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A precisely positioned MED12 activation helix stimulates CDK8 kinase activity

ABSTRACT: A precisely positioned MED12 activation helix stimulates CDK8 kinase activity

PROVIDER: PRJNA558963 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9910960.fastq.gz	Fastqsanger.gz
	SRR9910961.fastq.gz	Fastqsanger.gz
	SRR9910962.fastq.gz	Fastqsanger.gz
	SRR9910963.fastq.gz	Fastqsanger.gz
	SRR9910964.fastq.gz	Fastqsanger.gz

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Similar Datasets

A precisely positioned MED12 activation helix stimulates CDK8 kinase activity

Project description:The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA Polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, crosslinking coupled to mass spectrometry and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an "activation helix" close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Last, functional assays in presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type-II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but they also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.

2020-01-04 | GSE135458 | GEO

A precisely positioned MED12 activation helix stimulates CDK8 kinase activity

Project description:Chemical cross-linking coupled to mass spectrometry was used to study binary and ternary complexes involving cyclin-dependent kinase 8 (CDK8), cyclin-C, and subunit 12 of the Mediator complex (MED12). Cross-linking was performed using different cross-linking chemistries: (1) disuccinimidyl suberate (DSS); (2) a combination of pimelic acid dihydrazide (PDH) and the coupling reagent, DMTMM.

2020-01-16 | PXD015394 | Pride

Interactions of CDK19 with MED12 and cyclin-C assessed by chemical cross-linking mass spectrometry

Project description:Chemical cross-linking coupled to mass spectrometry was used to study binary and ternary complexes involving cyclin-dependent kinase 19 (CDK19), cyclin-C, and an N-terminal fragment of subunit 12 of the Mediator complex (MED12 1-100). Cross-linking was performed using disuccinimidyl suberate (DSS). These results were generated in the context of the study published as Klatt et al., A precisely positioned MED12 activation helix stimulates CDK8 kinase activity, Proc. Natl. Acad. Sci. USA 2020 (DOI: 10.1073/pnas.1917635117) with the associated data set PXD015394, but were not included in the article.

2020-05-18 | PXD019251 | Pride

A precisely positioned MED12 activation helix stimulates CDK8 kinase activity.

Project description:The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an "activation helix" close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.

| S-EPMC7022176 | biostudies-literature

CDK8 knockdown in HT-29 cells and CDK8 & MED12 knockdown in R1 cells

Project description:This SuperSeries is composed of the following subset Series: GSE30815: CDK8 knockdown in HT-29 human colon cancer cells GSE30816: CDK8 and MED12 knockdown in R1 mouse ES cells Refer to individual Series

2012-03-05 | E-GEOD-30817 | biostudies-arrayexpress

Cdk8, Cyclin C, Med12 or Med13 depletion effect on gene expression in Drosophila S2 cells

Project description:Expression profiling following depletion of Mediator Cdk8 module subunits Cdk8, Cyclin C (CycC), Med12 and Med13 72 hours after dsRNA treatment of Drosophila melanogaster S2 cells. Results provide insight into the role of individual Cdk8 module subunits in regulation of transcription. 22 samples. 2 Cdk8 dsRNA, 4 CycC dsRNA, 4 Med12 dsRNA, 4 Med13 dsRNA, 8 control samples including 4 Luciferase (Luc) dsRNA and 4 GFP dsRNA

2014-04-29 | E-GEOD-52343 | biostudies-arrayexpress

CDK8 and MED12 knockdown in R1 mouse ES cells

2012-03-05 | GSE30816 | GEO

CDK8 and MED12 knockdown in R1 mouse ES cells

Project description:Transcriptional profiling of R1 mouse ES cells infected with non-targeting control (NTC) shRNA and two different shRNA sequences against Cdk8 (shCdk8-1 and shCdk-2) and Med12 (shMed12-1 and shMed12-2). Multi-condition experiment comparing shNTC versus shCdk8-1 and shCdk8-2 after 8 days (3 biological replicates each) and shNTC versus shCdk8-1, shCdk8-2, shMed12-1, and shMed12-2 after 13 days (3 biological replicates each); in total 24 samples profiled.

2012-03-05 | E-GEOD-30816 | biostudies-arrayexpress

Cdk8, Cyclin C, Med12 or Med13 depletion effect on gene expression in Drosophila S2 cells

2014-04-29 | GSE52343 | GEO

Dual function of Med12 in PRC1-dependent gene repression and ncRNA-mediated transcriptional activation

Project description:Mediator is regarded a general co-activator of RNA-Polymerase II dependent transcription but not much is known about its function and regulation in mouse pluripotent embryonic stem cells (mESC). One means of controlling Mediator function is provided by binding of the Cdk8 module (Med12, Cdk8, Ccnc and Med13) to Mediator. Here we report that the Cdk8 module subunit Med12 operates together with PRC1 to silence developmental key genes in the pluripotent state. At the molecular level, PRC1 is required to assemble ncRNA containing Med12-Mediator complexes at promoters of repressed genes. In the course of cellular differentiation the H2A-ubiquitin binding protein Zrf1 abrogates PRC1-Med12 binding and facilitates the recruitment of Cdk8 into Mediator. Remodeling of the Mediator-associated protein complex converts Mediator into a transcriptional enhancer that mediates ncRNA-dependent activation of Polycomb target genes

2016-05-24 | GSE73352 | GEO

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