Activation of cardiac Cdk9 represses PGC-1 and confers a predisposition to heart failure.
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Hypertrophy allows the heart to adapt to workload but culminates in later pump failure; how it is achieved remains uncertain. Previously, we showed that hypertrophy is accompanied by activation of cyclin T/Cdk9, which phosphorylates the C-terminal domain of the large subunit of RNA polymerase II, stimulating transcription elongation and pre-mRNA processing; Cdk9 activity was required for hypertrophy in culture, whereas heart-specific activation of Cdk9 by cyclin T1 provoked hypertrophy in mice. Here, we report that alphaMHC-cyclin T1 mice appear normal at baseline yet suffer fulminant apoptotic cardiomyopathy when challenged by mechanical stress or signaling by the G-protein Gq. At pathophysiological levels, Cdk9 activity suppresses many genes for mitochondrial proteins including master regulators of mitochondrial function (peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), nuclear respiratory factor-1). In culture, cyclin T1/Cdk9 suppresses PGC-1, decreases mitochondrial membrane potential, and sensitizes cardiomyocytes to apoptosis, effects rescued by exogenous PGC-1. Cyclin T1/Cdk9 inhibits PGC-1 promoter activity and preinitiation complex assembly. Thus, chronic activation of Cdk9 causes not only cardiomyocyte enlargement but also defective mitochondrial function, via diminished PGC-1 transcription, and a resulting susceptibility to apoptotic cardiomyopathy. (+info)
Cyclin-dependent kinase-9: an RNAPII kinase at the nexus of cardiac growth and death cascades.
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Over the past decade and a half, the paradigm has emerged of cardiac hypertrophy and ensuing heart failure as fundamentally a problem in signal transduction, impinging on the altered expression or function of gene-specific transcription factors and their partners, which then execute the hypertrophic phenotype. Strikingly, RNA polymerase II (RNAPII) is itself a substrate for two protein kinases-the cyclin-dependent kinases Cdk7 and Cdk9--that are activated by hypertrophic cues. Phosphorylation of RNAPII in the carboxyl terminal domain (CTD) of its largest subunit controls a number of critical steps subsequent to transcription initiation, among them enabling RNAPII to overcome its stalling in the promoter-proximal region and to engage in efficient transcription elongation. Here, we summarize our current understanding of the RNAPII-directed protein kinases in cardiac hypertrophy. Cdk9 activation is essential in tissue culture for myocyte enlargement and sufficient in transgenic mice for hypertrophy to occur and yet is unrelated to the "fetal" gene program that is typical of pathophysiological heart growth. Although this trophic effect of Cdk9 appears benign superficially, pathophysiological levels of Cdk9 activity render myocardium remarkably susceptible to apoptotic stress. Cdk9 interacts adversely with Gq-dependent pathways for hypertrophy, impairing the expression of numerous genes for mitochondrial proteins, and, in particular, suppressing master regulators of mitochondrial biogenesis and function, perioxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1), and nuclear respiratory factor-1 (NRF-1). Given the dual transcriptional roles of Cdk9 in hypertrophic growth and mitochondrial dysfunction, we suggest the potential usefulness of Cdk9 as a target in heart failure drug discovery. (+info)
Cyclin-dependent kinase 9 is required for tumor necrosis factor-alpha-stimulated matrix metalloproteinase-9 expression in human lung adenocarcinoma cells.
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The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) promotes tumor progression through activation of matrix metalloproteinase (MMP) activity. MMP-9 is a gelatinase secreted by both cancer cells and surrounding stromal cells, and it contributes to TNF-alpha-stimulated tumor invasion and metastasis. Cyclin-dependent kinase 9 (CDK9), the catalytic component of positive transcription elongation factor-b, phosphorylates serine 2 residues in the C-terminal domain of RNA polymerase II for productive transcription elongation and is up-regulated upon exposure to various stresses. This study investigated roles of CDK9 in TNF-alpha-stimulated MMP-9 expression in human lung adenocarcinoma cells. CDK9 activity was inhibited using three different strategies, including the CDK9 pharmacological inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a dominant-negative CDK9, and a CDK9-specific small interfering RNA. All three approaches reduced TNF-alpha-mediated accumulation of MMP-9 in the conditioned media as demonstrated by gelatin zymography. In contrast, transforming growth factor-beta1-induced accumulation of MMP-2 was unaffected by DRB. Expression of the MMP-9 gene was examined using reverse transcription real time PCR and using a transient transfection assay to evaluate MMP-9 promoter activity. DRB reduced the TNF-alpha-induced increase in MMP-9 mRNA levels but did not effect transforming growth factor-beta1-induced MMP-2 mRNA expression. Consistently DRB and dominant-negative CDK9 completely abrogated TNF-alpha-stimulated human MMP-9 promoter activity. TNF-alpha did not regulate expression or localization of CDK9 or its regulatory partner Cyclin T. However, TNF-alpha stimulated CDK9 binding to Cyclin T and MMP-9 gene occupancy by both CDK9 and the serine 2-phosphorylated form of RNA polymerase II. Our findings indicate that CDK9 mediates TNF-alpha-induced MMP-9 transcription. Disruption of TNF-alpha signaling using CDK9 inhibitors could serve as a potential therapeutic strategy against tumor invasion and metastasis. (+info)
Granulin and granulin repeats interact with the Tat.P-TEFb complex and inhibit Tat transactivation.
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The cellular positive transcription elongation factor b (P-TEFb), containing cyclin T1 and cyclin-dependent kinase 9 (CDK9), interacts with the human immunodeficiency virus, type 1 (HIV-1) regulatory protein Tat to enable viral transcription and replication. Cyclin T1 is an unusually long cyclin and is engaged by cellular regulatory proteins. Previous studies showed that the granulin/epithelin precursor (GEP) binds the histidine-rich region of cyclin T1 and inhibits P-TEFb function. GEP is composed of repeats that vary in sequence and properties. GEP also binds directly to Tat. Here we show that GEP and some of its constituent granulin repeats can inhibit HIV-1 transcription via Tat without directly binding to cyclin T1. The interactions of granulins with Tat and cyclin T1 differ with respect to their binding sites and divalent cation requirements, and we identified granulin repeats that bind differentially to Tat and cyclin T1. Granulins DE and E bind Tat but do not interact directly with cyclin T1. These granulins are present in complexes with Tat and P-TEFb in which Tat forms a bridge between the cellular proteins. Granulins DE and E repress transcription from the HIV-1 LTR and gene expression from the viral genome, raising the possibility of developing granulin-based inhibitors of viral infection. (+info)
Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.
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Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat. (+info)
Cdk9 regulates neural differentiation and its expression correlates with the differentiation grade of neuroblastoma and PNET tumors.
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Cdk9 is a member of the Cdc2-like family of kinases. Its cyclin partners are members of the family of cyclin T (T1, T2a and T2b) and cyclin K. The Cdk9/Cyclin T complex appears to be involved in regulating several physiological processes. Recently, Cdk9 has been identified as a regulator of the differentiation program of several cell types, such as muscle cells, monocytes and lymphocytes, suggesting that it may have a function in controlling specific differentiative pathways. We analyzed whether Cdk9 and Cyclin T1 may be involved in the regulation of neuron and astrocyte differentiation. Cdk9 and Cyclin T1 expression levels were monitored during the differentiation program of neuroblastoma and astrocytoma cell lines. Our results suggest that Cdk9/Cyclin T1 complex may be required for neuron differentiation induced by retinoic acid, because the expression level of the complex varies during differentiation, but no significant changes were observed in its expression in the astrocytoma cell line. In addition, the expression of Cdk9 and Cyclin T1 was evaluated by immunohistochemistry in samples of neuroblastoma, PNET (Primary Neuroectodermal Tumor) and astrocytoma tumors of different grades, in order to assess whether there was a correlation between Cdk9 expression and tumor grading. Our results show that in neuroblastoma and PNET tumor samples Cdk9 is more expressed the more differentiated the tumor is. Conversely, no significant alteration of Cdk9 expression was observed in astrocytoma tumor samples of different grades, thus confirming the results obtained for the cell lines. (+info)
Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat.
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The active form of the positive transcription elongation factor b (P-TEFb) consists of cyclin T and the kinase Cdk9. P-TEFb stimulates transcription by phosphorylating the C-terminal domain of RNA polymerase II. It becomes inactivated when associated in a tetrameric complex with the abundant 7SK small nuclear RNA and the recently identified protein Hexim1. In this study, we identified a stable and soluble C-terminal domain (residues 255-359) in Hexim1 of 12.5-kDa size that binds the cyclin boxes of Cyclin T1. Functional assays in HeLa cells showed that this cyclin T-binding domain (TBD) is required for the binding of Hexim1 to P-TEFb and inhibition of transcriptional activity in vivo. Analytical gel filtration and GST pull-down experiments revealed that both full-length Hexim1 and the TBD are homodimers. Isothermal titration calorimetry yielded a weak multimer for the TBD with a multimerization constant of 1.3 x 10(3) m. The binding affinity between the TBD and cyclin T1 was analyzed with fluorescence spectroscopy methods, using a dansyl-based fluorescence label at position G257C. Equilibrium fluorescence titration and stopped flow fast kinetics yield a dissociation constant of 1.2 mum. Finally, we tested the effect of the HIV-1 Tat protein on the cyclin T1-TBD complex formation. GST pull-down experiments and size exclusion chromatography exhibit a mutually exclusive binding of the two effectors to cyclin T1. Our data suggest a model where HIV-1 Tat competes with Hexim1 for cyclin T1 binding, thus releasing P-TEFb from the inactive complex to stimulate the transcription of HIV-1 gene expression. (+info)
The carboxyl-terminal domain of RNA polymerase II is phosphorylated by a complex containing cdk9 and infected-cell protein 22 of herpes simplex virus 1.
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The infected-cell protein 22 (ICP22), a regulatory protein encoded by the alpha22 gene of herpes simplex virus 1, is required for the optimal expression of a set of late viral proteins that includes the products of the U(S)11, U(L)38, and U(L)41 genes. ICP22 has two activities. Thus, ICP22 and the U(L)13 protein kinase mediate the activation of cdc2 and degradation of its partners, cyclins A and B. cdc2 and its new partner, the DNA polymerase accessory factor (U(L)42), bind topoisomerase IIalpha in an ICP22-dependent manner. In addition, ICP22 and U(L)13 mediate an intermediate phosphorylation of the carboxyl terminus of RNA polymerase II (RNA POL II). Here we report another function of ICP22. Thus, ICP22 physically interacts with cdk9, a constitutively active cyclin-dependent kinase involved in transcriptional regulation. A protein complex containing ICP22 and cdk9 phosphorylates in vitro the carboxyl-terminal domain of RNA POL II in a viral U(S)3 protein kinase-dependent fashion. Finally, the carboxyl-terminal domain of RNA POL II fused to glutathione S-transferase is phosphorylated in reaction mixtures containing complexes pulled down with ICP22 or cdk9 immune precipitated from lysates of wild-type parent virus or deltaU(L)13 but not deltaU(S)3 mutant-infected cells. The experiments described here place ICP22 and cdk9 in a complex with the carboxyl-terminal domain of RNA POL II. At the same time we confirm the requirement of ICP22 and the U(L)13 protein kinase in the posttranslational modification of RNA POL II that alters its electrophoretic mobility, although U(S)3 kinase appears to play a role in a cell-type-dependent fashion. (+info)