scholarly journals A precisely positioned MED12 activation helix stimulates CDK8 kinase activity

2020 ◽  
Vol 117 (6) ◽  
pp. 2894-2905 ◽  
Author(s):  
Felix Klatt ◽  
Alexander Leitner ◽  
Iana V. Kim ◽  
Hung Ho-Xuan ◽  
Elisabeth V. Schneider ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Kinase Inhibitors ◽  
Terminal Segment ◽  
In Vivo Studies ◽  
Regulatory Subunit ◽  
Catalytic Core ◽  
Cyclin C ◽  
C Complex

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.

scholarly journals Current understanding of nonsurgical interventions for refractory differentiated thyroid cancer: a systematic review

2021 ◽  
pp. FSO738
Author(s):  
Heidi Jones ◽  
Victoria Green ◽  
James England ◽  
John Greenman
Keyword(s):  
Thyroid Cancer ◽  
Kinase Inhibitors ◽  
Oncolytic Viruses ◽  
In Vivo Studies ◽  
T Cell Therapy ◽  
Treatment Regimens ◽  
Number Of Patients ◽  
Related Mortality

Thyroid cancer incidence and related mortality is increasing year-on-year, and although treatment for early disease with surgery and radioiodine results in a 98% 5-year survival rate, recurrence and treatment refractory disease is evident in an unacceptable number of patients. Alternative treatment regimens have therefore been sought in the form of tyrosine kinase inhibitors, immunotherapy, vaccines, chimeric antigen receptor T-cell therapy and oncolytic viruses. The current review aims to consolidate knowledge and highlight the latest clinical trials using secondary therapies in thyroid cancer treatment, focusing on both in vitro and in vivo studies, which have investigated therapies other than radioiodine.

scholarly journals TORC1 signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

Genetics ◽  
2021 ◽  
Author(s):  
Riley Horvath ◽  
Nicole Hawe ◽  
Cindy Lam ◽  
Konstantin Mestnikov ◽  
Mariam Eji-Lasisi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Nuclear Localization ◽  
Protein Phosphatase ◽  
Regulatory Subunit ◽  
Gal Genes ◽  
Dependent Site ◽  
Insight Into

Abstract Cdk8 of the RNA Polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55, and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

scholarly journals Neospora caninum: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 382 ◽  
Author(s):  
Pablo Winzer ◽  
Nicoleta Anghel ◽  
Dennis Imhof ◽  
Vreni Balmer ◽  
Luis-Miguel Ortega-Mora ◽  
...  
Keyword(s):  
Drug Exposure ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Neospora Caninum ◽  
Farm Animals ◽  
In Vivo Studies ◽  
Drug Pressure ◽  
Drug Removal

Background: Bumped kinase inhibitors (BKIs) are potential drugs for neosporosis treatment in farm animals. BKI-1294 exposure results in the formation of multinucleated complexes (MNCs), which remain viable in vitro under constant drug pressure. We investigated the formation of BKI-1294 induced MNCs, the re-emergence of viable tachyzoites following drug removal, and the localization of CDPK1, the molecular target of BKIs. Methods: N. caninum tachyzoites and MNCs were studied by TEM and immunofluorescence using antibodies directed against CDPK1, and against NcSAG1 and IMC1 as markers for tachyzoites and newly formed zoites, respectively. Results: After six days of drug exposure, MNCs lacked SAG1 surface expression but remained intracellular, and formed numerous zoites incapable of disjoining from each other. Following drug removal, proliferation continued, and zoites lacking NcSAG1 emerged from the periphery of these complexes, forming infective tachyzoites after 10 days. In intracellular tachyzoites, CDPK1 was evenly distributed but shifted towards the apical part once parasites were extracellular. This shift was not affected by BKI-1294. Conclusions: CDPK1 has a dynamic distribution depending on whether parasites are located within a host cell or outside. During MNC-to-tachyzoite reconversion newly formed tachyzoites are generated directly from MNCs through zoites of unknown surface antigen composition. Further in vivo studies are needed to determine if MNCs could lead to a persistent reservoir of infection after BKI treatment.

scholarly journals Fedratinib Attenuates Bleomycin-Induced Pulmonary Fibrosis via the JAK2/STAT3 and TGF-β1 Signaling Pathway

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4491
Author(s):  
Hao Ruan ◽  
Jiaoyan Luan ◽  
Shaoyan Gao ◽  
Shuangling Li ◽  
Qiuyan Jiang ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Signaling Pathway ◽  
Kinase Inhibitors ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
In Vivo Studies ◽  
Matrix Deposition ◽  
Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease with multiple causes, characterized by excessive myofibrocyte aggregation and extracellular matrix deposition. Related studies have shown that transforming growth factor-β1 (TGF-β1) is a key cytokine causing fibrosis, promoting abnormal epithelial–mesenchymal communication and fibroblast-to-myofibroblast transition. Fedratinib (Fed) is a marketed drug for the treatment of primary and secondary myelofibrosis, targeting selective JAK2 tyrosine kinase inhibitors. However, its role in pulmonary fibrosis remains unclear. In this study, we investigated the potential effects and mechanisms of Fed on pulmonary fibrosis in vitro and in vivo. In vitro studies have shown that Fed attenuates TGF-β1- and IL-6-induced myofibroblast activation and inflammatory response by regulating the JAK2/STAT3 signaling pathway. In vivo studies have shown that Fed can reduce bleomycin-induced inflammation and collagen deposition and improve lung function. In conclusion, Fed inhibited inflammation and fibrosis processes induced by TGF-β1 and IL-6 by targeting the JAK2 receptor.

Apolipoprotein E-Mimetic Therapeutic Peptides Mediate CLL Cell Cytotoxicity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 359-359
Author(s):  
Dale J. Christensen ◽  
Karen M. Bond ◽  
Alicia D. Volkheimer ◽  
Jessica Oddo ◽  
Youwei Chen ◽  
...  
Keyword(s):  
B Cells ◽  
Apolipoprotein E ◽  
Colorimetric Assay ◽  
In Vivo Studies ◽  
Regulatory Subunit ◽  
Community Control ◽  
Mimetic Peptides ◽  
Therapeutic Peptides

Abstract Background and Significance : Even though we have treatments for CLL, it remains an incurable leuke mia. We need new and better treatments for this disease. The Akt kinase is usually constitutively acti vated (phosphorylated) in CLL, and it acts to maintain CLL cell viability. Chromosome deletion at 11q22–23 is frequently seen in CLL, and this cytogenetic abnormality portends a bad prognosis. The PPP2R1B gene that encodes the Ab constant regulatory subunit of the tumor suppressor protein phos phatase 2a (PP2a) is within the deleted segment in CLL patients with deleted 11q22–23. The resulting underexpression noted in those with deleted 11q22–23 leads to decreased PP2a activity in CLL cells. PP2a is important in deactivation of Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and nuclear factor kB (through IkK). We have developed apolipoprotein E-mimetic therapeutic peptides that potently decrease phosphorylation of Akt, decrease TNF and nitric oxide (NO) and NO synthase (NOS) expression, and display anti-inflammatory activity in vitro and in vivo. NOS is overexpressed in CLL, and its product NO inhibits CLL cell apoptosis. Methods : Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CD19+ CLL or normal B cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. The apoE-mimetic peptides were prepared by chemical synthesis. Results : The apoE-mimetic COG compounds are peptides of 10 to 34 amino acids derived from the ligand-binding region of the apolipoprotein E holoprotein. Samples from 6 early stage CLL patients and 11 normal individuals were examined. Five of 6 patients were Rai stage 0 at presentation, and one was stage 1. They had been followed 5.6 yr (median; range 1.9–10.6 yr). Five of 6 were CD38 negative, and 2 of 6 were Zap-70 positive. Of 5analyzed, all had mutated IgVH gene. Five of 6 patients had not been treated. Of 6 peptides examined, all displayed some cytotoxicity for CLL cells in vitro. Peptide COG 112 was the most potent, while the control peptide with an inverted sequence (COG 056) had very little or no activity (Table). CLL PBMC Agent ED50 (nM) ED50 range (n) ED50 (nM) ED50 range (n) COG 112 (active) 215 64 to 351 (6) 5,150 1,100 to >12,500 (6) COG 056 (control) 13,546 2771 to 20,418 (6) >25,000 20,470 to >25,500 (6) COG 112 induced cell death in a dose-dependent fashion in all patients’ samples.The ED50 of COG 112 for normal B cells was very high (> 24,400 nM). COG 112 was approximatel 24 to 116 fold more potent for killing of CLL cells compared to normal PBMC or purified B cells. In vivo studies in normal mice using COG 112 revealed no toxicity even with doses of 100 mg/kg. Conclusions : ApoE mimetic peptides kill CLL cells in vitro with high efficacy and potency (ED50s in the low nanomolar range). The cytotoxicity is very specific for CLL cells compared to normal PBMC and B cells (24 to 116 fold more potent for CLL cells). Preliminary studies show that the peptide is nontoxic in vivo in normal mice. In vivo trials with apoE peptides in patients with CLL should help determine the toxicity and efficacy in patients.

scholarly journals Rsp5 Ubiquitin-Protein Ligase Mediates DNA Damage-Induced Degradation of the Large Subunit of RNA Polymerase II in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (10) ◽  
pp. 6972-6979 ◽  
Author(s):  
Sylvie L. Beaudenon ◽  
Maria R. Huacani ◽  
Guangli Wang ◽  
Donald P. McDonnell ◽  
Jon M. Huibregtse
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
26S Proteasome ◽  
Regulatory Subunit ◽  
Ubiquitin Protein Ligase

ABSTRACT Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation ofSEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.

In vivo studies of the γ subunit of retinal cGMP-phophodiesterase with a substitution of tyrosine-84

2001 ◽  
Vol 353 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Stephen H. TSANG ◽  
Clyde K. YAMASHITA ◽  
Kentaro DOI ◽  
Daniel J. SALCHOW ◽  
Nicole BOUVIER ◽  
...  
Keyword(s):  
Regulatory Control ◽  
In Vivo Studies ◽  
Wild Type ◽  
Targeted Disruption ◽  
Catalytic Core ◽  
Γ Subunit ◽  
Biochemical Assays ◽  
Fold Reduction

The inhibitory rod cGMP phosphodiesterase γ subunit (PDEγ) is a major component of the photoresponse and is required to support rod integrity. Pdegtm1/Pdegtm1 mice (which lack PDEγ owing to a targeted disruption of the Pdeg gene) suffer from a very rapid and severe photoreceptor degeneration. The Y84G (Tyr84 → Gly) allele of PDEγ has previously been shown in experiments carried out in vitro to reduce the regulatory control of the PDE catalytic core (PDEαβ) exerted by the wild-type γ subunit. To determine the effects of this mutation on in vivo function, the murine opsin promoter was used to direct expression to the photoreceptors of +/Pdegtm1 mice of a mutant Y84G and a wild-type PDEγ control transgene. The transgenic mice were crossed with Pdegtm1/Pdegtm1 mice to generate animals able to synthesize only the transgenic PDEγ. Our results showed that wild-type PDEγ and Y84G transgenes could complement the Pdegtm1/Pdegtm1 mutant for photoreceptor survival. The mutation caused a significant biochemical defect in PDE activation by transducin. However, the Y84G mutation did not fully eliminate the control of PDEγ on the PDE catalytic core in vivo; the expression of the mutant subunit was associated with only a 10-fold reduction in the amplitude of the a-wave and a 1.5-fold decrease in the b-wave of the corneal electroretinogram. Unexpectedly, the mutation caused a much ‘milder’ phenotype in vivo than was predicted from the biochemical assays in vitro.

scholarly journals Drosophila Cdk8, a kinase partner of cyclin C that interacts with the large subunit of RNA polymerase II.

1996 ◽  
Vol 7 (4) ◽  
pp. 505-513 ◽  
Author(s):  
V Leclerc ◽  
J P Tassan ◽  
P H O'Farrell ◽  
E A Nigg ◽  
P Léopold
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Eukaryotic Cell ◽  
Knock Out ◽  
Cyclin C

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. Here we report 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. We also show that Cdk8 can interact in vivo with the large subunit of RNA polymerase II and that 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, we suggest that cyclin C and Cdk8 control RNA polymerase II function.

scholarly journals Molecular functions of nuclear actin in transcription

2006 ◽  
Vol 172 (7) ◽  
pp. 967-971 ◽  
Author(s):  
Piergiorgio Percipalle ◽  
Neus Visa
Keyword(s):  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
In Vivo Studies ◽  
Macromolecular Assemblies ◽  
In Vivo Experiments ◽  
Specific Subset

Actin is not only a major cytoskeletal component in all eukaryotic cells but also a nuclear protein that plays a role in gene transcription. We put together data from in vitro and in vivo experiments that begin to provide insights into the molecular mechanisms by which actin functions in transcription. Recent studies performed in vitro have suggested that actin, in direct contact with the transcription apparatus, is required in an early step of transcription that is common to all three eukaryotic RNA polymerases. In addition, there is evidence from in vivo studies that actin is involved in the transcription elongation of class II genes. In this case, actin is bound to a specific subset of premessenger RNA binding proteins, and the actin–messenger RNP complex may constitute a molecular platform for recruitment of histone-modifying enzymes. We discuss a general model for actin in RNA polymerase II transcription whereby actin works as a conformational switch in conjunction with specific adaptors to facilitate the remodeling of large macromolecular assemblies at the promoter and along the active gene.

Sign in / Sign up

Export Citation Format

Share Document