CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.
- MeSH
- cyklin-dependentní kinasy metabolismus genetika MeSH
- embryo savčí metabolismus MeSH
- embryonální vývoj * genetika MeSH
- fenotyp MeSH
- lebka embryologie patologie MeSH
- mentální retardace genetika MeSH
- modely nemocí na zvířatech * MeSH
- mutace genetika MeSH
- myši MeSH
- nervus trigeminus embryologie MeSH
- neurogeneze * genetika MeSH
- obličej embryologie abnormality MeSH
- protein doublecortin MeSH
- rozštěp patra genetika patologie embryologie MeSH
- rozštěp rtu genetika patologie embryologie MeSH
- vývojová regulace genové exprese * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.
- MeSH
- aktivace enzymů účinky léků MeSH
- chinolony farmakologie MeSH
- chromatin metabolismus MeSH
- cyklin-dependentní kinasy * antagonisté a inhibitory metabolismus MeSH
- fosfoproteiny * chemie metabolismus MeSH
- fosforylace MeSH
- malý jaderný ribonukleoprotein U2 * chemie metabolismus MeSH
- prekurzory RNA * genetika metabolismus MeSH
- sestřih RNA * účinky léků MeSH
- spliceozomy * účinky léků metabolismus MeSH
- threonin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Cycle-regulating and transcriptional cyclin-dependent kinases (CDKs) are attractive targets in cancer drug development. Several CDK inhibitors have already been obtained or are close to regulatory approval for clinical applications. OBJECTIVE: Phenylazopyrazole CAN508 has been described as the first selective CDK9 inhibitor with an IC50 of 350 nM. Since the azo-moiety is not a suitable functionality for drugs due to pharmacological reasons, the preparation of carbo-analogues of CAN508 with similar biological activities is desirable. The present work is focused on the synthesis of carbo-analogues similar to CAN508 and their CDK inhibition activity. METHODS: Herein, the synthesis of 21 novel carbo analogues of CAN508 and their intermediates is reported. Subsequently, target compounds 8a - 8u were evaluated for protein kinase inhibition (CDK2/cyclin E, CDK4/cyclin D, CDK9/cyclin T) and antiproliferative activities in cell lines (K562, MCF-7, MV4-11). Moreover, the binding mode of derivative 8s in the active site of CDK9 was modelled. RESULTS: Compounds 8a - 8u were obtained from key intermediate 7, which was prepared by linear synthesis involving Vilsmeier-Haack, Knoevenagel, Hunsdiecker, and Suzuki-Miyaura reactions. Styrylpyrazoles 8t and 8u were the most potent CDK9 inhibitors with IC50 values of approximately 1 μM. Molecular modelling suggested binding in the active site of CDK9. The flow cytometric analysis of MV4-11 cells treated with the most active styrylpyrazoles showed a significant G1-arrest. CONCLUSION: The prepared styrylpyrazoles showed inhibition activity towards CDKs and can provide a novel chemotype of kinase inhibitors.
Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle in eukaryotes. Assessing their activity is one of the basic methods used to analyze their function. This is particularly true in synchronized cultures of unicellular organisms, where the entire culture is in the same physiological state. In this chapter, I describe a simple biochemical method to assess CDK activity in algae. Although the results are easier to interpret in the context of synchronized cultures, the method is not limited to them. The protocol requires only standard laboratory equipment and access to a radioactivity working room. The method is applicable to any algal species, including newly developed ones, as it does not require any specific tools. The method can, therefore, be used to widen the portfolio of cell cycle regulatory models within algae.
To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.
- MeSH
- buněčný cyklus fyziologie MeSH
- cyklin-dependentní kinasy genetika metabolismus MeSH
- fosfoproteiny chemie metabolismus MeSH
- fosforylace MeSH
- glutamáty metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- izotopové značení metody MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- peptidy metabolismus MeSH
- peroxiredoxin VI chemie metabolismus MeSH
- proteolýza * MeSH
- proteom genetika metabolismus MeSH
- proteomika metody MeSH
- sekvence aminokyselin MeSH
- sestřihové faktory chemie metabolismus MeSH
- signální transdukce genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Replication-dependent histones (RDH) are required for packaging of newly synthetized DNA into nucleosomes during the S phase when their expression is highly upregulated. However, the mechanisms of this upregulation in metazoan cells remain poorly understood. Using iCLIP and ChIP-seq, we found that human cyclin-dependent kinase 11 (CDK11) associates with RNA and chromatin of RDH genes primarily in the S phase. Moreover, its amino-terminal region binds FLASH, an RDH-specific 3'-end processing factor, which keeps the kinase on the chromatin. CDK11 phosphorylates serine 2 (Ser2) of the carboxy-terminal domain of RNA polymerase II (RNAPII), which is initiated when RNAPII reaches the middle of RDH genes and is required for further RNAPII elongation and 3'-end processing. CDK11 depletion leads to decreased number of cells in S phase, likely owing to the function of CDK11 in RDH gene expression. Thus, the reliance of RDH expression on CDK11 could explain why CDK11 is essential for the growth of many cancers.
- MeSH
- chromatin genetika metabolismus MeSH
- cyklin-dependentní kinasy genetika metabolismus MeSH
- fosforylace MeSH
- genetická transkripce * MeSH
- histony genetika metabolismus MeSH
- lidé MeSH
- proteiny regulující apoptózu genetika metabolismus MeSH
- proteiny vázající vápník genetika metabolismus MeSH
- regulace genové exprese MeSH
- replikace DNA MeSH
- RNA genetika metabolismus MeSH
- S fáze MeSH
- serin metabolismus MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Temperature is one of the key factors affecting growth and division of algal cells. High temperature inhibits the cell cycle in Chlamydomonas reinhardtii. At 39 °C, nuclear and cellular divisions in synchronized cultures were blocked completely, while DNA replication was partly affected. In contrast, growth (cell volume, dry matter, total protein, and RNA) remained unaffected, and starch accumulated at very high levels. The cell cycle arrest could be removed by transfer to 30 °C, but a full recovery occurred only in cultures cultivated up to 14 h at 39 °C. Thereafter, individual cell cycle processes began to be affected in sequence; daughter cell release, cell division, and DNA replication. Cell cycle arrest was accompanied by high mitotic cyclindependent kinase activity that decreased after completion of nuclear and cellular division following transfer to 30 °C. Cell cycle arrest was, therefore, not caused by a lack of cyclin-dependent kinase activity but rather a blockage in downstream processes.
- MeSH
- bílkoviny řas metabolismus MeSH
- buněčné kultury metody MeSH
- Chlamydomonas reinhardtii cytologie fyziologie MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- down regulace MeSH
- fyziologický stres MeSH
- kontrolní body buněčného cyklu * MeSH
- regulace genové exprese u rostlin MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog-sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA-seq and ChIP-seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3'ends of predominantly long, poly(A)-signal-rich genes. CDK12 inhibition does not globally reduce levels of RNAPII-Ser2 phosphorylation. However, individual CDK12-dependent genes show a shift of P-Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.
- MeSH
- cyklin-dependentní kinasy genetika metabolismus MeSH
- fosforylace MeSH
- HCT116 buňky MeSH
- kontrolní body fáze G1 buněčného cyklu genetika fyziologie MeSH
- lidé MeSH
- oprava DNA genetika fyziologie MeSH
- replikace DNA genetika fyziologie MeSH
- RNA-polymerasa II genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA damage is a ubiquitous threat endangering DNA integrity in all living organisms. Responses to DNA damage include, among others, induction of DNA repair and blocking of cell cycle progression in order to prevent transmission of damaged DNA to daughter cells. Here, we tested the effect of the antibiotic zeocin, inducing double stranded DNA breaks, on the cell cycle of synchronized cultures of the green alga Chlamydomonas reinhardtii. After zeocin application, DNA replication partially occurred but nuclear and cellular divisions were completely blocked. Application of zeocin combined with caffeine, known to alleviate DNA checkpoints, decreased cell viability significantly. This was probably caused by a partial overcoming of the cell cycle progression block in such cells, leading to aberrant cell divisions. The cell cycle block was accompanied by high steady state levels of mitotic cyclin-dependent kinase activity. The data indicate that DNA damage response in C. reinhardtii is connected to the cell cycle block, accompanied by increased and stabilized mitotic cyclin-dependent kinase activity.
- MeSH
- bleomycin toxicita MeSH
- Chlamydomonas reinhardtii účinky léků genetika MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- cytostatické látky toxicita MeSH
- DNA rostlinná účinky léků MeSH
- dvouřetězcové zlomy DNA MeSH
- kofein farmakologie MeSH
- kontrolní body buněčného cyklu MeSH
- mutageny toxicita MeSH
- replikace DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21-/-, RPE p21-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.
- MeSH
- antitumorózní látky farmakologie terapeutické užití MeSH
- checkpoint kinasa 1 antagonisté a inhibitory MeSH
- cyklin-dependentní kinasy antagonisté a inhibitory metabolismus MeSH
- fosforylace účinky léků MeSH
- HCT116 buňky MeSH
- inhibitor p21 cyklin-dependentní kinasy genetika metabolismus MeSH
- kontrolní body fáze S buněčného cyklu účinky léků MeSH
- lidé MeSH
- malá interferující RNA genetika MeSH
- nádory farmakoterapie metabolismus MeSH
- poškození DNA účinky léků genetika MeSH
- proteiny buněčného cyklu antagonisté a inhibitory MeSH
- pyrazoly farmakologie terapeutické užití MeSH
- pyrimidinony farmakologie terapeutické užití MeSH
- transfekce MeSH
- tyrosinkinasy antagonisté a inhibitory MeSH
- viabilita buněk účinky léků genetika účinky záření MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
