Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Methods are described for assaying (Tetrahymena) histone acetyltransferase activity and (Drosophila) casein kinase II activity by spotting extracts on nitrocellulose filters. The methods are quantitative over a wide range of enzyme concentrations and are almost as sensitive as liquid assays. Examples are presented for illustrating the use of these methods for enzyme purification, concentration, and desalting, as well as for electrophoretic blotting from agarose gels. A simple method for autoradiographic enhancement of nitrocellulose filters is also described.
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PMID:Enzyme activity dot blots: a rapid and convenient assay for acetyltransferase or protein kinase activity immobilized on nitrocellulose. 346 11

Mounting evidence suggests that eukaryotic RNA polymerases preassociate with multiple transcription factors in the absence of DNA, forming RNA polymerase holoenzyme complexes. We have purified an apparent RNA polymerase I (Pol I) holoenzyme from Xenopus laevis cells by sequential chromatography on five columns: DEAE-Sepharose, Biorex 70, Sephacryl S300, Mono Q, and DNA-cellulose. Single fractions from every column programmed accurate promoter-dependent transcription. Upon gel filtration chromatography, the Pol I holoenzyme elutes at a position overlapping the peak of Blue Dextran, suggesting a molecular mass in the range of approximately 2 MDa. Consistent with its large mass, Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels reveal approximately 55 proteins in fractions purified to near homogeneity. Western blotting shows that TATA-binding protein precisely copurifies with holoenzyme activity, whereas the abundant Pol I transactivator upstream binding factor does not. Also copurifying with the holoenzyme are casein kinase II and a histone acetyltransferase activity with a substrate preference for histone H3. These results extend to Pol I the suggestion that signal transduction and chromatin-modifying activities are associated with eukaryotic RNA polymerases.
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PMID:Histone acetyltransferase and protein kinase activities copurify with a putative Xenopus RNA polymerase I holoenzyme self-sufficient for promoter-dependent transcription. 985 2

We previously reported that the activation of the M promoter of the human choline acetyltransferase (ChAT) gene by butyrate and trapoxin in transfected CHP126 cells is blocked by PD98059, a specific mitogen-activated protein kinase kinase (MEK) inhibitor (E. Espinos and M. J. Weber, Mol. Brain Res. 56:118-124, 1998). We now report that the transcriptional effects of histone deacetylase inhibitors are mediated by an H7-sensitive serine/threonine protein kinase. Activation of the ChAT promoter by butyrate and trapoxin was blocked by 50 microM H7 in both transient- and stable-transfection assays. Overexpression of p300, a coactivator protein endowed with histone acetyltransferase activity, stimulated the ChAT promoter and had a synergistic effect on butyrate treatment. These effects were blocked by H7 and by overexpressed adenovirus E1A 12S protein. Moreover, both H7 and PD98059 suppressed the activation of the Rous sarcoma virus (RSV) and simian virus 40 promoters by butyrate in transfection experiments. Similarly, the induction of the cellular histone H1(0) gene by butyrate in CHP126 cells was blocked by H7 and by PD98059. Previous data (L. Cuisset, L. Tichonicky, P. Jaffray, and M. Delpech, J. Biol. Chem. 272:24148-24153, 1997) showed that the induction of the H1(0) gene by butyrate is blocked by okadaic acid, an inhibitor of protein phosphatases. We now show that the activation of the ChAT and RSV promoters by butyrate in transfected CHP126 cells is also blocked by 200 nM okadaic acid. Western blotting and in vivo metabolic labeling experiments showed that butyrate has a biphasic effect on histone H3 phosphorylation, i.e., depression for up to 16 h followed by stimulation. The data thus strongly suggest that the transcriptional effects of histone deacetylase inhibitors are mediated through the activation of MEK1 and of an H7-sensitive protein kinase in addition to protein phosphatases.
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PMID:Cooperation between phosphorylation and acetylation processes in transcriptional control. 1020 71

We have previously identified a cDNA encoding a cellular protein, Tip60 (Tat interactive protein, 60 kDa), that specifically interacts with the Tat (transactivating transcriptional regulator) protein of the human immunodeficiency virus-1 (HIV-1). In this report, we have characterized cellular Tip and find that it is a 60 kDa nuclear protein expressed in a wide variety of differentiated cell lines from insects to man. To identify cellular functions of Tip, we have assayed the effects of Tip on cellular pathways that Tat has been reported to affect. Overexpression of Tip results in an almost complete block in activation of a Gal4-CREB (cAMP response element binding protein) fusion protein by cyclic AMP dependent protein kinase A (PKA). This inhibition appears to be mediated through direct interaction of Tip and CREB, since Tip directly binds to CREB protein in vitro. We show that amino acid substitutions of two conserved amino acids found in the putative acetyl coenzyme A binding motif of Tip completely abolishes the histone acetyltransferase (HAT) activity of recombinant Tip. Inhibition of CREB activation by Tip is not diminished in a HAT negative Tip mutant, indicating that Tip can negatively regulate gene expression independent of HAT activity. Recently, Tip has also been shown to be a transcriptional coactivator of nuclear hormone receptors; therefore, Tip can both activate transcription factors of one signaling pathway (nuclear hormone receptors) and bind to a different transcription factor (CREB) and inhibit activation of another signaling pathway.
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PMID:Tip60 inhibits activation of CREB protein by protein kinase A. 1072 Apr 89

The cyclic AMP (cAMP)-responsive factor CREB promotes cellular gene expression, following its phosphorylation at Ser133, via recruitment of the coactivator paralogs CREB-binding protein (CBP) and p300. CBP and p300, in turn, appear to mediate target gene induction via their association with RNA polymerase II complexes and via intrinsic histone acetyltransferase activities that mobilize promoter-bound nucleosomes. In addition to cAMP, a wide variety of stimuli, including hypoxia, UV irradiation, and growth factor addition, induce Ser133 phosphorylation with stoichiometry and kinetics comparable to those induced by cAMP. Yet a number of these signals are incapable of promoting target gene activation via CREB phosphorylation per se, suggesting the presence of additional regulatory events either at the level of CREB-CBP complex formation or in the subsequent recruitment of the transcriptional apparatus. Here we characterize a Tyr134Phe CREB mutant that behaves as a constitutive activator in vivo. Like protein kinase A (PKA)-stimulated wild-type CREB, the Tyr134Phe polypeptide was found to stimulate target gene expression via the Ser133-dependent recruitment of CBP and p300. Biochemical studies reveal that mutation of Tyr134 to Phe lowers the K(m) for PKA phosphorylation and thereby induces high levels of constitutive Ser133 phosphorylation in vivo. Consistent with its constitutive activity, Tyr134Phe CREB strongly promoted differentiation of PC12 cells in concert with suboptimal doses of nerve growth factor. Taken together, these results demonstrate that Ser133 phosphorylation is sufficient for cellular gene activation and that additional signal-dependent modifications of CBP or p300 are not required for recruitment of the transcriptional apparatus to the promoter.
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PMID:Characterization of a CREB gain-of-function mutant with constitutive transcriptional activity in vivo. 1082 95

The protein kinase ribosomal S6 kinase 2 (RSK2) has been implicated in phosphorylation of transcription factor CREB and histone H3 in response to mitogenic stimulation by epidermal growth factor. Binding of phospho-CREB to the coactivator CBP allows gene activation through recruitment of the basal transcriptional machinery. Acetylation of H3 by histone acetyltransferase (HAT) activities, such as the one carried by CBP, has been functionally coupled to H3 phosphorylation. While various lines of evidence indicate that coupled histone acetylation and phosphorylation may act in concert to induce chromatin remodeling events facilitating gene activation, little is known about the coupling of the two processes at the signaling level. Here we show that CBP and RSK2 are associated in a complex in quiescent cells and that they dissociate within a few minutes upon mitogenic stimulus. CBP preferentially interacts with unphosphorylated RSK2 in a complex where both RSK2 kinase activity and CBP acetylase activity are inhibited. Dissociation is dependent on phosphorylation of RSK2 on Ser227 and results in stimulation of both kinase and HAT activities. We propose a model in which dynamic formation and dissociation of the CBP-RSK2 complex in response to mitogenic stimulation allow regulated phosphorylation and acetylation of specific substrates, leading to coordinated modulation of gene expression.
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PMID:Mitogen-regulated RSK2-CBP interaction controls their kinase and acetylase activities. 1156 91

Transcriptional factors binding to cAMP-responsive elements (CREs) in the promoters of various genes belong to the basic domain-leucine zipper superfamily and are composed of three genes in mammals, CREB, CREM, and ATF-1. A large number of CREB, CREM, and ATF-1 proteins are generated by posttranscriptional events, mostly alternative splicing, and regulate gene expression by acting as activators or repressors. Activation is classically brought about by signaling-dependent phosphorylation of a key acceptor site (Ser133 in CREB) by a number of possible kinases, including PKA, CamKIV, and Rsk-2. Phosphorylation is the prerequisite for the interaction of CBP (CREB-binding protein), a co-activator that has also histone acetyltransferase activity. Repression may involve dynamic dephosphorylation of the activators and thus decreased association with CBP. Another pathway of transcriptional repression on CRE sites implicates the inducible repressor ICER (inducible cAMP early repressor), a product of the CREM gene. Being an inducible repressor, ICER is involved in autoregulatory feedback loops of transcription that govern the down-regulation of early response genes, such as the proto-oncogene c-fos. The liver represents a remarkable physiological setting where cAMP-responsive signaling plays a major role. Indeed, a finely tuned program of gene expression is triggered by partial hepatectomy, so that through specific checkpoints a coordinated regeneration of the tissue is obtained. Temporal kinetics of transcriptional activation after hepatectomy reveals a pattern of early induction for several genes, some of them controlled by the CREB/CREM transcription factors. An important role of CREM in liver physiology was suggested by the robust induction of ICER after partial hepatectomy. The delay in tissue regeneration in CREM-deficient mice confirmed the important function of this factor in regulating hepatocyte proliferation. As gene induction is accompanied by critical changes in chromatin organization, the deciphering of the specific modification codes that histones display during liver regeneration and physiology will provide exciting new insights into the dynamics of chromatin architecture.
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PMID:Coupling cAMP signaling to transcription in the liver: pivotal role of CREB and CREM. 1196 86

Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that function as activators or repressors. Although both transcription factors share a common domain involved in forming multimers, we demonstrate that Sp1 and Sp3 form separate complexes in estrogen-dependent human breast cancer cells. Sp1 and Sp3 complexes associate with histone deacetylases (HDACs) 1 and 2. Although most HDAC2 is not phosphorylated in the breast cancer cells, HDAC2 bound to Sp1 and Sp3 and cross-linked to chromatin in situ is highly enriched in a phosphorylated form that has a reduced mobility in SDS-polyacrylamide gels. We show that protein kinase CK2 is associated with and phosphorylates HDAC2. Alkaline phosphatase treatment of HDAC2 and Sp1 and Sp3 complexes reduced the associated HDAC activity. Protein kinase CK2 is up-regulated in several cancers including breast cancer, and Sp1 and Sp3 have key roles in estrogen-induced proliferation and gene expression in estrogen-dependent breast cancer cells. CK2 phosphorylation of HDAC2 recruited by Sp1 or Sp3 could regulate HDAC activity and alter the balance of histone deacetylase and histone acetyltransferase activities and dynamic chromatin remodeling of estrogen-regulated genes.
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PMID:The transcriptional repressor Sp3 is associated with CK2-phosphorylated histone deacetylase 2. 1217 73

Here we show that the phosphorylation of histone acetyltransferase Tip60, a target of human immunodeficiency virus, type 1-encoded transactivator Tat, plays a crucial role in the control of its catalytic activity. Baculovirus-based expression and purification of Tip60 combined with mass spectrometry allowed the identification of serines 86 and 90 as two major sites of phosphorylation in vivo. The phosphorylation of Tip60 was found to modulate its histone acetyltransferase activity. One of the identified phosphorylated serines, Ser-90, was within a consensus cyclin B/Cdc2 site. Ser-90 was specifically phosphorylated in vitro by the cyclin B/Cdc2 complex. Accordingly, the phosphorylation of Tip60 was enhanced after drug-induced arrest of cells in G(2)/M. This G(2)/M-dependent phosphorylation of Tip60 was abolished by treating cells with a specific inhibitor of the cyclin-dependent kinase, roscovitin. All together, these results strongly suggest a G(2)/M-dependent control of Tip60 activity.
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PMID:Tip60 acetyltransferase activity is controlled by phosphorylation. 1246 30

CBP/p300 recruitment to enhancer-bound complexes is a key determinant in promoter activation by many transcription factors. We present a novel mechanism of activating such complexes and show that pre-assembled Elk-1-p300 complexes become activated following Elk-1 phosphorylation by changes in Elk-1-p300 interactions rather than recruitment. It is known that Elk-1 binds to promoter in the absence of stimuli. However, it is unclear how activation of Elk-1 by mitogen-acivated protein kinase (MAPK)-mediated phosphorylation leads to targeted gene transactivation. We show that Elk-1 can interact with p300 in vitro and in vivo in the absence of a stimulus through the Elk-1 C-terminus and the p300 N-terminus. Phosphorylation on Ser383 and Ser389 of Elk-1 by MAPK enhances this basal binding but, most importantly, Elk-1 exhibits new interactions with p300. These interaction changes render a strong histone acetyltransferase activity in the Elk-1-associated complex that could play a critical role in chromatin remodeling and gene activation. The pre-assembly mechanism may greatly accelerate transcription activation, which is important in regulation of expression of immediate-early response genes, in particular those involved in stress responses.
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PMID:MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300. 1251 34


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