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1.
Int J Mol Sci ; 25(10)2024 May 15.
Article in English | MEDLINE | ID: mdl-38791449

ABSTRACT

Dysregulation of cyclin-dependent kinase 8 (CDK8) activity has been associated with many diseases, including colorectal and breast cancer. As usual in the CDK family, the activity of CDK8 is controlled by a regulatory protein called cyclin C (CycC). But, while human CDK family members are generally activated in two steps, that is, the binding of the cyclin to CDK and the phosphorylation of a residue in the CDK activation loop, CDK8 does not require the phosphorylation step to be active. Another peculiarity of CDK8 is its ability to be associated with CycC while adopting an inactive form. These specificities raise the question of the role of CycC in the complex CDK8-CycC, which appears to be more complex than the other members of the CDK family. Through molecular dynamics (MD) simulations and binding free energy calculations, we investigated the effect of CycC on the structure and dynamics of CDK8. In a second step, we particularly focused our investigation on the structural and molecular basis of the protein-protein interaction between the two partners by finely analyzing the energetic contribution of residues and simulating the transition between the active and the inactive form. We found that CycC has a stabilizing effect on CDK8, and we identified specific interaction hotspots within its interaction surface compared to other human CDK/Cyc pairs. Targeting these specific interaction hotspots could be a promising approach in terms of specificity to effectively disrupt the interaction between CDK8. The simulation of the conformational transition from the inactive to the active form of CDK8 suggests that the residue Glu99 of CycC is involved in the orientation of three conserved arginines of CDK8. Thus, this residue may assume the role of the missing phosphorylation step in the activation mechanism of CDK8. In a more general view, these results point to the importance of keeping the CycC in computational studies when studying the human CDK8 protein in both the active and the inactive form.


Subject(s)
Cyclin C , Cyclin-Dependent Kinase 8 , Molecular Dynamics Simulation , Protein Binding , Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinase 8/chemistry , Cyclin C/metabolism , Cyclin C/chemistry , Humans , Phosphorylation , Thermodynamics , Binding Sites
2.
Pharmacol Rep ; 76(3): 535-556, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38602606

ABSTRACT

BACKGROUND: Genetic alterations are well characterized as contributors to the pathogenesis of cancers. Epigenetic abnormalities can lead to perturbations of the expression of genes in cancer cells without structural defects. Deregulation of proteins of the transcription machinery may result in perturbations of target genes. Mediator, a multiprotein component of the transcription machinery facilitates the function of RNA polymerase II, which transcribes most human genes. A part of the mediator with kinase activity, called the Mediator kinase module shows genetic alterations in a sub-set of colorectal cancers. METHODS: Data from publicly available genomic series of colorectal cancer patients were examined to determine alterations of Mediator kinase module component genes, including MED12, MED12L, MED13, MED13L, CDK8, CDK19, and CCNC. The prevalence of alterations in genomically defined colorectal cancer sub-sets was also interrogated. The effect of Mediator kinase module member gene expression on colorectal cancer relapse-free survival was investigated. RESULTS: Mutations in genes of the Mediator kinase module were present in a small percentage of colorectal cancers, ranging between 2 to 10% for MED12 and MED13 and alternative units MED12L and MED13L and below 2% for kinases CDK8 and CDK19 and cyclin C. Amplifications of the CDK8 gene were observed in 3% to 5% of colorectal cancers. The highest prevalence of mutations was observed in MSI cancers and the equivalent CMS1 group, with other genomic groups showing much lower frequency. An association of higher expression of MED12 with inferior relapse-free survival was observed. In contrast, higher expression of cyclin C was associated with improved survival. Colorectal cancer cell lines with CDK8 amplifications displayed sensitivity to several small molecule inhibitors of the KRAS/PI3K pathway but not to BET inhibitors. CONCLUSION: The Mediator kinase module is deregulated in a sub-set of colorectal cancers with differences observed in genomically defined groups. These variations may result in differences in sensitivity to targeted therapies and may have to be taken into consideration as such therapies are developed.


Subject(s)
Colorectal Neoplasms , Cyclin C , Cyclin-Dependent Kinase 8 , Cyclin-Dependent Kinases , Gene Expression Regulation, Neoplastic , Mediator Complex , Mutation , Humans , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Mediator Complex/genetics , Cyclin-Dependent Kinase 8/genetics , Cyclin-Dependent Kinases/genetics , Cyclin C/genetics
3.
J Med Chem ; 66(7): 4932-4951, 2023 04 13.
Article in English | MEDLINE | ID: mdl-36930701

ABSTRACT

The CDK8-cyclin C complex is an important anti-tumor target, but unlike CDK8, cyclin C remains undruggable. Modulators regulating cyclin C activity directly are still under development. Here, a series of hydrophobic tagging-based degraders of the CDK8-cyclin C complex were designed, synthesized, and evaluated to identify the first dual degrader, LL-K8-22, which induced selective and synchronous degradation of CDK8 and cyclin C. Proteomic and immunoblot studies exhibited that LL-K8-22 significantly degraded CDK8 without reducing CDK19 and did not degrade other cyclin proteins except cyclin C. Moreover, LL-K8-22 showed enhanced anti-proliferative effects over its parental molecule, BI-1347, with potency increased by 5-fold in MDA-MB-468 cells. LL-K8-22 exhibited more pronounced effects on CDK8-cyclin C downstream signaling than BI-1347, suppressing STAT1 phosphorylation more persistently. RNA-sequencing analysis revealed that LL-K8-22 inhibited E2F- and MYC-driven carcinogenic transcriptional programs. Overall, LL-K8-22 is the first-in-class degrader of cyclin C and would be useful for studying the unknown functions of cyclin C.


Subject(s)
Cyclin C , Cyclin-Dependent Kinases , Cyclin C/metabolism , Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , Phosphorylation , Proteomics , Signal Transduction
4.
Science ; 378(6620): eabn5647, 2022 Nov 11.
Article in English | MEDLINE | ID: mdl-36356142

ABSTRACT

T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC, components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor-engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12-deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers-most notably for STAT and AP-1 transcription factors-and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.


Subject(s)
Cyclin C , Mediator Complex , Neoplasms , Receptors, Chimeric Antigen , T-Lymphocytes , Humans , Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , Mediator Complex/genetics , T-Lymphocytes/immunology , Transcription Factors/genetics , Genome-Wide Association Study , Cyclin C/genetics , Genetic Testing , Immunotherapy, Adoptive , Neoplasms/immunology , Neoplasms/therapy
5.
Mol Metab ; 64: 101548, 2022 10.
Article in English | MEDLINE | ID: mdl-35863637

ABSTRACT

OBJECTIVE: Cyclin C (CCNC) is the most conserved subunit of the Mediator complex, which is an important transcription cofactor. Recently, we have found that CCNC facilitates brown adipogenesis in vitro by activating C/EBPα-dependent transcription. However, the role of CCNC in brown adipose tissue (BAT) in vivo remains unclear. METHODS: We generated conditional knock-out mice by crossing Ccncflox/flox mice with Myf5Cre, Ucp1Cre or AdipoqCre transgenic mice to investigate the role of CCNC in BAT development and function. We applied glucose and insulin tolerance test, cold exposure and indirect calorimetry to capture the physiological phenotypes and used immunostaining, immunoblotting, qRT-PCR, RNA-seq and cell culture to elucidate the underlying mechanisms. RESULTS: Here, we show that deletion of CCNC in Myf5+ progenitor cells caused BAT paucity, despite the fact that there was significant neonatal lethality. Mechanistically different from in vitro, CCNC deficiency impaired the proliferation of embryonic brown fat progenitor cells without affecting brown adipogenesis or cell death. Interestingly, CCNC deficiency robustly reduced age-dependent lipid accumulation in differentiated brown adipocytes in all three mouse models. Mechanistically, CCNC in brown adipocytes is required for lipogenic gene expression through the activation of the C/EBPα/GLUT4/ChREBP axis. Consistent with the importance of de novo lipogenesis under carbohydrate-rich diets, high-fat diet (HFD) feeding abolished CCNC deficiency -caused defects of lipid accumulation in BAT. Although insulin sensitivity and response to acute cold exposure were not affected, CCNC deficiency in Ucp1+ cells enhanced the browning of white adipose tissue (beiging) upon prolonged cold exposure. CONCLUSIONS: Together, these data indicate an important role of CCNC-Mediator in the regulation of BAT development and lipid accumulation in brown adipocytes.


Subject(s)
Adipocytes, Brown , Cyclin C , Adipocytes, Brown/metabolism , Adipose Tissue, Brown/metabolism , Adipose Tissue, White/metabolism , Animals , Cyclin C/metabolism , Lipids , Mice , Mice, Knockout , Mice, Transgenic
6.
Clin Transl Med ; 12(4): e833, 2022 04.
Article in English | MEDLINE | ID: mdl-35475325

ABSTRACT

The resistance to cisplatin-based chemotherapy is a common cause of poor prognosis in cancer patients. Cisplatin stimulation causes cyclin C translocating to mitochondria, and in turn induces mitochondrial fission. However, little is known about the role of cyclin C in mitochondrial dysfunction in cancer cells challenged with cisplatin. In the present commentary, we bring to the attention of readers the recent report by Jiang et al which revealed the importance of ubiquitylation and translocation of cyclin C in gastric cancer cells in response to cisplatin stimulation for mitochondrial stability. This finding provides new insights into exploring the novel mechanisms of chemoresistance and developing the new chemotherapy synergistic agents in the era of precision oncology.


Subject(s)
Antineoplastic Agents , Cyclin C , Stomach Neoplasms , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis , Cisplatin/pharmacology , Cisplatin/therapeutic use , Drug Resistance, Neoplasm , Humans , Precision Medicine , Stomach Neoplasms/drug therapy , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology
7.
Clin Transl Med ; 12(3): e770, 2022 03.
Article in English | MEDLINE | ID: mdl-35343092

ABSTRACT

BACKGROUND: Cyclin C (CCNC) was reported to take part in regulating mitochondria-derived oxidative stress under cisplatin stimulation. However, its effect in gastric cancer is unknown. This study aimed to investigate the role of cyclin C and its ubiquitylation in regulating cisplatin resistance in gastric cancer. METHODS: The interaction between HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1 (HACE1) and cyclin C was investigated by GST pull-down assay, co-immunoprecipitation and ubiquitylation assay. Mitochondria-derived oxidative stress was studied by MitoSOX Red assay, seahorse assay and mitochondrial membrane potential measurement. Cyclin C-associated cisplatin resistance was studied in vivo via xenograft. RESULTS: HACE1 catalysed the ubiquitylation of cyclin C by adding Lys11-linked ubiquitin chains when cyclin C translocates to cytoplasm induced by cisplatin treatment. The ubiquitin-modified cyclin C then anchor at mitochondira, which induced mitochondrial fission and ROS synthesis. Depleting CCNC or mutation on the ubiquitylation sites decreased mitochondrial ROS production and reduced cell apoptosis under cisplatin treatment. Xenograft study showed that disrupting cyclin C ubiquitylation by HACE1 conferred impairing cell apoptosis response upon cisplatin administration. CONCLUSIONS: Cyclin C is a newly identified substrate of HACE1 E3 ligase. HACE1-mediated ubiquitylation of cyclin C sheds light on a better understanding of cisplatin-associated resistance in gastric cancer patients. Ubiquitylation of cyclin C by HACE1 regulates cisplatin-associated sensitivity in gastric cancer. With cisplatin-induced nuclear-mitochondrial translocation of cyclin C, its ubiquitylation by HACE1 increased mitochondrial fission and mitochondrial-derived oxidative stress, leading to cell apoptosis.


Subject(s)
Cisplatin , Stomach Neoplasms , Cisplatin/pharmacology , Cyclin C/genetics , Humans , Stomach Neoplasms/drug therapy , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
8.
Nutrients ; 13(8)2021 Jul 21.
Article in English | MEDLINE | ID: mdl-34444642

ABSTRACT

Fatty acids play a significant role in maintaining cellular and DNA protection and we previously found an inverse relationship between blood levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and DNA damage. The aim of this study was to explore differences in proteomic profiles, for 117 pro-inflammatory proteins, in two previously defined groups of individuals with different DNA damage and EPA and DHA levels. Healthy children and adolescents (n = 140) aged 9 to 13 years old in an urban area of Brazil were divided by k-means cluster test into two clusters of DNA damage (tail intensity) using the comet assay (cluster 1 = 5.9% ± 1.2 and cluster 2 = 13.8% ± 3.1) in our previous study. The cluster with higher DNA damage and lower levels of DHA (6.2 ± 1.6 mg/dL; 5.4 ± 1.3 mg/dL, p = 0.003) and EPA (0.6 ± 0.2 mg/dL; 0.5 ± 0.1 mg/dL, p < 0.001) presented increased expression of the proteins CDK8-CCNC, PIK3CA-PIK3R1, KYNU, and PRKCB, which are involved in pro-inflammatory pathways. Our findings support the hypothesis that low levels of n-3 long-chain PUFA may have a less protective role against DNA damage through expression of pro-inflammatory proteins, such as CDK8-CCNC, PIK3CA-PIK3R1, KYNU, and PRKCB.


Subject(s)
DNA Damage , Docosahexaenoic Acids/blood , Eicosapentaenoic Acid/blood , Fatty Acids, Omega-3/blood , Adolescent , Brazil , Child , Class I Phosphatidylinositol 3-Kinases/blood , Class Ia Phosphatidylinositol 3-Kinase/blood , Cross-Sectional Studies , Cyclin C/blood , Cyclin-Dependent Kinase 8/blood , Female , Humans , Hydrolases/blood , Inflammation/metabolism , Male , Protein Kinase C beta/blood , Proteomics
9.
Nucleic Acids Res ; 49(13): 7476-7491, 2021 07 21.
Article in English | MEDLINE | ID: mdl-34197614

ABSTRACT

Poly (ADP-ribose) polymerase inhibitor (PARPi)-based therapies initially reduce tumor burden but eventually lead to acquired resistance in cancer patients with BRCA1 or BRCA2 mutation. To understand the potential PARPi resistance mechanisms, we performed whole-genome CRISPR screens to discover genetic alterations that change the gene essentiality in cells with inducible depletion of BRCA2. We identified that several RNA Polymerase II transcription Mediator complex components, especially Cyclin C (CCNC) as synthetic survival targets upon BRCA2 loss. Total mRNA sequencing demonstrated that loss of CCNC could activate the transforming growth factor (TGF)-beta signaling pathway and extracellular matrix (ECM)-receptor interaction pathway, however the inhibition of these pathways could not reverse cell survival in BRCA2 depleted CCNC-knockout cells, indicating that the activation of these pathways is not required for the resistance. Moreover, we showed that the improved survival is not due to restoration of homologous recombination repair although decreased DNA damage signaling was observed. Interestingly, loss of CCNC could restore replication fork stability in BRCA2 deficient cells, which may contribute to PARPi resistance. Taken together, our data reveal CCNC as a critical genetic determinant upon BRCA2 loss of function, which may help the development of novel therapeutic strategies that overcome PARPi resistance.


Subject(s)
BRCA2 Protein/genetics , Cyclin C/genetics , BRCA2 Protein/metabolism , CRISPR-Cas Systems , Cell Survival , DNA Damage , DNA Replication , Gene Expression Regulation , Gene Knockout Techniques , HEK293 Cells , Humans , Mediator Complex/genetics , Mediator Complex/physiology , Recombinational DNA Repair , Stress, Physiological/genetics
10.
Nucleic Acids Res ; 49(15): 8665-8683, 2021 09 07.
Article in English | MEDLINE | ID: mdl-34329458

ABSTRACT

The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR-Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.


Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , Cyclin C/genetics , Cyclin-Dependent Kinase 8/genetics , Transcription, Genetic , Animals , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Cell Line, Tumor , DNA Damage/drug effects , DNA Repair/drug effects , DNA Replication/drug effects , Humans , Mice , Mouse Embryonic Stem Cells/drug effects , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects
11.
Biomater Sci ; 9(4): 1325-1333, 2021 Feb 23.
Article in English | MEDLINE | ID: mdl-33355563

ABSTRACT

Cell membrane-based nanoparticles have garnered increasing attention owing to their inherent biomimetic properties, such as homotypic targeting, prolong circulation, and immune escaping mechanisms. However, most of these biomimetic nanoparticles appear as an orientated core-shell unit because of the lack of the full utilization and direction control of membranes. Different from those single-unit delivery systems, we reported a multiple-unit nanocluster by randomly reuniting multiple PAMAM polymeric core units into a single nanocluster via simple electrostatic interactions between 4T1 cell membrane fragments and PAMAM. Similar to tumor cell clusters, the doxorubicin (DOX)-loaded nanoclusters (CCNCs) could actively metastasis towards cancer cells after getting access to the systemic circulation due to their specific homotypic targeting ability. In this study, CCNCs showed significantly higher tumor inhibition efficacy in 4T1 tumor-bearing mice compared with that of free DOX and PAMAM@DOX-treated groups. Furthermore, the quantitative analysis showed that the number of pulmonary metastatic nodules remarkably reduced, indicating the potential anti-metastasis effect of CCNCs. Overall, these tumor cell membrane fragment reunited PAMAM polymer units could disguise as tumor cell clusters for encouraging tumor homotypic targeting and anti-metastasis treatment.


Subject(s)
Nanoparticles , Neoplasms , Animals , Cell Line, Tumor , Cell Membrane , Cyclin C , Doxorubicin , Drug Delivery Systems , Mice , Polymers
12.
Biochem Genet ; 59(1): 114-133, 2021 Feb.
Article in English | MEDLINE | ID: mdl-32780225

ABSTRACT

The full-length cDNA of cyclin C of the giant tiger shrimp Penaeus monodon (PmCyC) was isolated by RACE-PCR. It was 1443 bp in length containing an open reading frame (ORF) of 804 bp and 267 deduced amino acids. Tissue distribution analysis indicated that PmCyC was more abundantly expressed in ovaries and testes than other tissues of female and male juveniles (P < 0.05). A pair of primers was designed, and an amplification product of 403 bp containing an intron of 123 bp was obtained. Polymorphism of amplified PmCyC gene segments of the 5th (3-month-old G5, N = 30) and 7th (5-month-old G7, N = 18) generations of domesticated juveniles was analyzed. Four conserved SNPs (T>C134, T>C188, G>A379, and T>C382) were found within the examined sequences. A TaqMan genotyping assay was developed for detection of a T>C134 SNP. Association analysis indicated that this SNP displayed significant association with body weight (P < 4.2e-10) and total length (P < 2e-09) of the examined G7 P. monodon (N = 419) with an allele substitution effect of 5.02 ± 0.78 g and 1.41 ± 0.19 cm, respectively. Juveniles with C/C134 (22.80 ± 2.51 g and 12.97 ± 0.53 cm, N = 19) and T/C134 (20.41 ± 0.93 g and 12.77 ± 0.21 cm, N = 129) genotypes exhibited a significantly greater average body weight and total length than those with a T/T134 genotype (14.72 ± 0.53 g and 11.37 ± 0.13 cm, N = 271) (P < 0.05).


Subject(s)
Cyclin C/genetics , Penaeidae/genetics , Polymorphism, Single Nucleotide , Animals , DNA, Complementary/metabolism , Female , Genotype , Introns , Male , Open Reading Frames , Ovary/metabolism , Penaeidae/growth & development , Phylogeny , Polymerase Chain Reaction , Sequence Analysis, DNA , Testis/metabolism , Tissue Distribution
13.
J Biol Chem ; 295(48): 16280-16291, 2020 11 27.
Article in English | MEDLINE | ID: mdl-32934007

ABSTRACT

The Cdk8 kinase module (CKM) is a detachable Mediator subunit composed of cyclin C and one each of paralogs Cdk8/Cdk19, Med12/Med12L, and Med13/Med13L. Our previous RNA-Seq studies demonstrated that cyclin C represses a subset of hydrogen peroxide-induced genes under normal conditions but is involved in activating other loci following stress. Here, we show that cyclin C directs this transcriptional reprograming through changes in its promoter occupancy. Following peroxide stress, cyclin C promoter occupancy increased for genes it activates while decreasing at loci it represses under normal conditions. Promoter occupancy of other CKM components generally mirrored cyclin C, indicating that the CKM moves as a single unit. It has previously been shown that some cyclin C leaves the nucleus following cytotoxic stress to induce mitochondrial fragmentation and apoptosis. We observed that CKM integrity appeared compromised at a subset of repressed promoters, suggesting a source of cyclin C that is targeted for nuclear release. Interestingly, mTOR inhibition induced a new pattern of cyclin C promoter occupancy indicating that this control is fine-tuned to the individual stress. Using inhibitors, we found that Cdk8 kinase activity is not required for CKM movement or repression but was necessary for full gene activation. In conclusion, this study revealed that different stress stimuli elicit specific changes in CKM promoter occupancy correlating to altered transcriptional outputs. Finally, although CKM components were recruited or expelled from promoters as a unit, heterogeneity was observed at individual promoters, suggesting a mechanism to generate gene- and stress-specific responses.


Subject(s)
Cell Nucleus/metabolism , Cyclin C/metabolism , Mitochondria/metabolism , Oxidative Stress , Promoter Regions, Genetic , Transcription, Genetic , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/genetics , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Transformed , Cell Nucleus/genetics , Cyclin C/genetics , Cyclin-Dependent Kinase 8/genetics , Cyclin-Dependent Kinase 8/metabolism , Hydrogen Peroxide/pharmacology , Mice , Mice, Knockout , Mitochondria/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
14.
PLoS Genet ; 16(5): e1008832, 2020 05.
Article in English | MEDLINE | ID: mdl-32463833

ABSTRACT

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFß, or Transforming Growth Factor-ß) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFß signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.


Subject(s)
Cyclin C/genetics , Cyclin-Dependent Kinase 8/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Transcription Factors/metabolism , Animals , Cyclin C/metabolism , Cyclin-Dependent Kinase 8/metabolism , Drosophila , Gene Expression Regulation, Developmental , Haploinsufficiency , Imaginal Discs/growth & development , Imaginal Discs/metabolism , Signal Transduction , Transcription, Genetic
15.
J Am Heart Assoc ; 9(7): e014366, 2020 04 07.
Article in English | MEDLINE | ID: mdl-32248761

ABSTRACT

Background Nuclear-to-mitochondrial communication regulating gene expression and mitochondrial function is a critical process following cardiac ischemic injury. In this study, we determined that cyclin C, a component of the Mediator complex, regulates cardiac and mitochondrial function in part by modifying mitochondrial fission. We tested the hypothesis that cyclin C functions as a transcriptional cofactor in the nucleus and a signaling molecule stimulating mitochondrial fission in response to stimuli such as cardiac ischemia. Methods and Results We utilized gain- and loss-of-function mouse models in which the CCNC (cyclin C) gene was constitutively expressed (transgenic, CycC cTg) or deleted (knockout, CycC cKO) in cardiomyocytes. The knockout and transgenic mice exhibited decreased cardiac function and altered mitochondria morphology. The hearts of knockout mice had enlarged mitochondria with increased length and area, whereas mitochondria from the hearts of transgenic mice were significantly smaller, demonstrating a role for cyclin C in regulating mitochondrial dynamics in vivo. Hearts from knockout mice displayed altered gene transcription and metabolic function, suggesting that cyclin C is essential for maintaining normal cardiac function. In vitro and in vivo studies revealed that cyclin C translocates to the cytoplasm, enhancing mitochondria fission following stress. We demonstrated that cyclin C interacts with Cdk1 (cyclin-dependent kinase 1) in vivo following ischemia/reperfusion injury and that, consequently, pretreatment with a Cdk1 inhibitor results in reduced mitochondrial fission. This finding suggests a potential therapeutic target to regulate mitochondrial dynamics in response to stress. Conclusions Our study revealed that cyclin C acts as a nuclear-to-mitochondrial signaling factor that regulates both cardiac hypertrophic gene expression and mitochondrial fission. This finding provides new insights into the regulation of cardiac energy metabolism following acute ischemic injury.


Subject(s)
Cyclin C/metabolism , Energy Metabolism , Mitochondria, Heart/metabolism , Mitochondrial Dynamics , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/metabolism , Animals , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/metabolism , Cells, Cultured , Cyclin C/deficiency , Cyclin C/genetics , Disease Models, Animal , Energy Metabolism/drug effects , Humans , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/drug effects , Mitochondria, Heart/pathology , Mitochondrial Dynamics/drug effects , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/prevention & control , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Protein Kinase Inhibitors/pharmacology , Protein Transport , Rats, Wistar , Signal Transduction
16.
Mol Biol Cell ; 31(10): 1015-1031, 2020 05 01.
Article in English | MEDLINE | ID: mdl-32160104

ABSTRACT

Environmental stress elicits well-orchestrated programs that either restore cellular homeostasis or induce cell death depending on the insult. Nutrient starvation triggers the autophagic pathway that requires the induction of several Autophagy (ATG) genes. Cyclin C-cyclin-dependent kinase (Cdk8) is a component of the RNA polymerase II Mediator complex that predominantly represses the transcription of stress-responsive genes in yeast. To relieve this repression following oxidative stress, cyclin C translocates to the mitochondria where it induces organelle fragmentation and promotes cell death prior to its destruction by the ubiquitin-proteasome system (UPS). Here we report that cyclin C-Cdk8, together with the Ume6-Rpd3 histone deacetylase complex, represses the essential autophagy gene ATG8. Similar to oxidative stress, cyclin C is destroyed by the UPS following nitrogen starvation. Removing this repression is important as deleting CNC1 allows enhanced cell growth under mild starvation. However, unlike oxidative stress, cyclin C is destroyed prior to its cytoplasmic translocation. This is important as targeting cyclin C to the mitochondria induces both mitochondrial fragmentation and cell death following nitrogen starvation. These results indicate that cyclin C destruction pathways are fine tuned depending on the stress and that its terminal subcellular address influences the decision between initiating cell death or cell survival pathways.


Subject(s)
Cyclin C/metabolism , Nitrogen/deficiency , Proteasome Endopeptidase Complex/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Ubiquitin/metabolism , Autophagy/drug effects , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/metabolism , Cell Survival/drug effects , Cyclin-Dependent Kinase 8/metabolism , Gene Expression Regulation, Fungal/drug effects , Hydrogen Peroxide/toxicity , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Dynamics/drug effects , Models, Biological , Oxidative Stress/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sirolimus/pharmacology , Stress, Physiological/drug effects
17.
Proc Natl Acad Sci U S A ; 117(6): 2894-2905, 2020 02 11.
Article in English | MEDLINE | ID: mdl-31988137

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.


Subject(s)
Cyclin-Dependent Kinase 8/metabolism , Mediator Complex/metabolism , Catalytic Domain , Cyclin C/genetics , Cyclin C/metabolism , Cyclin-Dependent Kinase 8/chemistry , Cyclin-Dependent Kinase 8/genetics , Enzyme Activation , Humans , Mediator Complex/genetics , Protein Binding , Protein Conformation, alpha-Helical , Protein Domains
18.
Plant Physiol ; 182(3): 1375-1386, 2020 03.
Article in English | MEDLINE | ID: mdl-31964799

ABSTRACT

Control of flowering time has been a major focus of comparative genetic analyses in plant development. This study reports on a forward genetic approach to define previously uncharacterized components of flowering control pathways in the long-day legume, pea (Pisum sativum). We isolated two complementation groups of late-flowering mutants in pea that define two uncharacterized loci, LATE BLOOMER3 (LATE3) and LATE4, and describe their diverse effects on vegetative and reproductive development. A map-based comparative approach was employed to identify the underlying genes for both loci, revealing that that LATE3 and LATE4 are orthologs of CYCLIN DEPENDENT KINASE8 (CDK8) and CYCLIN C1 (CYCC1), components of the CDK8 kinase module of the Mediator complex, which is a deeply conserved regulator of transcription in eukaryotes. We confirm the genetic and physical interaction of LATE3 and LATE4 and show that they contribute to the transcriptional regulation of key flowering genes, including the induction of the florigen gene FTa1 and repression of the floral repressor LF Our results establish the conserved importance of the CDK8 module in plants and provide evidence for the function of CYCLIN C1 orthologs in the promotion of flowering and the maintenance of normal reproductive development.


Subject(s)
Flowers/metabolism , Mediator Complex/metabolism , Pisum sativum/metabolism , Cyclin C/metabolism , Cyclin-Dependent Kinase 8/metabolism , Gene Expression Regulation, Plant
19.
EMBO Rep ; 20(9): e47425, 2019 09.
Article in English | MEDLINE | ID: mdl-31385392

ABSTRACT

Intrinsic apoptosis requires mitochondrial outer membrane disruption triggered by recruitment, activation, and oligomerization of the Bcl-2 homology protein Bax. Following oxidative stress, we demonstrated that the transcriptional regulator cyclin C is released into the cytosol where it directs mitochondrial fragmentation and efficient apoptotic induction. This study reveals that cytoplasmic cyclin C is required for both normal Bax activation and its efficient mitochondrial localization. This activity appears direct as cyclin C co-immunoprecipitates with active Bax in stressed cells and binds recombinant Bax in vitro. In addition, stable cyclin C-Bax association requires the fission complex. Pharmacologically stimulating cyclin C nuclear release is sufficient for Bax association and their mitochondrial localization in the absence of any stress signals. However, these cells do not undergo cell death as Bax fails to oligomerize. These data support a model that cyclin C association defines an initial step in Bax mitochondrial recruitment and provides a physical connection between the fission and apoptotic factors. This strategy allows the cell to discriminate stress-induced fission able to recruit Bax from other types of mitochondrial divisions.


Subject(s)
Cyclin C/metabolism , Mitochondria/metabolism , bcl-2-Associated X Protein/metabolism , Animals , Apoptosis/physiology , Blotting, Western , Cell Line , Fluorescent Antibody Technique , HeLa Cells , Humans , Immunoprecipitation , Mice , Mice, Knockout , Mitochondrial Membranes/metabolism , Protein Transport/physiology , Signal Transduction/physiology
20.
Cells ; 8(8)2019 08 03.
Article in English | MEDLINE | ID: mdl-31382571

ABSTRACT

CDK8 and CDK19 Mediator kinases are transcriptional co-regulators implicated in several types of cancer. Small-molecule CDK8/19 inhibitors have recently entered or are entering clinical trials, starting with breast cancer and acute myeloid leukemia (AML). To identify other cancers where these novel drugs may provide benefit, we queried genomic and transcriptomic databases for potential impact of CDK8, CDK19, or their binding partner CCNC. sgRNA analysis of a panel of tumor cell lines showed that most tumor types represented in the panel, except for some central nervous system tumors, were not dependent on these genes. In contrast, analysis of clinical samples for alterations in these genes revealed a high frequency of gene amplification in two highly aggressive subtypes of prostate cancer and in some cancers of the GI tract, breast, bladder, and sarcomas. Analysis of survival correlations identified a group of cancers where CDK8 expression correlated with shorter survival (notably breast, prostate, cervical cancers, and esophageal adenocarcinoma). In some cancers (AML, melanoma, ovarian, and others), such correlations were limited to samples with a below-median tumor mutation burden. These results suggest that Mediator kinases are especially important in cancers that are driven primarily by transcriptional rather than mutational changes and warrant an investigation of their role in additional cancer types.


Subject(s)
Cyclin C/physiology , Cyclin-Dependent Kinase 8/physiology , Cyclin-Dependent Kinases/physiology , Neoplasms/metabolism , Cell Line, Tumor , Cyclin C/antagonists & inhibitors , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Databases, Genetic , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/drug therapy , Neoplasms/genetics
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