations in this gene that predict absence or truncation of cyclin M are associated with STAR syndrome, whose features include toe syndactyly, telecanthus, and anogenital and renal malformations in heterozygous females (10). However, both the functions of cyclin M and the pathogenesis of STAR syndrome remain unknown. We show that a recombinant CDK10/cyclin M heterodimer is an active protein kinase that phosphorylates ETS2 in v...

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Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics

Cited by 94 publications

References 54 publications

The malarial CDK Pfmrk and its effector PfMAT1 phosphorylate DNA replication proteins and co-localize in the nucleus

The malarial CDK Pfmrk and its effector PfMAT1 phosphorylate DNA replication proteins and co-localize in the nucleus

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome

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