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1.
Mol Cell Biol ; 20(13): 4849-58, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10848610

ABSTRACT

CREB binding protein (CBP) is a 270-kDa nuclear protein required for activated transcription of a large number of cellular genes. Although CBP was originally discovered through its interaction with phosphorylated CREB (pCREB), it is utilized by a multitude of cellular transcription factors and viral oncoproteins. Both CREB and the tumor suppressor p53 have been shown to directly interact with the KIX domain of CBP. Although coactivator competition is an emerging theme in transcriptional regulation, we have made the fortuitous observation that protein kinase A-phosphorylated CREB strongly enhances p53 association with KIX. Phosphorylated CREB also facilitates interaction of a p53 mutant, defective for KIX binding, indicating that CREB functions in a novel way to bridge p53 and the coactivator. This is accomplished through direct interaction between the bZIP domain of CREB and the amino terminus of p53; a protein-protein interaction that is also detected in vivo. Consistent with our biochemical observations, we show that stimulation of the intracellular cyclic AMP (cAMP) pathway, which leads to CREB phosphorylation, strongly enhances both the transcriptional activation and apoptotic properties of p53. We propose that phosphorylated CREB mediates recruitment of CBP to p53-responsive promoters through direct interaction with p53. These observations provide evidence for a novel pathway that integrates cAMP signaling and p53 transcriptional activity.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Tumor Suppressor Protein p53/metabolism , CREB-Binding Protein , Colforsin/metabolism , Colforsin/pharmacology , Cyclic AMP Response Element-Binding Protein/chemistry , Cyclic AMP-Dependent Protein Kinases/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Humans , Jurkat Cells/drug effects , Jurkat Cells/metabolism , Leucine Zippers , Phosphorylation , Promoter Regions, Genetic , Signal Transduction , Tumor Suppressor Protein p53/genetics
2.
J Biol Chem ; 274(37): 26321-8, 1999 Sep 10.
Article in English | MEDLINE | ID: mdl-10473588

ABSTRACT

The pleiotropic cellular coactivator CREB binding protein (CBP) plays a critical role in supporting p53-dependent tumor suppressor functions. p53 has been shown to directly interact with a carboxyl-terminal region of CBP for recruitment of the coactivator to p53-responsive genes. In this report, we identify the KIX domain as a new p53 contact point on CBP. We show that both recombinant and endogenous forms of p53 specifically interact with KIX. We demonstrate that the activation domain of p53 participates in KIX binding and provide evidence showing that this interaction is critical for p53 transactivation function. The human T-cell leukemia virus, type-I-encoded oncoprotein Tax is a well established repressor of p53 transcription function. Like p53, Tax also binds to KIX. The finding that both transcription factors bind to a common region of CBP suggests that coactivator competition may account for the observed repression. We demonstrate reciprocal repression between Tax and p53 in transient transfection assays, supporting the idea of intracellular coactivator competition. We biochemically confirm coactivator competition by directly showing that both transcription factors bind to KIX in a mutually exclusive fashion. These data provide molecular evidence for the observed intracellular competition and suggest that Tax inhibits p53 function by abrogating a novel p53-KIX interaction. Thus, Tax competition for the p53-KIX complex may be a pivotal event in the human T-cell leukemia virus, type I transformation pathway.


Subject(s)
Human T-lymphotropic virus 1/physiology , Leukemia, T-Cell/virology , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Tumor Suppressor Protein p53/metabolism , Binding, Competitive , CREB-Binding Protein , Cloning, Molecular , Gene Products, tax/metabolism , Humans , Jurkat Cells , Nuclear Proteins/genetics , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Trans-Activators/genetics
3.
J Mol Biol ; 281(3): 395-400, 1998 Aug 21.
Article in English | MEDLINE | ID: mdl-9698555

ABSTRACT

The oncoprotein Tax, encoded by the human T-cell leukemia virus type I (HTLV-I), is required for high-level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Recent evidence suggests that Tax stimulates HTLV-I transcription through recruitment of the cellular coactivator protein CBP to the HTLV-I promoter, promoting high-level viral replication via the transcriptional activation properties associated with CBP. Tax directly contacts the KIX domain of CBP to stably anchor the coactivator to nucleoprotein complexes at the promoter. Here, we identify KIX amino acid residues 588 to 683 as the minimal region sufficient for interaction with Tax. This region is similar to the minimal KIX amino acid residues necessary for strong interaction with phosphorylated CREB, and is composed of a structural domain that forms an extensive hydrophobic core. We further show that a double point mutation in KIX differentially affects the binding of Tax and phosphorylated CREB, suggesting that these transcription factors may recognize unique amino acid residues within the KIX domain. These observations suggest that Tax directly contacts the hydrophobic core of KIX, and provides a structural framework to further define the molecular interactions between Tax and CBP.


Subject(s)
Gene Products, tax/metabolism , Human T-lymphotropic virus 1/metabolism , Nuclear Proteins/metabolism , Trans-Activators/metabolism , CREB-Binding Protein , Humans , Nuclear Proteins/genetics , Phosphorylation , Point Mutation , Protein Binding , Recombinant Fusion Proteins , Sequence Deletion , Trans-Activators/genetics , Transcriptional Activation/physiology
4.
Mol Cell Biol ; 18(2): 721-31, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9447968

ABSTRACT

Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5'-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter.


Subject(s)
DNA, Viral/metabolism , DNA/metabolism , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/genetics , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Trans-Activators , Transcription Factors/metabolism , Binding Sites , CREB-Binding Protein , Chromomycin A3/pharmacology , Humans , Nucleic Acid Synthesis Inhibitors/pharmacology , Transcriptional Activation
5.
Mol Cell Biol ; 17(9): 5156-64, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9271393

ABSTRACT

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates viral transcription through interaction with the cellular transcription factor CREB (cyclic AMP response element [CRE] binding protein). Although Tax stabilizes the binding of CREB to the Tax-responsive viral CREs in the HTLV-1 promoter, the precise molecular mechanism by which Tax mediates strong transcriptional activation through CREB remains unclear. In this report, we show that Tax promotes high-affinity binding of the KIX domain of CREB binding protein (CBP) to CREB-viral CRE complexes, increasing the stability of KIX in these nucleoprotein complexes by up to 4.4 kcal/mol. Comparable KIX binding affinities were measured for both phosphorylated and unphosphorylated forms of CREB, and in all cases high-affinity binding was dependent upon both Tax and the viral CRE. Tax also promoted association of KIX to a truncated form of CREB containing only the 73-amino-acid basic leucine zipper (bZIP) domain, indicating that the entire amino-terminal CBP-interacting domain of CREB is nonessential in the presence of Tax. Functional studies upheld the binding studies, as expression of the bZIP domain of CREB was sufficient to support Tax transactivation of HTLV-1 transcription in vivo. Finally, we show that transfection of a KIX expression plasmid, which lacks activation properties, inhibited Tax transactivation in vivo. This suggests that KIX occupies the CBP binding site on Tax, and therefore CBP is likely a cofactor in mediating Tax stimulation of HTLV-1 transcription. Together, these data support a model in which Tax anchors CBP to the HTLV-1 promoter, with strong transcriptional activation resulting from the CBP-associated activities of nucleosome remodeling and recruitment of the general transcription machinery.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins , Gene Products, tax/genetics , Human T-lymphotropic virus 1/genetics , Promoter Regions, Genetic , Transcriptional Activation , Activating Transcription Factor 1 , Cell Line , DNA/metabolism , Half-Life , Humans , Kinetics , Macromolecular Substances , Protein Binding , Protein Conformation , Transcription Factors/metabolism
6.
J Biol Chem ; 270(48): 28503-6, 1995 Dec 01.
Article in English | MEDLINE | ID: mdl-7499359

ABSTRACT

The human T-cell leukemia virus type I oncoprotein Tax transcriptionally deregulates a wide variety of viral and cellular genes. Tax deregulation of gene expression is mediated through interaction with a variety of structurally unrelated cellular transcription factors, as Tax does not bind DNA in a sequence-specific manner. Although most of these cellular transcription factors have been shown to mediate activation by Tax, we have recently demonstrated that members of the basic helix-loop-helix (bHLH) family of transcription factors, which play a critical role in progression through the cell cycle, mediate repression by Tax. In this report, we examined whether Tax might repress transcription of the tumor suppressor p53, as the p53 gene has recently been demonstrated to be regulated by the bHLH protein c-Myc. Furthermore, loss or inactivation of the p53 gene has been shown to be causally associated with oncogenic transformation. We show that Tax represses transcription of the p53 gene and that this repression is dependent upon the bHLH recognition element in the p53 promoter. Together, these results suggest that Tax may promote malignant transformation through repression of p53 transcription.


Subject(s)
DNA-Binding Proteins , Gene Products, tax/physiology , Genes, p53 , Transcription, Genetic , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Humans , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/physiology , Transcription Factors/physiology , Upstream Stimulatory Factors
7.
Nature ; 376(6541): 606-8, 1995 Aug 17.
Article in English | MEDLINE | ID: mdl-7637812

ABSTRACT

Tax protein activates transcription of the human T-cell leukaemia virus type I (HTLV-I) genome through three imperfect cyclic AMP-responsive element (CRE) target sites located within the viral promoter. Previous work has shown that Tax interacts with the bZIP element of proteins that bind the CRE target site to promote peptide dimerization, suggesting an association between Tax and bZIP coiled coil. Here we show that the site of interaction with Tax is not the coiled coil, but the basic segment. This interaction increases the stability of the GCN4 bZIP dimer by 1.7 kcal mol-1 and the DNA affinity of the dimer by 1.9 kcal mol-1. The differential effect of Tax on several bZip-DNA complexes that differ in peptide sequence or DNA conformation suggests a model for Tax action based on stabilization of a distinct DNA-bound protein structure. This model may explain how Tax interacts with transcription factors of considerable sequence diversity to alter patterns of gene expression.


Subject(s)
DNA-Binding Proteins , DNA/metabolism , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/metabolism , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Base Sequence , Cyclic AMP/metabolism , DNA Probes , Fungal Proteins/metabolism , Leucine Zippers , Models, Biological , Molecular Sequence Data , Protein Binding , Protein Kinases/metabolism
8.
J Biol Chem ; 270(21): 12814-22, 1995 May 26.
Article in English | MEDLINE | ID: mdl-7759537

ABSTRACT

The human T-cell leukemia virus type I (HTLV-I) is the causative agent of an aggressive T-cell malignancy in humans. While the virus appears to maintain a state of latency in most infected cells, high level virion production is an essential step in the HTLV-I life cycle. The virally-encoded Tax protein, a potent activator of gene expression, is believed to control the switch from latency to replication. Tax stimulation of HTLV-I transcription is mediated through cellular activating transcription factor/cAMP response element binding proteins, which bind the three 21-base pair (bp) repeat viral enhancer elements. In this report, we show that viral latency may result from a highly unstable interaction between CREB and the HTLV-I 21-bp repeats, resulting in rapid dissociation of CREB from the viral promoter. In the presence Tax, the dissociation rate of CREB from a 21-bp repeat element is decreased. This stabilization is highly specific, requiring the amino-terminal region of CREB and appropriate 21-bp repeat sequences. We suggest that Tax stabilization of CREB binding to the viral promoter leads to an increase in gene expression, possibly providing the switch from latency to high level replication of the virus.


Subject(s)
Gene Products, tax/metabolism , Human T-lymphotropic virus 1/genetics , Transcription Factors , Transcription, Genetic , Transcriptional Activation , Virus Latency/genetics , Activating Transcription Factor 2 , Base Sequence , Binding Sites , Consensus Sequence , Cyclic AMP Response Element-Binding Protein/metabolism , DNA, Viral/metabolism , Human T-lymphotropic virus 1/growth & development , Humans , Kinetics , Molecular Sequence Data , Protein Binding , Repetitive Sequences, Nucleic Acid
9.
Proc Natl Acad Sci U S A ; 90(15): 7303-7, 1993 Aug 01.
Article in English | MEDLINE | ID: mdl-8346248

ABSTRACT

The Tax protein, encoded by the human T-cell leukemia virus type I, is a potent activator of viral and cellular gene transcription. Tax does not bind DNA directly but appears to trans-activate through an interaction with host-cell transcription factors that recognize sequences within the promoters of Tax-responsive genes. Cellular transcriptional activators implicated in mediating Tax trans-activation include members of the activating transcription factor/cAMP response element binding protein (ATF/CREB) family of proteins, serum response factor, Fos-Jun, and NF-kappa B. Recent evidence suggests that Tax may stimulate human T-cell leukemia virus type I transcription, at least in part, through enhanced binding of ATF/CREB proteins to their recognition elements within the Tax-responsive 21-bp repeats of the viral promoter. In this report, we demonstrate that Tax also enhances the site-specific DNA binding activity of serum response factor and Fos-Jun and modestly enhances the binding of the NF-kappa B subunits, p50 and p65. We also show that Tax increases the DNA binding activity of the eukaryotic transcription factors ATF-1, Sp1, and GAL4. These results are consistent with the finding that Tax is highly pleiotropic and suggest that Tax trans-activation may involve enhancement in the DNA binding activity of target transcriptional regulatory proteins. In addition, we show that the mechanism of Tax-enhanced DNA binding activity does not involve an alteration in the redox state of the target protein.


Subject(s)
Gene Expression Regulation, Viral , Gene Products, tax/physiology , Transcription Factors/metabolism , Base Sequence , DNA-Binding Proteins/metabolism , In Vitro Techniques , Molecular Sequence Data , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Oligodeoxyribonucleotides/chemistry , Oxidation-Reduction , Proto-Oncogene Proteins c-jun/metabolism , RNA, Messenger/genetics , Recombinant Proteins/metabolism , Serum Response Factor , Transcriptional Activation
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