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A precisely positioned MED12 activation helix stimulates CDK8 kinase activity.
Proc Natl Acad Sci U S A. 2020 02 11; 117(6):2894-2905.PN

Abstract

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.

Authors+Show Affiliations

Gene Regulation by Non-Coding RNA, Elite Network of Bavaria and University of Bayreuth, 95447 Bayreuth, Germany.Department of Biology, Institute of Molecular Systems Biology, Eidgenössiche Technische Hochschule Zürich, 8093 Zürich, Switzerland.Gene Regulation by Non-Coding RNA, Elite Network of Bavaria and University of Bayreuth, 95447 Bayreuth, Germany.Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany.Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. Proteros Biostructures GmbH, 82152 Martinsried, Germany.Gene Regulation by Non-Coding RNA, Elite Network of Bavaria and University of Bayreuth, 95447 Bayreuth, Germany.Gene Regulation by Non-Coding RNA, Elite Network of Bavaria and University of Bayreuth, 95447 Bayreuth, Germany.Gene Regulation by Non-Coding RNA, Elite Network of Bavaria and University of Bayreuth, 95447 Bayreuth, Germany.Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; huber@biochem.mpg.de claus.kuhn@uni-bayreuth.de. Center of Medical Biotechnology, University of Duisburg-Essen, 45117 Essen, Germany. Department of Chemistry, Technical University of Munich, 85747 Garching, Germany.Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany.Gene Regulation by Non-Coding RNA, Elite Network of Bavaria and University of Bayreuth, 95447 Bayreuth, Germany; huber@biochem.mpg.de claus.kuhn@uni-bayreuth.de.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

31988137

Citation

Klatt, Felix, et al. "A Precisely Positioned MED12 Activation Helix Stimulates CDK8 Kinase Activity." Proceedings of the National Academy of Sciences of the United States of America, vol. 117, no. 6, 2020, pp. 2894-2905.
Klatt F, Leitner A, Kim IV, et al. A precisely positioned MED12 activation helix stimulates CDK8 kinase activity. Proc Natl Acad Sci USA. 2020;117(6):2894-2905.
Klatt, F., Leitner, A., Kim, I. V., Ho-Xuan, H., Schneider, E. V., Langhammer, F., Weinmann, R., Müller, M. R., Huber, R., Meister, G., & Kuhn, C. D. (2020). A precisely positioned MED12 activation helix stimulates CDK8 kinase activity. Proceedings of the National Academy of Sciences of the United States of America, 117(6), 2894-2905. https://doi.org/10.1073/pnas.1917635117
Klatt F, et al. A Precisely Positioned MED12 Activation Helix Stimulates CDK8 Kinase Activity. Proc Natl Acad Sci USA. 2020 02 11;117(6):2894-2905. PubMed PMID: 31988137.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A precisely positioned MED12 activation helix stimulates CDK8 kinase activity. AU - Klatt,Felix, AU - Leitner,Alexander, AU - Kim,Iana V, AU - Ho-Xuan,Hung, AU - Schneider,Elisabeth V, AU - Langhammer,Franziska, AU - Weinmann,Robin, AU - Müller,Melanie R, AU - Huber,Robert, AU - Meister,Gunter, AU - Kuhn,Claus-D, Y1 - 2020/01/27/ PY - 2020/1/29/pubmed PY - 2020/5/28/medline PY - 2020/1/29/entrez KW - CDK8 KW - MED12 KW - Mediator KW - Mediator kinase module KW - kinase inhibitors SP - 2894 EP - 2905 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 6 N2 - 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. SN - 1091-6490 UR - https://www.unboundmedicine.com/medline/citation/31988137/A_precisely_positioned_MED12_activation_helix_stimulates_CDK8_kinase_activity_ L2 - http://www.pnas.org/cgi/pmidlookup?view=long&pmid=31988137 DB - PRIME DP - Unbound Medicine ER -