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Biochemical characterization, regulation, and inhibition of human transcription kinases CDK12 and CDK13 and human cell cycle-related kinase CDK14

dc.contributor.advisorGeyer, Matthias
dc.contributor.authorDust, Sofia
dc.date.accessioned2020-04-27T01:11:02Z
dc.date.available2020-12-01T23:00:17Z
dc.date.issued27.11.2019
dc.identifier.urihttp://hdl.handle.net/20.500.11811/8116
dc.description.abstractThe transcription process is the first step in the molecular information flow from genome to proteome. In eukaryotes, the RNA polymerase II (RNAPII) transcribes genes that code for proteins by synthesizing pre-mature messenger RNAs (mRNAs). The Cyclin-dependent kinases CDK12 and CDK13 in association with Cyclin K are transcription regulating kinases by modulating the function of RNAPII. Both CDK/Cyclin pairs regulate transcriptional elongation as well as processes occurring co-transcriptionally through phosphorylation of the C-terminal domain (CTD) of RNAPII. They selectively affect the expression of genes involved in DNA damage response (DDR) and mRNA processing, respectively. CDK12 and CDK13 are involved in numerous types of cancer, representing potential targets for novel cancer treatments. Small molecule inhibitors that selectively target CDK12 and CDK13 in an ATP-competitive manner are used to investigate the consequences of their inhibition in healthy cells and cancer cells.
In this thesis, using a protein structure-based approach, dinaciclib, a small molecule inhibitor of CDKs 1, 2 and 5, was identified as a potent inhibitor of CDK12 and CDK13. In comparison to flavopiridol, a known transcription-regulating CDK inhibitor, inhibition of CDK12 by dinaciclib was significantly more potent than by flavopiridol. By performing mutagenesis experiments of residues in the C-terminal extension helix of CDK12, we show that the histidine residue of the DCHEL motif causes a sterical hindrance that may reduce the inhibitory activity of flavopiridol. We also conclude that dinaciclib may readily accommodate its pyridine ring at the histidine binding site of the kinase leading to highly potent inhibition of CDK12 and CDK13. This might be achieved by a displacement mode of binding rather than by performing a base stacking interaction with the histidine imidazole ring. In a further approach, non-covalent reversible and covalent irreversible ATP-competitive small molecule inhibitors were characterized at different ATP concentrations and pre-incubation times. The result indicates that the potency of irreversible inhibitors cannot be easily characterized by conventional IC50-values as the kinetics of the covalent interaction involves a time dependency. In order to understand how small molecule compounds selectively target the kinase hinge region in CDK12, we determined the three-dimensional complex structure of CDK12/CycK co-crystallized with an irreversible ATP-competitive small molecule inhibitor at 2.6 Ångstrom resolution.
In a validation study, our findings derived from in vitro kinase activity assays and peptide mass finger print analyses confirmed that CDK12 and CDK13 in complex with Cyclin K directly phosphorylate the pre-mRNA processing proteins CDC5L, SF3B1, CSTF64 and SPT6H. The validation of pre-mRNA processing factors as new CDK12 and CDK13 substrates sheds light on the diverse regulatory function of the kinases in the process of co-transcriptional RNA processing. Structural and biochemical analysis of melanoma-associated CDK13 mutations supported the assumption that mutations near the kinase ATP-binding site may represent the leading cause for the development of cutaneous melanoma by inactivating the catalytic center of the kinase. The zincfinger containing protein ZC3H14, which was supposed as a CDK13 interacting protein in human melanoma cells, could not be confirmed as a CDK13/CycK phosphorylation substrate in our kinase assay. However, using CDK9 in complex with Cyclin T1, phosphorylation activity was present against ZC3H14. This suggests that in melanoma cells, Cyclin T1 may be required to generate an active CDK13 kinase complex. This assumption is further supported by the fact that Cyclin T1 was identified as the cyclin binding partner for CDK13 in human melanoma and was demonstrated to be required for the CDK13-mediated melanomagenesis.
Besides the CDKs that regulate progression through the cell cycle and different stages of the transcription process, there is a third CDK subfamily consisting of the understudied kinases CDK14-CDK18. Human CDK14 belongs to the family of cell division cycle 2 (CDC2)-related kinases and is associated with Cyclin Y, which contains an N-terminal myristoylation signal for plasma membrane targeting. The kinase complex of CDK14/CycY is implicated in the Wnt/ß-catenin signaling pathway by phosphorylation of the transmembrane receptor protein involved in Wnt signaling. The present structural and biochemical analysis of the human CDK14/CycY kinase complex identifies 14-3-3 proteins as CDK14/CycY binding proteins which are essential for the kinase activity. Our data provides a possible new mechanistic insight into the activation of CDK14 by proposing a phosphorylation-dependent regulatory interplay between CDK14, Cyclin Y, and 14-3-3 proteins that enhances the kinase-cyclin association and thus facilitates the kinase activity.
en
dc.language.isoeng
dc.rightsIn Copyright
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/
dc.subjectTranskription
dc.subjectGenetik
dc.subjectCyclin-abhängige Kinasen
dc.subjectPhosphorylierung
dc.subjectRNS-Polymerase II
dc.subjectRegulation
dc.subjectInhibitor
dc.subject.ddc500 Naturwissenschaften
dc.titleBiochemical characterization, regulation, and inhibition of human transcription kinases CDK12 and CDK13 and human cell cycle-related kinase CDK14
dc.typeDissertation oder Habilitation
dc.publisher.nameUniversitäts- und Landesbibliothek Bonn
dc.publisher.locationBonn
dc.rights.accessRightsopenAccess
dc.identifier.urnhttps://nbn-resolving.org/urn:nbn:de:hbz:5-56641
ulbbn.pubtypeErstveröffentlichung
ulbbnediss.affiliation.nameRheinische Friedrich-Wilhelms-Universität Bonn
ulbbnediss.affiliation.locationBonn
ulbbnediss.thesis.levelDissertation
ulbbnediss.dissID5664
ulbbnediss.date.accepted2019-11-13
ulbbnediss.instituteMedizinische Fakultät / Institute : Institut für Angeborene Immunität
ulbbnediss.fakultaetMathematisch-Naturwissenschaftliche Fakultät
dc.contributor.coRefereeImhof, Diana


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