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CREB interaction with CREB-binding protein (CBP)

Two closely related proteins, p300 and CBP, are functional homologs and global transcriptional coactivators that are involved in the regulation of various DNA-binding transcription factors. p300/CBP interacts with nuclear receptors, CREB, c-Jun, C-Myb (see Drosophila Myb oncogene-like), c-Fos, and MyoD. DNA-binding factors recruit p300/CBP by not only direct but also indirect interactions through cofactors. p300/CBP is not only a transcriptional adaptor but also a histone acetyltransferase. The p300/CBP-histone acetyltransferase domain has no obvious sequence similarity to GCN5, another protein with known histone acetyltransferase activity, or to other previously described acetyltransferases. P300 acetylates all core histones in mononucleosomes and the four lysines in the Histone H4 N-terminal tail. These observations suggest that p300/CBP is not a simple adaptor between DNA binding factors and cellular p300/CBP associated factor (PCAF) or transcription factors; rather, p300/CBP per se may contribute directly to transcriptional regulation via targeted acetylation of chromatin (Ogryzko, 1996 and references).

A phosphoserine binding domain has been characterized in the coactivator CREB-binding protein (CBP) which interacts with the protein kinase A-phosphorylated (and hence activated) form of the cyclic AMP-responsive factor CREB. The CREB binding domain, referred to as KIX, is alpha helical and binds to an unstructured kinase-inducible domain in CREB following phosphorylation of CREB at Ser-133. Phospho-Ser-133 forms direct contacts with residues in KIX, and these contacts are further stabilized by hydrophobic residues in the kinase-inducible domain, which flanks phospho-Ser-133. Like the src homology 2 (SH2) domains which bind phosphotyrosine-containing peptides, phosphoserine 133 appears to coordinate with a single arginine residue (Arg-600) in KIX, which is conserved in the CBP-related protein P300. Since mutagenesis of Arg-600 to Gln severely reduces CREB-CBP complex formation, these results demonstrate that, as in the case of tyrosine kinase pathways, signal transduction through serine/threonine kinase pathways may also require protein interaction motifs that are capable of recognizing phosphorylated amino acids (Parker, 1996).

Human activating transcription factor 4 (hATF4), a member of the cAMP-responsive element-binding protein (CREB) family of transcription factors, is a potent transcriptional activator in both mammalian cells and yeast. The N-terminal 113 amino acids of hATF4 activate transcription efficiently; unexpectedly, the C-terminal bZip DNA binding domain of hATF4 also activates transcription, albeit weakly. These results indicate that hATF4 interacts with several general transcription factors: TATA-binding protein, TFIIB, and the RAP30 subunit of TFIIF. In addition, hATF4 interacts with the coactivator CREB-binding protein (CBP) at four regions: (1) the KIX domain, (2) a region that contains the third zinc finger and the E1A-interacting domain, (3) a C-terminal region that contains the p160/SRC-1-interacting domain, and (4) the recently identified histone acetyltransferase domain. Interestingly, both the N-terminal and C-terminal regions of hATF4 interact with the above general transcription factors and CBP, providing a mechanistic explanation for their ability to activate transcription. Consistent with its role as a coactivator, CBP potentiates the ability of hATF4 to activate transcription (Liang, 1997).

Transcriptional activation of the c-jun gene is a critical event in the differentiation of F9 cells. An element [differentiation response element (DRE)] in the c-jun promoter is both necessary and sufficient to confer the capacity for differentiation-dependent up-regulation. This element binds the differentiation regulatory factor (DRF) complex, of which one component is the adenovirus E1A-associated protein p300, whose sequence is closely related to Creb binding protein. Activation transcription factor-2 (ATF-2) is identified as a DNA-binding subunit of the DRF complex. ATF-2 is a member of the ATF/CREB family of basic region-leucine zipper (bZip domain) transcription factors. p300 and ATF-2 interact with each other in vivo and in vitro. The bromodomain and the C/H2 domain of p300 mediate the binding to ATF-2, which in turn requires a proline-rich region between amino acids 112 and 350 for its interaction with p300. The phosphorylation of the serine residue at position 121 of ATF-2 appears to be induced by protein kinase Calpha (PKCalpha) after treatment of cells with retinoic acid (RA) or induction with E1A. In cotransfection assays, wild-type ATF-2 enhances the transcription of an E2/tk-luciferase construct, in conjunction with p300-E2. However, this is not the case for a mutant form of ATF-2, with a mutation at position 121 (pCMVATF-2Ser121-Ala). These results suggest that ATF-2 and p300 cooperate in the control of transcription by forming a protein complex that is responsive to differentiation-inducing signals, such as RA or E1A, and moreover, that the phosphorylation of ATF-2 by PKC is probably a signaling event in the pathway that leads to the transactivation of the c-jun gene in F9 cells (Kawasaki, 1998).

The efficient replication of human T-cell leukemia virus type 1 (HTLV-1) and viral gene expression are both dependent on 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 has been 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, high resolution footprinting was used to show that Tax extends the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint is enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increases the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences does not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 binds to the GC-rich flanking sequences and inhibits the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-links to the viral CRE in the 5'-flanking sequence, and this cross-link is 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 (Lenzmeier, 1998).

The nuclear factor CREB activates transcription of target genes in part through direct interactions with the KIX domain of the coactivator CBP, in a phosphorylation-dependent manner. The CREB transactivation domain is bipartite, consisting of kinase-inducible and constitutive domains termed KID and Q2, respectively, which function synergistically in response to hormonal stimulation. The Z2 domain has been shown to stimulate transcription via its association with the hTAFII130 subunit of TFIID. The KID region has been found to regulate target gene expression by interaction with CBP and P300 in a phosphoSer-133 dependent manner. The solution structure of the complex formed by the phosphorylated kinase-inducible domain (pKID) of CREB with KIX reveals that pKID undergoes a coil-->helix folding transition upon binding to KIX, forming two alpha helices. The amphipathic helix alphaB of pKID interacts with a hydrophobic groove defined by helices alpha1 and alpha3 of KIX. The other pKID helix, alphaA, contacts a different face of the alpha3 helix. The phosphate group of the critical phosphoserine residue of pKID forms a hydrogen bond to the side chain of Tyr-658 of KIX. The structure provides a model for interactions between other transactivation domains and their targets (Radhakrishnan, 1997).

How does the CREB stimulate target gene expression following CREB's phosphorylation at Ser-133. Two signals are required for target gene activation: a phospho(Ser-133)-dependent interaction of CREB with RNA polymerase II via the coactivator CBP and a glutamine-rich domain interaction with TFIID via hTAFII130. The adenovirus E1A oncoprotein is found to inhibit phospho(Ser-133) CREB activity by binding to CBP, specifically blocking recruitment of RNA Pol II to the promoter. These results suggest that the recruitment of CBP-RNA Pol II complexes per se is not sufficient for transcriptional activation and that activator-mediated recruitment of TFIID is additionally required for induction of signal-dependent genes (Nakajima, 1997a).

The coactivator CBP has been proposed to stimulate the expression of certain signal-dependent genes via its association with RNA polymerase II complexes. Complex formation between CBP and RNA polymerase II requires RNA helicase A (RHA), a nuclear DNA/RNA helicase that is related to the Drosophila male dosage compensation factor Mle. In transient transfection assays, RHA is found to cooperate with CBP in mediating target gene activation via the CAMP responsive factor CREB. Since a mutation in RHA that compromises its helicase activity correspondingly reduces CREB-dependent transcription, it is proposed that RHA may induce local changes in chromatin structure that promote engagement of the transcriptional apparatus on signal responsive promoters. The involvement of a DNA helicase such as RHA in signal-dependent transcription is intriguiing because it suggests that recruitment of CBP complexes may promote local unwinding of promoter DNA via RHA and thereby permit engagement of the transcriptional apparatus (Nakajima, 1997b).

CREB-mediated constitutive transcription requires only CREB-binding sites and a minimal promoter region (containing the TATA through start sequences), indicating that CREB interacts directly with components of the general transcription machinery. Human CREB specifically binds to TFIIB. CREB binds TFIID complexes containing TBP, but does not directly interact with TBP. TFIIB interacts with TBP but CREB does not form a stable ternary complex with TFIIB and TBP. CREB interacts independently with TFIIB and TFIID, but not directly with TBP. Both wild-type CREB and a protein kinase A phosphorylation site mutant of CREB exhibit equivalent interactions with TFIIB, indicating that this phosphorylation is not required for interaction. Consistent with the role of CREB in promoting constitutive or basal transcription, the constitutive activation domain of CREB is sufficient for interaction with both TFIIB and TFIID (Xing, 1995).

Recruitment of the coactivator, CREB binding protein (CBP), by signal-regulated transcription factors, such as CREB [adenosine 3',5'-monophosphate (cAMP) response element binding protein], is critical for stimulation of gene expression. The mouse pituitary cell line AtT20 was used to show that the CBP recruitment step (CREB phosphorylation on serine-133) can be uncoupled from CREB/CBP-activated transcription. CBP contains a signal-regulated transcriptional activation domain that is controlled by nuclear calcium and calcium/calmodulin-dependent (CaM) protein kinase IV and by cAMP. Cytoplasmic calcium signals that stimulate the Ras mitogen-activated protein kinase signaling cascade or expression of the activated form of Ras provide the CBP recruitment signal but does not increase CBP activity and fails to activate CREB- and CBP-mediated transcription. These results identify CBP as a signal-regulated transcriptional coactivator and define a regulatory role for nuclear calcium and cAMP in CBP-dependent gene expression (Chawla, 1998).

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, it is shown 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; in all cases high-affinity binding is 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 upholds the binding studies, as expression of the bZIP domain of CREB is sufficient to support Tax transactivation of HTLV-1 transcription in vivo. Transfection of a KIX expression plasmid, which lacks activation properties, inhibits Tax transactivation in vivo. This suggests that KIX occupies the CBP binding site on Tax, and therefore CBP is likely to be 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 (Giebler, 1997).

A fragment of the mixed-lineage leukemia (MLL) gene (Mll, HRX, ALL-1) was identified in a yeast genetic screen designed to isolate proteins that interact with the CREB-CREB-binding protein (CBP) complex. When tested for binding to CREB or CBP individually, this MLL fragment interacts directly with CBP, but not with CREB. In vitro binding experiments refined the identification of the minimal region of interaction to amino acids 2829 to 2883 of MLL, a potent transcriptional activation domain, and amino acids 581 to 687 of CBP (the CREB-binding or KIX domain). The transactivation activity of MLL is dependent on CBP, since either adenovirus E1A expression, which inhibits CBP activity, or alteration of MLL residues important for CBP interaction prove effective at inhibiting MLL-mediated transactivation. Single amino acid substitutions within the MLL activation domain reveal that five hydrophobic residues, potentially forming a hydrophobic face of an amphipathic helix, are critical for the interaction of MLL with CBP. Using purified components, it has been found that the MLL activation domain facilitates the binding of CBP to phosphorylated CREB. In contrast with paradigms in which factors compete for limiting quantities of CBP, these results reveal that two distinct transcription factor activation domains can cooperatively target the same motif on CBP (Ernst, 2001).

One prediction of these studies is that an overlap in MLL- and CREB-dependent target genes exists such that the cooperative interaction of MLL and CREB with CBP would play a role in regulating these genes. Murine knockout models for both CREB and MLL may be instructive in identifying potential shared target genes for these two transcriptional activators. One possible function of the MLL protein at such predicted target genes might be to convert the acute, signal-induced activation of CREB into a sustained, developmentally maintained response analogous to the maintenance role of Trithorax-group proteins during Drosophila embryogenesis (Ernst, 2001).

Fusion of the MLL gene to either CBP or p300 has been reported in several cases of myeloid leukemia or myeloid dysplasia\. The data presented here suggest that MLL utilizes CBP to activate transcription. This finding raises intriguing issues regarding the mechanism of MLL-CBP or MLL-p300 fusions in leukemogenesis. One hypothesis for the oncogenic mechanism of the MLL-fusion gene products is that the acquisition of a new C terminus would impart an enhanced or new activity to the MLL molecule (gain-of-function or neomorphic activity). The MLL-CBP physical interaction presented in this study may be regulated during normal hematopoietic development to modulate the maintenance of endogenous MLL target genes. One testable prediction is that fusion of MLL to CBP would result in a form of MLL that could not be uncoupled from CBP, leading to the temporally inappropriate maintenance of MLL target gene expression. Constitutive expression of certain MLL target genes would consequently contribute to leukemogenesis. Attractive candidates for such target genes are HOX genes, which have been shown to play a role in normal or aberrant hematopoiesis through either loss- or gain-of-function experiments (Ernst, 2001).

Table of contents

CrebB-17A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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