Cyclin C: Biological Overview | References
Gene name - Cyclin C
Cytological map position - 88D6-88D6
Function - signaling protein
Keywords - regulatory partner of CDK8, mediator complex component, cofactor for EcR-dependent transcription, links nutrient intake to developmental transitions and fat metabolism, negative regulator of the lipogenic pathway, regulation of Serpent-dependent transcription and innate immunity, involved in small nuclear RNA (snRNA) 3' end processing, recruited to phosphorylate the Notch ICD and coordinate activation with turnover
Symbol - CycC
FlyBase ID: FBgn0004597
Genetic map position - chr3R:14,889,004-14,890,205
Classification - Cyclin
Cellular location - nuclear
EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition (Xie, 2015).
In animals, the amount of juvenile growth is controlled by the coordinated timing of maturation and growth rate, which are strongly influenced by the environmental factors such as nutrient availability. This is particularly evident in arthropods, such as insects, arachnids and crustaceans, which account for over 80% of all described animal species on earth. Characterized by their jointed limbs and exoskeletons, juvenile arthropods have to replace their rigid cuticles periodically by molting. In insects, the larval-larval and larval-pupal transitions are controlled by the interplay between juvenile hormone (JH) and steroid hormone ecdysone. Drosophila has been a powerful system for deciphering the conserved mechanisms that regulate hormone signaling, sugar and lipid homeostasis, and the molecular mechanisms underlying the nutritional regulation of development. In Drosophila, all growth occurs during the larval stage when larvae constantly feed, and as a result their body mass increases approximately 200-fold within 4 d, largely due to de novo lipogenesis. At the end of the third instar, pulses of ecdysone, combined with a low level of JH, trigger the larval-pupal transition and metamorphosis. During this transition, feeding is inhibited, and after pupariation, feeding is impossible, thus the larval-pupal transition marks when energy metabolism is switched from energy storage by lipogenesis in larvae to energy utilization by lipolysis in pupae (Xie, 2015).
The molecular mechanisms of ecdysone-regulated metamorphosis and developmental timing have been studied extensively in Drosophila. Ecdysone binds to the Ecdysone Receptor (EcR), which heterodimerizes with Ultraspiracle (USP), an ortholog of the vertebrate Retinoid X Receptor (RXR). By activating the expression of genes whose products are required for metamorphosis, ecdysone and EcR-USP are essential for the reorganization of flies' body plans before emerging from pupal cases as adults. Despite the tremendous progress in understanding of the physiological and developmental effects of EcR-USP signaling, the molecular mechanism of how the EcR-USP transcription factor interacts with the general transcription machinery of RNA polymerase II (Pol II) and stimulates its target gene expression remains mysterious. EcR is colocalized with Pol II in Bradysia hygida and Chironomus tentans. Although a number of proteins, such as Alien, Bonus, Diabetes and Obesity Regulated (dDOR), dDEK, Hsc70, Hsp90, Rigor mortis (Rig), Smrter (Smr), Taiman, and Trithorax-related (TRR), have been identified as regulators or cofactors of EcR-mediated gene expression, it is unknown how these proteins communicate with the general transcription machinery and whether additional cofactors are involved in EcR-mediated gene expression. In addition, it remains poorly understood how EcR activates transcription correctly after integrating nutritional and developmental cues (Xie, 2015).
The multisubunit Mediator complex serves as a molecular bridge between transcriptional factors and the core transcriptional machinery, and is thought to regulate most (if not all) of Pol II-dependent transcription. Biochemical analyses have identified two major forms of the Mediator complexes: the large and the small Mediator complexes. In addition to a separable 'CDK8 submodule', the large Mediator complex contains all but one (MED26) of the subunits of the small Mediator complex. The CDK8 submodule is composed of MED12, MED13, CDK8, and CycC. CDK8 is the only enzymatic subunit of the Mediator complex, and CDK8 can both activate and repress transcription depending on the transcription factors with which it interacts (Taatjes, 2010; Galbraith, 2010). Amplification and mutation of genes encoding CDK8, CycC, and other subunits of Mediator complex have been identified in a variety of human cancers, however, the function and regulation of CDK8-CycC in non-disease conditions remain poorly understood. CDK8 and CycC are highly conserved in eukaryotes, thus analysis of the functional regulation of CDK8-CycC in Drosophila is a viable approach to understand their activities (Xie, 2015).
Previous, work has shown that CDK8-CycC negatively regulates the stability of sterol regulatory element-binding proteins (SREBPs) by directly phosphorylating a conserved threonine residue (Zhao, 2012). This study now reports that CDK8-CycC also regulates developmental timing in Drosophila by linking nutrient intake with EcR-activated gene expression. Homozygous cdk8 or cycC mutants resemble EcR mutants in both pupal morphology and retarded developmental transitions. Despite the elevation of both EcR and USP proteins in cdk8 or cycC mutants, genome-wide gene expression profiling analyses reveal systematic down-regulation of EcR-target genes, suggesting the CDK8-CycC defect lies between the receptor complex and transcriptional activation. CDK8-CycC is required for EcR-USP transcription factor binding to EcR target genes. Mass spectrometry analysis for proteins that co-immunoprecipitate with EcR and USP has identified multiple Mediator subunits, including CDK8 and CycC, and yeast two-hybrid assays have revealed that CDK8 and Med14 can directly interact with the EcR-AF1 domain. Furthermore, the dynamic changes of CDK8, EcR, USP, and SREBP correlated with the fundamental roles of SREBP in regulating lipogenesis and EcR-USP in regulating metamorphosis during the larval–pupal transition. Importantly, it was shown that starving the early third instar larvae causes precocious increase of CDK8, EcR and USP proteins, as well as premature inactivation of SREBP; whereas refeeding of the starved larvae reduces CDK8, EcR, and USP proteins, but potently stimulates SREBP activity. These results suggest a dual role of CDK8-CycC, linking nutrient intake to de novo lipogenesis (by inhibiting SREBP) and developmental signaling (by regulating EcR-dependent transcription) during the larval–pupal transition (Xie, 2015).
Through EcR-USP, ecdysone plays pivotal roles in controlling developmental timing in Drosophila. This study shows that cdk8 or cycC mutants resemble EcR-B1 mutants and CDK8-CycC is required for proper activation of EcR-target genes. Molecular and biochemical analyses suggest that CDK8-CycC and the Mediator complexes are directly involved in EcR-dependent gene activation. In addition, the protein levels of CDK8 and CycC are up-regulated at the onset of the wandering stage, closely correlated with the activation of EcR-USP and down-regulation of SREBP-dependent lipogenesis during the larval–pupal transition. Remarkably, starvation of the feeding larvae leads to premature up-regulation of CDK8 and EcR-USP, and precocious down-regulation of SREBP, while refeeding of the starved larvae results in opposite effects on the CDK8-SREBP/EcR network. Thus, it is proposed that CDK8-CycC serves as a key mediator linking food consumption and nutrient intake to EcR-dependent developmental timing and SREBP-dependent lipogenesis during the larval–pupal transition (Xie, 2015).
The Mediator complex is composed of up to 30 different subunits, and biochemical analyses of the Mediator have identified the small Mediator complex and the large Mediator complex, with the CDK8 submodule being the major difference between the two complexes (Poss, 2013; Canaway, 2011; Malik, 2005). Several reports link EcR and certain subunits of the Mediator complex. For example, Med12 and Med24 were shown to be required for ecdysone-triggered apoptosis in Drosophila salivary glands. It was recently reported that ecdysone and multiple Mediator subunits could regulate cell-cycle exit in neuronal stem cells by changing energy metabolism in Drosophila, and specifically, EcR was shown to co-immunoprecipitate with Med27 (Hamem, 2014). However, exactly how Mediator complexes are involved in regulating EcR-dependent transcription remains unknown. The current data suggest that CDK8 and CycC are required for EcR-activated gene expression. Loss of either CDK8 or CycC reduced USP binding to EcR target promoters, diminished EcR target gene expression, and delayed developmental transition, which are reminiscent of EcR-B1 mutants. Importantly, mass spectrometry analysis for proteins that co-immunoprecipitate with EcR or USP has identified multiple Mediator subunits, including all four subunits of the CDK8 submodule (Xie, 2015).
Taken together, previous works and the present work highlight a critical role of the Mediator complexes including CDK8-CycC in regulating EcR-dependent transcription. How does CDK8-CycC regulate EcR-activated gene expression? Biochemical analyses show that CDK8 can interact with EcR and USP in vivo and that CDK8 can directly interact with EcR-AF1. These observations, together with the current understanding of how nuclear receptors and Mediator coordinately regulate transcription, suggest that CDK8-CycC may positively and directly regulate EcR-dependent transcription. Yeast two-hybrid analysis indicates that the recruitment of CDK8-CycC to EcR-USP can occur via interactions between CDK8 and the AF1 domain of EcR. Interestingly, this assay also revealed a direct interaction between EcR-AF1 and a fragment of Med14 that contains the LXXLL motif. In future work, it will be interesting to determine whether CDK8 and Med14 compete with each other in binding with the EcR-AF1, whether they interact with EcR-AF1 sequentially in activating EcR-dependent transcription, and how the Mediator complexes coordinate with other known EcR cofactors in regulating EcR-dependent gene expression (Xie, 2015).
In cdk8 or cycC mutants, the binding of USP to the promoters of the EcR target genes is significantly compromised, even though nuclear protein levels of both EcR and USP are increased. It is unclear how CDK8-CycC positively regulates EcR-USP binding to EcREs near promoters. CDK8 can directly phosphorylate a number of transcription factors, such as Notch intracellular domain, E2F1, SMADs, SREBP, STAT1, and p53. Interestingly, the endogenous EcR and USP are phosphorylated at multiple serine residues, and treatment with 20E enhances the phosphorylation of USP. Protein kinase C has also been proposed to phosphorylate USP. It will be interesting to determine whether CDK8 can also directly phosphorylate either EcR or USP, thereby potentiating expression of EcR target genes and integrating signals from multiple signaling pathways (Xie, 2015).
Although a direct role for CDK8-CycC to regulate EcR-USP activated gene expression is favored, it was not possible to exclude the potential contribution of impaired biosynthesis of 20E to the developmental defects in cdk8 or cycC mutants. For example, the expression of genes involved in synthesis of 20E, such as sad and spok, is significantly reduced in cdk8 or cycC mutant larvae. Indeed, the ecdysteroid titer is significantly lower in cdk8 mutants than control from the early L3 to the WPP stages, and feeding the cdk8 mutant larvae with 20E can partially reduce the retardation in pupariation. Nevertheless, impaired ecdysone biosynthesis alone cannot explain developmental defects that were characterized in this report for the following reasons. First, feeding cdk8 or cycC mutants with 20E cannot rescue the defects in pupal morphology, developmental delay, and the onset of pupariation. Second, the expression of EcRE-lacZ reporter in cdk8 or cycC mutant salivary glands cannot be as effectively stimulated by 20E treatment as in control. Third, knocking down of either cdk8 or cycC in PG did not lead to obvious defects in developmental timing. Therefore, the most likely scenario is that the cdk8 or cycC mutants are impaired not only in 20E biosynthesis in the PG, but also in EcR-activated gene expression in peripheral tissues. Defects in either ecdysone biosynthesis or EcR transcriptional activity will generate the same outcome: diminished expression of the EcR target genes, thereby delayed onset of pupariation (Xie, 2015).
How CDK8-CycC regulates biosynthesis of ecdysone in PG remains unknown. Several signaling pathways have been proposed to regulate ecdysone biosynthesis in Drosophila PG, including PTTH and Drosophila insulin-like peptides (dILPs)-activated receptor tyrosine kinase pathway and Activins/TGFβ signaling pathway. Interestingly, CDK8 has been reported to regulate the transcriptional activity of SMADs, transcription factors downstream of the TGFβ signaling pathway, in both Drosophila and mammalian cells. Thus, it is conceivable that the effect of cdk8 or cycC mutation on ecdysone biosynthesis may due to dysregulated TGFβ signaling in the PG (Xie, 2015).
An effort to explore the potential role of food consumption and nutrient intake on CDK8-CycC has resulted an unexpected observation that the protein level of CDK8 is strongly influenced by starvation and refeeding: starvation potently increased CDK8 level, while refeeding has opposite effect, and both occur post-transcriptionally. The importance of this observation is highlighted in two aspects. First, considering the generally repressive role of CDK8 on Pol II-dependent gene expression, up-regulation of CDK8 may provide an efficient way to quickly tune down most of the Pol II-dependent transcription in response to starvation; while down-regulation of CDK8 in response to refeeding may allow many genes to express when nutrients are abundant. Second, it will be necessary to test whether the effects of nutrient intake on CDK8-CycC is conserved in mammals. If so, considering that both CDK8 and CycC are dysregulated in a variety of human cancers, the effects of nutrient intake on CDK8 may have important implications in not only understanding of the effects of nutrients on tumorigenesis, but also providing nutritional guidance for patients with cancer (Xie, 2015).
Major dietary components including carbohydrates, lipids, and proteins, can strongly influence the developmental timing in Drosophila. Excessive dietary carbohydrates repress growth and potently retard the onset of pupariation. One elegant model proposed to explain how high sugar diet delays developmental timing is that high sugar diet reduces the activity of the Target of Rapamycin (TOR) in the PG, thereby reducing the secretion of ecdysone and delaying the developmental transition. Previously, it was reported that insulin signaling could down-regulate CDK8-CycC, and that ectopic expression of CycC could antagonize the effect of insulin stimulation in mammalian cells, as well as the effect of refeeding on the expression of dFAS in Drosophila (Zhao, 2012). Although the mRNA levels of TOR and insulin receptor (InR) are not significantly affected in cdk8 or cycC mutants, it is necessary to further study whether and how different dietary components may regulate CDK8-CycC in the future (Xie, 2015).
Developmental genetic analyses of the cdk8 and cycC mutants have revealed major defects in fat metabolism and developmental timing. De novo lipogenesis, which is stimulated by insulin signaling, contributes significantly to the rapid increase of body mass during the constant feeding larval stage. This process is terminated by pulses of ecdysone that trigger the wandering behavior at the end of the L3 stage, followed by the onset of the pupariation. Insulin and ecdysone signaling are known to antagonize each other, and together determine body size of Drosophila. The genetic interaction is established, but the detailed molecular mechanisms are not. The SREBP family of transcription factors controls the expression of lipogenic enzymes in metazoans and the expression of cholesterogenic enzymes in vertebrates. Previous work shows that CDK8 directly phosphorylates the nuclear SREBP proteins on a conserved threonine residue and promotes the degradation of nuclear SREBP proteins. Consistent with the lipogenic role of SREBP and the inhibitory role of insulin to CDK8-CycC (Zhao, 2012), the transcriptional activity of SREBP is high while the levels of CDK8-CycC and EcR-USP are low prior to the onset of wandering stage. Subsequently during the wandering and non-mobile, non-feeding pupal stage, the transcriptional activity of SREBP is dramatically reduced, accompanied by the significant accumulation of CDK8-CycC and EcR-USP (Xie, 2015).
The causal relationship of these phenomena was further tested by starvation and refeeding experiments. On the one hand, it was observed that the levels of CDK8, EcR and USP are potently induced by starvation, while the mature SREBP level and the transcriptional activity of SREBP are reduced by starvation. Starvation of larvae prior to the two nutritional checkpoints in early L3, known as minimum viable weight and critical weight, which are reached almost simultaneously in Drosophila, will lead to larval lethality; while starvation after larvae reach the critical weight will lead to early onset of pupariation and formation of small pupae. Thus, this nutritional checkpoint ensures the larvae have accumulated sufficient growth before metamorphosis initiation. If the status with high CDK8, EcR, and USP is regarded as an older or later stage, these results indicate that starvation shifts the regulatory network precociously (see Model for the CDK8-SREBP/EcR regulatory network). On the other hand, the current analyses of refed larvae show that refeeding potently reduced the levels of CDK8, EcR and USP. If the status with low CDK8, EcR, and USP is considered as a younger or earlier stage, these results indicate that refeeding delays the activation of this network, which is consistent with the model and delayed pupariation as observed. Taken together, these results based on starved and refed larvae suggest that CDK8-CycC is a key regulatory node linking nutritional cues with de novo lipogenesis and developmental timing (Xie, 2015).
The larval-pupal transition is complex and dynamic. Although the expression of SREBP target genes fit well with the predicted effects of starvation and refeeding, the expression of EcR targets during the stage that was analyzed in this study does not reflect the changes in the protein levels of EcR and USP. It is reasonable to consider that CDK8-CycC and EcR-USP are necessary, but not sufficient, for the activation of EcR target genes. One possibility is that there is a delay on synthesis of 20E or other cofactors that are required for EcR-activated gene expression in response to starvation. Indeed, the 20E levels were measured during the first 16 hr of starvation, and no significant difference was observed between fed and starved larvae. It will be necessary to further analyze the effect of starvation on 20E synthesis at later time points in the future (Xie, 2015).
Taken together, a model is proposed whereby CDK8-CycC functions as a regulatory node that coordinates de novo lipogenesis during larval stage and EcR-dependent pupariation in response to nutritional cues. It is likely that pulses of 20E synthesized in the PG, and subsequent behavioral change from feeding to wandering, ultimately trigger the transition from SREBP-dependent lipogenesis to EcR-dependent pupariation. The opposite effects of CDK8-CycC on SREBP- and EcR-dependent gene expression suggest that the role of CDK8 on transcription is context-dependent (Xie, 2015).
In conclusion, this study illustrates how CDK8-CycC regulates EcR-USP-dependent gene expression, and the results suggest that CDK8-CycC may function as a regulatory node linking fat metabolism and developmental timing with nutritional cues during Drosophila development (Xie, 2015).
The Cdk8 (cyclin-dependent kinase 8) module of Mediator integrates regulatory cues from transcription factors to RNA polymerase II. It consists of four subunits where Med12 and Med13 link Cdk8 and Cyclin C (CycC) to core Mediator. This study has investigated the contributions of the Cdk8 module subunits to transcriptional regulation using RNA interference in Drosophila cells. Genome-wide expression profiling demonstrated separation of Cdk8-CycC and Med12-Med13 profiles. However, transcriptional regulation by Cdk8-CycC was dependent on Med12-Med13. This observation also revealed that Cdk8-CycC and Med12-Med13 often have opposite transcriptional effects. Interestingly, Med12 and Med13 profiles overlapped significantly with that of the GATA factor Serpent. Accordingly, mutational analyses indicated that GATA sites are required for Med12-Med13 regulation of Serpent-dependent genes. Med12 and Med13 were also found to be required for Serpent-activated innate immunity genes in defense to bacterial infection. The results reveal a novel role for the Cdk8 module in Serpent-dependent transcription and innate immunity (Kuuluvainen, 2014).
In this genome-wide study on transcription regulation by the Cdk8 module, a striking pairwise similarity was noted between Cdk8 and CycC as well as Med12 and Med13. Co-regulation by all four subunits was surprisingly limited, clearly differing from yeast where depletion of any Cdk8 module subunit results in similar effects on transcription. Importantly, the lack of Med13-specific genes indicates that Med13 does not regulate transcription without Med12, although structural and biochemical analysis of the Cdk8 module suggests this might be possible. The results thus suggest that the previously identified Med13 regulatory mechanisms are likely to be directed toward functions of either Med12-Med13 or the entire Cdk8 module (Kuuluvainen, 2014).
The dependence of gene regulation by Cdk8-CycC on Med12-Med13 noted in this study and in a genetic study on leg bristles (Loncle, 2007) supports the suggested structural hierarchy where Cdk8-CycC is linked to the core Mediator through Med12 and Med13 (Adler, 2012; Chen, 2012). Identification of the dependence of Cdk8-CycC on Med12-Med13 also revealed that these pairs often have opposite transcriptional effects. This indicates that opposite regulation by Cdk8-CycC and Med12-Med13 should be considered as a possibility on all Cdk8-CycC-regulated genes and functions previously identified to be Med12-Med13-independent (Kuuluvainen, 2014).
Cdk8-CycC dependence on Med12-Med13 highlights the importance of investigating the possible involvement of Cdk8-CycC in Med12-Med13-dependent phenotypes. Interestingly, suppression of Shh signaling in cells with the FG and Lujan syndrome mutations in Med12 was recently shown to be a result of dissociation of Cdk8 but not Med12 on Gli3 target promoters (Zhou, 2012). Furthermore, the finding that Cdk8-CycC can act opposite to Med12-Med13 although being Med12-Med13-dependent indicates that, for example, loss of Med12 could lead to similar phenotypes as gain of Cdk8; both genetic alterations have been noted in human colorectal cancer. Taken together, these results are consistent with the notion that Cdk8-CycC mediates gene regulation primarily through interaction with Mediator through Med12-Med13, whereas Med12-Med13 can regulate transcription independently of Cdk8-CycC (Kuuluvainen, 2014).
Med12-Med13 was found in this study to be important for Srp-dependent transcription, and the previously identified physical interaction between Srp and Cdk8 module components provides a plausible mechanism for this. In addition to Mtk and DptB, the IMD target CecA1 is also a target of Srp and dependent on the Cdk8 module components. Consistent with this, induction of the A. gambiae homolog of CecA1, Cec1, was recently shown to require Med12 and Med13. Multiple known (e.g., Eater, Sr-CI, Pxn) and novel (e.g., CG14629, CG10962) Srp-dependent genes found in this study to be Med12-Med13-dependent implicate Med12-Med13 in various Srp-regulated functions. Besides its role in AMP gene induction, Srp is required in hematopoietic differentiation. In some instances, this may be modulated by the Cdk8 module, suggested by the requirement of Drosophila Med12-Med13 and zebrafish Med12 in differentiation of specific blood cell lineages. Based on this, it will be interesting to study the possible involvement of Med12-Med13 in mammalian GATA-dependent hematopoiesis (Kuuluvainen, 2014).
It appears that transcription regulation by the Cdk8 module is largely context-dependent. In this regard, it was intriguing to identify several genes involved in neuronal functions (Epac, Fie, ogre, and PQBP-1) as strongly regulated by Med12-Med13 in S2 cells. It will be interesting to analyze whether these genes are also regulated by Med12-Med13 in neural tissues, where Med12 and one of it targets identified here, PQBP-1, present related phenotypes (Kuuluvainen, 2014).
Formation of the 3' end of RNA polymerase II-transcribed small nuclear RNA (snRNAs) requires a poorly understood group of proteins called the Integrator complex. A fluorescence-based read-through reporter was used that expresses GFP in response to snRNA misprocessing; a genome-wide RNAi screen in Drosophila S2 cells was performed to identify novel factors required for snRNA 3'-end formation. In addition to the known Integrator complex members, Asunder and CG4785 were identified as additional Integrator subunits. Functional and biochemical experiments revealed that Asunder and CG4785 are additional core members of the Integrator complex. Also, a conserved requirement was identified in both fly and human snRNA 3'-end processing for cyclin C and Cdk8 that is distinct from their function in the Mediator Cdk8 module. Moreover, biochemical association was observed between Integrator proteins and cyclin C/Cdk8, and overexpression of a kinase-dead Cdk8 was seen to cause snRNA misprocessing. These data functionally define the Drosophila Integrator complex and demonstrate an additional function for cyclin C/Cdk8 unrelated to its function in Mediator (Chen, 2012).
Altered lipid metabolism underlies several major human diseases, including obesity and type 2 diabetes. However, lipid metabolism pathophysiology remains poorly understood at the molecular level. Insulin is the primary stimulator of hepatic lipogenesis through activation of the SREBP-1c transcription factor. This study identified cyclin-dependent kinase 8 (CDK8) and its regulatory partner cyclin C (CycC) as negative regulators of the lipogenic pathway in Drosophila, mammalian hepatocytes, and mouse liver. The inhibitory effect of CDK8 and CycC on de novo lipogenesis was mediated through CDK8 phosphorylation of nuclear SREBP-1c at a conserved threonine residue. Phosphorylation by CDK8 enhanced SREBP-1c ubiquitination and protein degradation. Importantly, consistent with the physiologic regulation of lipid biosynthesis, CDK8 and CycC proteins are rapidly downregulated by feeding and insulin, resulting in decreased SREBP-1c phosphorylation. Moreover, overexpression of CycC efficiently suppresses insulin and feeding-induced lipogenic gene expression. Taken together, these results demonstrate that CDK8 and CycC function as evolutionarily conserved components of the insulin signaling pathway in regulating lipid homeostasis (Zhao, 2012).
In contrast to the sterol regulation of SREBP-2, insulin functions as the major activator of SREBP1C gene expression and inducer of SREBP-1c precursor processing to the mature nuclear form. Numerous studies have established a key role of nuclear SREBP-1c on lipogenic gene expression and de novo lipogenesis. In this regard, SREBP-1c levels are elevated in mice and humans under a variety of pathophysiologic states, including obesity, insulin resistance associated with hyperinsulinemia, high carbohydrate diet, excessive alcohol consumption, and non-alcoholic fatty liver disease (NAFLD) -- in the case of the latter being directly shown to increase the proportion of liver VLDL triglyceride production by de novo lipogenesis from 2%-5% to 20%-30%. In those disease states, it is generally thought that the increased de novo lipogenesis results from high levels of insulin driving SREBP-1c transcription and precursor processing through PI3K- and mTORC1-dependent pathways, despite the marked insulin resistance to gluconeogenesis. However, these studies have only focused on the mechanisms of transcriptional activation of SREBP-1c expression and have not considered changes in the transcriptional activity or protein stability of nuclear SREBP-1c protein (Zhao, 2012).
This study has identified a highly conserved role for CDK8 in the control of fatty acid and triglyceride metabolism. In Drosophila larvae, loss of CDK8 or CycC increased SREBP-dependent neutral lipid accumulation in fat body and lipogenic gene expression. Similarly, RNAi knockdown in hepatocytes in vitro or mouse liver in vivo also increased triglyceride levels, along with increasing SREBP target gene expression and de novo lipogenesis. Surprisingly, CycC and CDK8 levels were negatively regulated by food intake in mouse liver and by insulin in cultured cells. Thus, a model is proposed whereby insulin stimulates de novo lipogenesis by downregulating the CDK8-CycC complex, which normally inhibits de novo lipogenesis through promoting nuclear SREBP-1c degradation. This new mechanism of insulin-induced lipogenesis is in addition to the well-documented function of insulin in stimulating SREBP-1c at the transcriptional level. Consistent with this model, overexpression of CycC blocks insulin-induced stabilization of nuclear SREBP-1 and lipogenic gene expression in vitro and in vivo. These observations establish an important and physiological role of CDK8-CycC in regulating fatty acid biosynthesis (Zhao, 2012).
CDK8 and CycC are best known for their functions as the subunits of the Mediator complex, which serves as a transcription cofactor complex. Along with MED12 and MED13, they form the CDK8 submodule of the Mediator complex. However, unlike that of CDK8 and CycC, fat body-specific knockdown of MED12 and MED13 did not cause statistically significant changes in lipid levels in Drosophila larvae, although there was a trend toward a decrease in lipids in MED13-knockdown larvae. Similarly, MED12 or MED13 knockdown had no effect on SREBP-1 in mammalian cells. These results support a specific role of CDK8-CycC in lipid metabolism and are consistent with a previous report demonstrating functions for the CDK8-CycC complex distinct from those of the MED12-MED13 complex in regulating developmental patterns in Drosophila (Loncle, 2007). The current data also support a model whereby specific subunits of the Mediator complex are involved in only specific biological pathways, although the Mediator complex collectively may regulate most of the RNAPII-controlled gene expression. Alternatively, CDK8-CycC may function independent of the Mediator complex. Mechanistically, CDK8-CycC inhibits lipid accumulation largely by repressing SREBP-mediated gene expression. Biochemical analyses have revealed that SREBP-1c can be directly phosphorylated by CDK8 at a conserved T402 residue (for human SREBP-1c) in vitro and in cultured mammalian cells. Furthermore, it was observed that phosphorylation of SREBP-1 at T402 promotes its degradation. Thus, this phosphorylation event provides a critical control mechanism to regulate the level of nuclear SREBP-1c protein by increasing ubiquitination and subsequent rate of degradation without affecting the levels of the precursor SREBP-1c protein. These results suggest a simple explanation for the increased fat accumulation on CDK8 or CycC mutants or in cells with reduced levels of CDK8 and CycC. That is, reduction of CDK8 or CycC will result in hypophosphorylation of SREBP-1, which increases the stability of the nuclear SREBP proteins, thereby allowing increased expression of SREBP target genes involved in de novo lipogenesis (Zhao, 2012).
It has been known that phosphorylation of nuclear SREBP proteins controls their stability. GSK-3β, which functions downstream of insulin signaling, was previously reported to be involved in phosphorylation of SREBP proteins at several conserved sites, including T426 of SREBP-1a (the site corresponding to T402 of SREBP-1c). This study identified that CDK8 can directly phosphorylate the T402 site of SREBP-1c. It is not uncommon that the same threonine or serine residue of a protein can be phosphorylated by multiple kinases. The fact that the T402 site of SREBP-1c can be targeted by both GSK-3β and CDK8 is consistent with the key role of phosphorylation on this site for the binding of the E3 ligase SCFFbw7b (Zhao, 2012).
The importance of these findings is underscored by the following two points. First, the observed high rates of lipogenesis and enhanced SREBP-target gene expression in dyslipidemic states in CDK8 and CycC mutants cannot be solely accounted for by changes in SREBP-1c transcription or maturation. It will be important to determine whether the control over nuclear SREBP-1c protein levels through the CDK8-CycC complex can provide a mechanistic basis for the enigmatic enhancement of lipogenesis that occurs during insulin resistance and diabetes. Second, CDK8 has been recently identified as an oncoprotein in melanoma and colorectal cancers. Aberrant lipid and carbohydrate metabolism is a universal feature of human cancer cells; however, the mechanisms linking such aberrant metabolism and tumorigenesis remain poorly understood. The current results suggest that dysregulation of CDK8 may not only promote tumorigenesis, but also disrupt the cellular lipid homeostasis by lifting its repressive effect on SREBP-dependent transcription. It will be important to investigate in the future whether such a mechanism is functional in different types of human cancer cells (Zhao, 2012).
Spatial and temporal gene regulation relies on a combinatorial code of sequence-specific transcription factors that must be integrated by the general transcriptional machinery. A key link between the two is the mediator complex, which consists of a core complex that reversibly associates with the accessory kinase module. Genes activated by Notch signaling at the dorsal-ventral boundary of the Drosophila wing disc fall into three classes that are affected differently by the loss of kinase module subunits. One class requires all four kinase module subunits for activation, while the others require only Med12 and Med13, either for activation or for repression. These distinctions do not result from different requirements for the Notch coactivator Mastermind or the corepressors Hairless and Groucho. It is proposed that interactions with the kinase module through distinct cofactors allow the DNA-binding protein Suppressor of Hairless to carry out both its activator and repressor functions (Janody, 2011).
Intercellular signaling pathways drive many processes during development. Their activation results in changes in transcription factor activity that lead to the activation or repression of specific target genes. An important goal is to understand the transcriptional regulatory codes that allow the combinations of proteins bound to enhancer elements to direct precise patterns of gene expression. One well-characterized developmental paradigm is the specification of the Drosophila wing margin by Notch signaling. The Notch receptor is specifically activated at the dorsal-ventral boundary of the larval wing imaginal disc, due to the restricted expression of its ligands Delta and Serrate and of the glycosyltransferase Fringe. Notch activation results in expression of the target genes Enhancer of split m8 (E(spl)m8), cut, wingless (wg), and vestigial (vg), the last through a specific enhancer element known as the boundary enhancer (vgBE). Wg signaling then leads to the differentiation of characteristic sensory bristles adjacent to the margin of the adult wing (Janody, 2011).
Upon ligand binding, Notch is cleaved by the γ-secretase complex, and its intracellular domain (Nintra) enters the nucleus, where it interacts with the DNA-binding protein Suppressor of Hairless (Su(H)). In the absence of Notch activation, Su(H) represses target gene expression through interactions with the corepressor Hairless (H), which binds to Groucho (Gro) and C-terminal binding protein (CtBP). Nintra displaces these corepressors from Su(H) and recruits coactivators such as Mastermind (Mam). It has been proposed that only a subset of Notch target genes require Su(H) to recruit coactivators, while others require Notch signaling only to relieve Su(H)-mediated repression, allowing transcription to be activated by other factors. However, the mechanisms by which Su(H) directs both activation and repression are not fully understood (Janody, 2011).
The mediator complex is thought to promote transcriptional activation by recruiting RNA polymerase II (Pol II), the general transcriptional machinery, and the histone acetyltransferase p300 to promoters, and by stimulating transcriptional elongation by Pol II molecules paused downstream of the promoter. The 'head' and 'middle' modules of the core complex bind to Pol II and general transcription factors, while the 'tail' module consists largely of adaptor subunits that bind to sequence-specific transcription factors. This core complex reversibly associates with a fourth 'kinase' module that consists of the four subunits Med12, Med13, Cdk8, and Cyclin C (CycC). Several studies have implicated the kinase module in transcriptional repression, which can be mediated by phosphorylation of Pol II and other factors by Cdk8, by histone methyltransferase recruitment, and by occlusion of the Pol II binding site. However, this module also appears to function in activation in some contexts; for example, it promotes Wnt target gene expression during Drosophila and mouse development, in mammalian cells, and in colon cancer. Although all four subunits have very similar mutant phenotypes in yeast, loss of Med12 or Med13 has more severe effects on Drosophila development than loss of Cdk8 or CycC, suggesting that Med12 and Med13 have evolved additional functions in higher eukaryotes (Janody, 2011).
This study shows that Notch target genes at the wing margin can be divided into three classes based on their requirements for kinase module subunits. An E(spl)m8 reporter requires all four subunits for its activation, cut requires only Med12 and Med13 (known as Kohtalo [Kto] and Skuld [Skd], respectively, in Drosophila) for its activation, and wg and the vgBE enhancer require Med12 and Med13 for their repression in cells close to the wing margin. Because Med12 and Med13 coimmunoprecipitate with Su(H), regulate an artificial reporter driven by Su(H) binding sites, and can be replaced by a VP16 activation domain or a WRPW repression signal fused to Su(H), it is proposed that the kinase module directly regulates Notch target genes. All four Notch target genes fail to be expressed in the absence of Mam and are similarly affected by the loss of Hairless or Gro, suggesting that other more specific cofactors might recruit kinase module subunits to these genes (Janody, 2011).
The kinase module of the mediator complex is conserved throughout eukaryotes, yet its functions in transcription remain poorly understood. In yeast, loss of any of the four subunits has a very similar effect. In Drosophila, however, loss of Med12 or Med13 has more dramatic effects than loss of Cdk8 or CycC. The kinase module was originally thought to be primarily important for transcriptional repression, mediated by the kinase activity of Cdk8. However, Med12 and Med13 appear to directly activate genes regulated by Wnt signaling in Drosophila and mammalian systems, and also play a positive role in gene activation by the Gli3 and Nanog transcription factors. The data presented in this study confirm that Med12 and Med13 have functions distinct from Cdk8 and CycC. In addition, evidence is provided that all four kinase module subunits contribute to the activation of E(spl)m8 (Janody, 2011).
The human Mastermind homologue MAM has been shown to recruit Cdk8 and CycC to promoters of Notch target genes, where Cdk8 phosphorylates the intracellular domain of Notch, leading to its ubiquitination by the Fbw7 ligase and degradation (Fryer, 2004). This mechanism would be expected to reduce Notch target gene expression, consistent with the increase in E(spl)mβ expression seen in clones lacking the Drosophila Fbw7 homologue Archipelago (Nicholson, 2011); thus it cannot explain the positive effects of Cdk8 and CycC on E(spl)m8. A function for Cdk8 and CycC in Notch-mediated activation would be analogous to recent findings showing that Cdk8 phosphorylation of Smad transcription factors and of histone H3 promotes activation. Cdk8 phosphorylation of RNA polymerase II (Pol II) is also important for transcriptional elongation (Janody, 2011).
Of interest, the current data also suggest that Med12 and Med13 are involved in the repression of wg and the vgBE enhancer in the absence of Notch signaling. The kinase module has been proposed to inhibit transcription through steric hindrance of Pol II binding, independently of Cdk8 kinase activity. Removal of this module on the C/EBP promoter is thought to convert the mediator complex to its active form. In contrast, this study find that wg and vgBE require Med12 and Med13 for their repression but not their activation, while cut and E(spl)m8 require Med12 and Med13 only for their activation, arguing that the two functions occur on different promoters. It cannot be ruled out that Med12 and Med13 have only indirect effects on some of the genes examined; however, their physical association with Su(H) and the requirement for Su(H) binding sites for misexpression of an artificial reporter in skd and kto mutant clones are consistent with a direct effect of Med12 and Med13 on the Su(H) complex (Janody, 2011).
Med12 and Med13 are found associated with both active and inactive promoters in genome-wide chromatin immunoprecipitation studies, suggesting that they can have different effects on transcription when bound to distinct interaction partners. Although both are very large proteins, they contain no domains predicted to have enzymatic activity, and may instead act as scaffolds for the assembly of transcriptional complexes (Janody, 2011).
It has been proposed that Notch target genes could be categorized into two classes: permissive genes, for which the primary function of Notch is to relieve repression by the Su(H) complex, and instructive genes, for which Notch plays an essential role in activation by recruiting specific coactivators. These differences presumably depend on the combinatorial code of transcription factors that regulate each promoter. This study shows that vgBE, an enhancer previously placed in the permissive category, as well as wg, require Med12 and Med13 for their repression but not their activation. During eye development, the proneural gene atonal is likewise regulated permissively by Notch, and ectopically expressed in skd or kto mutant clones. Unexpectedly, this study found that Gro, previously thought to be a cofactor through which Hairless mediates repression, is not required for the repression of vgBE or wg. Hairless may repress target genes at the wing margin through CtBP, its other binding partner. Alternatively, Gro may affect the expression of other upstream regulators of wing margin fate, masking its repressive effect on the genes that were examined (Janody, 2011).
It was also show in this study that instructive Notch target genes can be further subdivided into two classes based on their requirement for kinase module subunits; E(spl)m8 requires all four subunits, while cut requires Med12 and Med13, but not Cdk8 and CycC. Cdk8 and CycC may simply increase the ability of the mediator complex to recruit Pol II or promote transcriptional initiation; this model would suggest that E(spl)m8 has a higher activation threshold than cut. Alternatively, Cdk8 and CycC might enhance the function of a transcription factor that is specifically required for the expression of E(spl)m8 but not cut. Good candidates for such factors would be the proneural proteins Achaete or Scute or their partner Daughterless (Janody, 2011).
The mechanism by which the kinase module is recruited to promote the activation of instructive target genes is not yet clear. Although Mam proteins are well-characterized coactivators for Nintra, this study found that Mam is necessary for the activation of both instructive and permissive genes. It may thus have a general function in transcriptional activation, such as recruiting histone acetyltransferases or stabilizing the Notch-Su(H) complex. A coactivator that recruits Med12 and Med13 specifically to instructive target genes to promote activation may remain to be identified. The current results, like recent reports demonstrating that the arrangement of Su(H) binding sites can affect the interactions between Notch and its coactivators, highlight the complexity in the mechanisms through which promoter elements respond to Notch signaling (Janody, 2011).
Wnt target gene transcription is mediated by nuclear translocation of stabilized β-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. The Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of β-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. It is proposed that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13 (Carrera, 2008).
The mediator complex was first defined in yeast as a large multisubunit complex required for transcription of RNA polymerase II (PolII)-dependent genes. Since then, its composition and function have been shown to be conserved in Drosophila, mouse, and human cells. The mediator complex can directly bind to Pol II and recruit it to target promoters, but it also appears to function at a step subsequent to Pol II assembly into the preinitiation complex. Several mediator subunits have been shown to act as adaptors for specific transcription factors, linking them to the mediator complex and allowing them to activate transcription (Carrera, 2008 and references therein).
Four subunits, Med12, Med13, Cdk8, and Cyclin C (CycC), form an accessory subcomplex known as the kinase module. Genetic and microarray analyses in yeast implicate the kinase module primarily in transcriptional repression. Many of its effects have been attributed to the Cdk8 kinase, which phosphorylates the C-terminal domain of Pol II, the Cyclin H component of the TFIIH general transcription factor, and other subunits of the mediator complex, as well as specific transcription factors. The large Med12 and Med13 proteins are required for specific developmental processes in Drosophila, zebrafish, and Caenorhabditis elegans, but their biochemical functions are not understood (Carrera, 2008 and references therein).
Secreted proteins of the Wnt family play important roles in both development and oncogenesis. Transcription of Wnt target genes is mediated by nuclear translocation of stabilized Armadillo (Arm)/β-catenin and its binding to the HMG box transcription factor TCF. The adaptor protein Legless (Lgl)/Bcl-9 links Armβ-catenin to Pygopus (Pygo); the N-terminal homology domain (NHD) of Pygo is essential for Wnt-regulated transcriptional activation and is thought to interact with unknown general transcriptional regulators. This study shows that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo (kto) and skuld (skd), are essential for the transcription of Wingless (Wg) target genes in vivo and a Wg-responsive reporter in cultured cells. skd and kto act downstream of Arm stabilization and are required for the function of the NHD of Pygo when fused to an exogenous DNA-binding domain. Skd and Kto interact with Pygo in vivo through the NHD. It is suggested that this interaction recruits the mediator complex to allow for the transcription of Wg target genes (Carrera, 2008).
Two domains of Arm/α-catenin are important for the activation of Wnt target genes: (1) Arm repeats 1-4, which act by binding Lgs and thus recruiting Pygo, and (2) a C-terminal transcriptional activation domain. The C-terminal domain has been shown to bind to the histone acetyltransferases p300 and CBP, Hyrax/Parafibromin, which recruits histone modification complexes, and directly to the Med12 mediator complex subunit. However, this domain is insufficient for target gene activation in vivo, which requires Lgs, Pygo, and an amino acid in Arm that is critical for Lgs binding. In addition, although the C-terminal domain is a strong activator in cell culture, it is not sufficient to replace the function of Arm in vivo when fused to dTCF, whereas the activation domain of Pygo is. It has been proposed that Pygo interacts with unidentified general transcriptional regulators through its NHD. The current results suggest that the Pygo NHD recruits the mediator complex through the Kto/Med12 and Skd/Med13 subunits and that these subunits are essential for its activation function (Carrera, 2008).
An alternative view of the role of Pygo is that it acts as a nuclear anchor for Lgs and Arm. This model has been further refined by recent data showing that Pygo is constitutively localized to Wg target genes in a manner dependent on its NHD and on TCF, and it might function there to capture Arm. However, the finding that PygoDeltaPHD-GAL4 is sufficient to activate UAS-GFP expression in all cells in vivo strongly supports an additional activation function for Pygo. It is suggested that this function reflects its ability to recruit the mediator complex. Interestingly, the C. elegans Med12 and Med13 homologues have been implicated in the transcriptional repression of Wnt target genes although these effects have not been shown to be direct. Their dispensability for Wnt target gene activation may reflect the absence of pygo homologues in the worm genome (Carrera, 2008).
The kinase module of the mediator complex is commonly thought to have a repressive function; it has been shown to sterically hinder recruitment of Pol II, and Ras signaling promotes transcriptional elongation by inducing loss of this module from the mediator complex bound to C/EBP-regulated promoters. However, recent results suggest that the kinase module can play a role in transcriptional activation as well as repression. An exclusively repressive function would be difficult to reconcile with the observation that the genome-wide occupancy profiles of Cdk8 and Med13 characterized by ChIP match that of the core mediator complex. The current results support an essential and direct function for the Med12 and Med13 subunits in the activation of Wg target genes. The transcriptional and phenotypic profiles of mutants in the four subunits of the yeast kinase module are very similar. However, Drosophila cdk8 and cycC are required for only a subset of the functions of skd and kto that does not include Wg target gene activation. Therefore, Med12 and Med13 may have gained additional functions during the evolution of higher eukaryotes. The identical defects of the two mutants may reflect the requirement for Skd to stabilize the Kto protein. Similarly, Med24 stabilizes Med16 and Med23 and promotes their incorporation into the mediator complex (Carrera, 2008 and references therein).
Several mediator complex subunits act as adaptors that link specific transcription factors to the mediator complex. For example, Med1 interacts with nuclear receptors; Med23 interacts with phosphorylated Elk-1, the adenovirus E1A protein, and Heat shock factor; Med16 interacts with differentiation-inducing factor; and Med15 interacts with Smad2/3 and Sterol regulatory element-binding protein. The current results show that, despite their location in a module that is not part of the core mediator complex, Med12 and Med13 act as adaptors for Pygo. These subunits also are likely to act as adaptors for additional transcription factors because mutations in Drosophila and other organisms have other phenotypes that cannot be explained by loss of Wg signaling. Indeed, Med12 has been shown to interact with both Sox9 and Gli3. The yeast Med13 homologue is a target for Ras-regulated PKA phosphorylation, suggesting the interesting possibility that Wg or other signals might directly regulate the activity of Med12 or Med13. Finally, because skd and kto are not essential for normal cell proliferation or survival, they may provide targets for the treatment of Wnt-driven cancers (Carrera, 2008).
Mediator (MED) is a conserved multisubunit complex bridging transcriptional activators and repressors to the general RNA polymerase II initiation machinery. In yeast, MED is organized in three core modules and a separable 'Cdk8 module' consisting of the cyclin-dependent kinase Cdk8, its partner CycC, Med12 and Med13. This regulatory module, specifically required for cellular adaptation to environmental cues, is thought to act through the Cdk8 kinase activity. This study has investigated the functions of the four Cdk8 module subunits in the metazoan model Drosophila. Physical interactions detected among the four fly subunits provide support for a structurally conserved Cdk8 module. The in vivo functions of this module were analyzed using null mutants for Cdk8, CycC, Med12 and Med13. Each gene is required for the viability of the organism but not of the cell. Cdk8-CycC and Med12-Med13 act as pairs, which share some functions but also have distinct roles in developmental gene regulation. These data reveal functional attributes of the Cdk8 module, apart from its regulated kinase activity, that may contribute to the diversification of genetic programs (Loncle, 2007).
The observation that fly Cdk8 and CycC proteins interact with Med12 and Med13 in vitro and/or in yeast cells reinforces the notion of a conserved Cdk8 module deduced from whole-genome analyses. Genetic data provide evidence that the four proteins composing the fly Cdk8 module can act together in vivo, as seen for the regulation of sens in sensory organ development. However, their divergent effects in regulating the target genes bab2, dac and dpp during tarsal and eye differentiation lead to an inference of functional diversification of Cdk8-CycC and Med12-Med13 into two distinct pairs possessing diverging functions within the confines of a shared MED module. Interestingly, whereas Med12 and Med13 are specifically required for a temporally restricted regulation of dac and dpp in eye disc cells, the same patterning genes are unaffected in leg disc mutant cells. This indicates that Med12-Med13 activity depends both on the target genes and the developmental context (Loncle, 2007).
The prevailing view of Cdk8 module action, based on the functional comparison of its four components in the budding yeast, holds that this MED module serves exclusively to ensure regulated kinase activity. Interestingly, recent work has raised the possibility of a Cdk8-independent Med13 activity situated downstream of the S. cerevisiae Ras/PKA signaling pathway. Comparative analysis in Drosophila has revealed that Drosophila Med12-Med13 can act independently of Cdk8-CycC. One possible explanation is that another Cdk and/or cyclin may partially replace these proteins within the fly 'Cdk8' module. However, whereas purified mammalian MED complexes contain the Cdk8-related subunit Cdk11 and human Cdk3 can interact with CycC in cultured cells, neither Cdk11 nor Cdk3 has a counterpart in the D. melanogaster genome. Furthermore, the equivalent mutant phenotypes of Cdk8 and CycC in the in vivo analysis support the idea that Cdk8 or CycC do not associate with another cyclin or Cdk. It is concluded that Drosophila Med12-Med13 likely function independently of a Cdk kinase activity in vivo (Loncle, 2007).
The preceding considerations lead to a deduction that Med12 and Med13 are able to associate with the core MED independently of Cdk8 or CycC. Conversely, it has been proposed that S. cerevisiae Med12 and Med13 are required for the stable association of the Cdk8-CycC pair to core MED. Accordingly, MED from human cells depleted for Med12 also exhibits diminished Cdk8. In the present work, both the observed binding of fly Med12 and Med13 to Cdk8 and CycC in vitro and the genetic dependency of Cdk8 on Med12 in vivo reinforce this model. Importantly, although the data are consistent with a role of Med12-Med13 proteins in associating the Cdk8-CycC pair to core MED, they above all highlight that Med12-Med13 also act independently of Cdk8 or CycC (Loncle, 2007).
The biochemical nature of this Cdk-independent activity of Med12-Med13 remains to be deciphered. It seems likely that the large Med12-Med13 pair (~500 kDa in metazoans) directly contributes to the extensive structural rearrangements occurring within MED on binding to specific activators. Consistent with this idea, mammalian Med12 physically interacts with diverse transcriptional regulators. Such Cdk-independent regulatory activity of Med12 and Med13 may directly impact the interaction of core MED with PolII. It is speculated that kinase-independent Med12-Med13 activities may have contributed extensively during evolution to regulate and diversify cell differentiation processes (Loncle, 2007).
Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. It has previously been reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. This study shows, in cultured HeLa cells, that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes (Fryer, 2004).
A number of cyclins have been described, most of which act together with their catalytic partners, the cyclin-dependent kinases (Cdks), to regulate events in the eukaryotic cell cycle. Cyclin C was originally identified by a genetic screen for human and Drosophila cDNAs that complement a triple knock-out of the CLN genes in Saccharomyces cerevisiae. Unlike other cyclins identified in this complementation screen, there has been no evidence that cyclin C has a cell-cycle role in the cognate organism. This study reports that cyclin C is a nuclear protein present in a multiprotein complex. It interacts both in vitro and in vivo with Cdk8, a novel protein-kinase of the Cdk family, structurally related to the yeast Srb10 kinase. Cdk8 can interact in vivo with the large subunit of RNA polymerase II and a kinase activity that phosphorylates the RNA polymerase II large subunit is present in Cdk8 immunoprecipitates. Based on these observations and sequence similarity to the kinase/cyclin pair Srb10/Srb11 in S. cerevisiae, it is suggested that cyclin C and Cdk8 control RNA polymerase II function (Leclerc, 1996).
Anti-cyclin C antibodies identified cyclin C as a nuclear protein whose level does not oscillate during the blastoderm cell cycles in Drosophila embryos. Because analysis of the same embryos with cyclin A and cyclin B antisera revealed mitotic destruction of these cyclins, it appears that cyclin C is not subject to the degradation that drives oscillation of these cyclins. Absence of mitotic destruction was further supported by cyclin C immunolocalization in the embryo: antigen levels were constant throughout interphase nuclei, and at mitosis there was striking localization of the antigen to mitotic chromosomes. The protein, which migrates in SDS-PAGE with an apparent molecular mass of 28 kDa, sediments on native sucrose gradients with an estimated mass of ~500 kDa, which indicates that cyclin C is part of a large multiprotein complex. This unusually large size allowed the co-sedimentation of members of the Cdk family and other cell cycle regulators with cyclin C fractions to be tested. Neither DmCdc2, DmCdc2c (alias DmCdk2), cyclin A, nor cyclin B co-sedimented with cyclin C. They were present in complexes in the 100- to 200-kDa range as expected from sedimentation results. Consequently, the co-sedimentation of cyclin C and a unique 51-kDa band recognized by anti-human Cdk8 antibodies strongly suggested in vivo interaction between these two molecules; this was confirmed later by co-immunoprecipitation of DmCdk8 and cyclin C from nuclear extracts. It also falls in line with the recent finding that human Cdk8 interacts specifically with cyclin C in HeLa cell lysates (Leclerc, 1996).
Based on sequence data for a human Cdk8 and the yeast Ume5/SrblO protein, Drosophila sequences were amplified by PCR, and a cDNA clone for the DmCDK8 gene was isolated and sequenced. The human and Drosophila Cdk8 aminoacid sequences show numerous identities, and comparison to SrblO and other Cdks leads to a few remarks. The classical PSTAIRE sequence that is involved in Cdk-cyclin interactions is replaced in both Cdk8 and SrblO by a S(M/Q)SACRE sequence, whose conservation could reflect the presence of a contact region for related cyclins like Srbl 1 and cyclin C. The 'T-loop' region of the Cdks is regulatory: in the inactive structure it virtually blocks the active site, while the active site is unmasked in the cyclin-bound structure. Full activation of characterized Cdks requires phosphorylation of the T-loop. Although the structure of the phosphorylated enzyme has not yet been solved, based on an analogy to the structure of the cyclic AMP-induced kinase, it has been proposed that phosphorylation introduces new interactions that stabilize the active conformation of the T-loop. Cdk8 of human and Drosophila appear to lack a requirement for this activating phosphorylation because the T-loops of these kinases lack candidate residues for phosphorylation (S, T, or Y). Although SrblO has a T within its T-loop, it is not clear whether it is analogous to the phosphorylated residue in other Cdks. The ambiguity arises because both Cdk8's and SrblO have a three-aminoacid insertion within the T-loop, and the level of homology is not sufficient to define the correct alignment. Most likely the change in the T-loop occurred at the site that is usually phosphorylated because structural comparison indicates that adjacent residues are involved in highly conserved structure. It is suggested that the Cdk8 kinases as well as the SrblO kinase do not require activating phosphorylation and that the Asp residue that has been introduced roughly at the position normally occupied by Thr provides a negative charge to substitute for phosphorylation (Leclerc, 1996).
Two results showed that the cloned Drosophila Cdk8 is specifically recognized by the antibody to the human Cdk8: in vitro-translated DmCdk8 protein was recognized by anti-human Cdk8 antibodies and comigrated on SDS-PAGE with a 51-kDa band seen in extracts. Western blots using this antibody showed that DmCdk8 co-sediments with cyclin C in sucrose gradients and immunoprecipitations showed that cyclin C co-precipitates with DmCdk8, suggesting that these proteins might work in association to produce an active kinase complex. Indeed, a kinase activity was found in anti-Cdk8 immunoprecipitate that phosphorylates a 240-kDa band that co-migrates with form IIo of RNA Pol II. It was confirmed that this band corresponded to form IIo of RNA Pol II by four independent lines of evidence. (1) RNA Pol II is present in the immunoprecipitates. (2) Depleting the extracts before immunoprecipitation with anti-Cdk8 antibodies suppresses the appearance of the 240-kDa phosphorylated band. (3) The 240-kDa phosphoprotein co-migrates exactly with form IIo of RNA Pol II run on the same gel. (4) Cdk8 immunocomplexes are capable of phosphorylating a CTD peptide in vitro (Leclerc, 1996).
Attempts to precipitate the cyclin C/DmCdk8 complex with two different antibodies to RNA polymerase failed. In the case of the ARNA3 antibodies, the failure can be simply explained because under the experimental conditions used no precipitation of the large subunit of RNA Pol II was seen. Anti-CTD antibodies efficiently immunoprecipitated Pol II in the extracts but no trace of Cdk8 or cyclin C were detected in the immunoprecipitate. Failure to co-immunoprecipitate both Cdk8 and cyclin C using anti-CTD antibodies might be due to a variety of reasons, but it might reflect a competition effect between antibody molecules and the proteins interacting with the CTD epitopes. A similar effect was previously suggested to explain the separation of the RNA Pol II core enzyme and a group of associated proteins called 'the mediator' (and among them, SrblO and Srbll) during affinity purification experiments using anti-CTD antibodies (Leclerc, 1996).
These experiments do not resolve whether phosphorylation of Pol II is due to the activity of the Cdk8 kinase itself. Other kinase activities might be present in the immune complex. For this reason, the presence was tested of a kinase previously implicated in phosphorylation of the CTD. Transcription factor TFIIH, which is involved in the phosphorylation of the CTD, contains Cdk7 and cyclin H. This pair of proteins is responsible for the in vitro CTD-kinase activity of the TFIIH complex. No trace of Drosophila Cdk7 protein was detected in the anti-Cdk8 immune complex using affinity- purified anti-DmCdk7 antibodies. This suggests either that the experimental conditions do not allow the stabilization of a complex comprising Cdk7 and Cdk8, or that the two kinases associate separately with the RNA Pol II enzyme. It indicates, furthermore, that the kinase activity present in the anti-Cdk8 immune precipitate is not due to the Cdk7 kinase (Leclerc, 1996).
In conclusion, this study has identified Cdk8, a kinase partner of cyclin C, and has demonstrated that it can interact in vivo with the large subunit of RNA Pol II. Furthermore, DmCdk8 is associated either directly or indirectly with a kinase activity that can phosphorylate the large subunit of RNA Pol II in vitro. The structural identity between Cdk8/cyclin C in Drosophila and SRB10/SRB11 in yeast, as well as the in vivo interaction between RNA Pol II and Cdk8 lead to the suggestion of a possible functional homology between the two kinase/cyclin pairs. Recent results assign a role for SRB10/SRB11 in transcriptional regulation in vivo as well as CTD phosphorylation in vitro. Drosophila genetics as well as additional biochemical studies will help to define the precise function of Cdk8/cyclin C. The possible involvement of cyclin C and Cdk8 in transcription suggests that the basis of the original isolation of cyclin C as a gene capable of complementing deficiencies in Gl cyclins in yeast should be re-evaluated. Although it is possible that cyclin C can play a cell cycle role in a foreign context, it is also possible that cyclin C function bypassed the Gl block by disturbing transcriptional controls in yeast (Leclerc, 1996).
Search PubMed for articles about Drosophila Cyclin C
Adler, A. S., McCleland, M. L., Truong, T., Lau, S., Modrusan, Z., Soukup, T. M., Roose-Girma, M., Blackwood, E. M. and Firestein, R. (2012). CDK8 maintains tumor dedifferentiation and embryonic stem cell pluripotency. Cancer Res 72: 2129-2139. PubMed ID: 22345154
Carrera, I., Janody, F., Leeds, N., Duveau, F. and Treisman, J. E. (2008). Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13. Proc. Natl. Acad. Sci. 105(18): 6644-9. PubMed ID: 18451032
Chen, J., Ezzeddine, N., Waltenspiel, B., Albrecht, T. R., Warren, W. D., Marzluff, W. F. and Wagner, E. J. (2012). An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3'-end formation. RNA 18: 2148-2156. PubMed ID: 23097424
Fryer, C. J., White, J. B. and Jones, K. A. (2004). Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol. Cell 16: 509-520. PubMed ID: 15546612
Galbraith, M. D., Donner, A. J. and Espinosa, J. M. (2010). CDK8: a positive regulator of transcription. Transcription 1: 4-12. PubMed ID: 21327159
Homem, C. C., Steinmann, V., Burkard, T. R., Jais, A., Esterbauer, H. and Knoblich, J. A. (2014). Ecdysone and mediator change energy metabolism to terminate proliferation in Drosophila neural stem cells. Cell 158: 874-888. PubMed ID: 25126791
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date revised: 15 January 2015
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