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EVOLUTIONARY HOMOLOGS

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CBP interactions with p53 and Mdm2

The structurally related transcriptional coactivators p300 and CBP possess histone acetyltransferase activity and associate with P/CAF, which is also a histone acetyltransferase. CBP and p300 have properties of tumor suppressor proteins; their interaction with P/CAF is disrupted by the adenoviral E1A oncoprotein, and the genes encoding CBP and p300 are mutated in human cancer. A physical interaction is observed between the transactivation domain of the p53 tumor suppressor protein and CBP. Furthermore, CBP and P/CAF enhanced the ability of p53 to activate expression of the endogenous p21(cip1/waf1) gene, whereas E1A and dominant negative CBP mutants suppress p53-dependent p21(cip1/waf1) expression. These studies link two tumor suppressor families and provide a framework for understanding the molecular mechanism by which p53 activates transcription (Scolnick, 1997).

The adenovirus E1A and SV40 large-T-antigen oncoproteins bind to members of the p300/CBP transcriptional coactivator family. Binding of p300/CBP is implicated in the transforming mechanisms of E1A and T-antigen oncoproteins. A common region of the T antigen is critical for binding both p300/CBP and the tumour suppressor p53, suggesting a link between the functions of p53 and p300. p300/CBP binds to p53 in the absence of viral oncoproteins, and p300 and p53 colocalize within the nucleus and coexist in a stable DNA-binding complex. Consistent with its ability to bind to p300, E1A disrupts functions mediated by p53. It reduces p53-mediated activation of the p21 and bax promoters, and suppresses p53-induced cell-cycle arrest and apoptosis. It is concluded that members of the p300/CBP family are transcriptional adaptors for p53, modulating its checkpoint function in the G1 phase of the cell cycle and its induction of apoptosis. Disruption of p300/p53-dependent growth control may be part of the mechanism by which E1A induces cell transformation. These results help to explain how p53 mediates growth and checkpoint control, and how members of the p300/CBP family affect progression from G1 to S phase in the cell cycle (Lill, 1997).

The tumour suppressor p53 is a transcriptional regulator whose ability to inhibit cell growth is dependent upon its transactivation function. The transcription factor CBP, which is also implicated in cell proliferation and differentiation, acts as a p53 coactivator and potentiates its transcriptional activity. The amino-terminal activation domain of p53 interacts with the carboxy-terminal portion of the CBP protein both in vitro and in vivo. In transfected SaoS-2 cells, CBP potentiates activation of the mdm-2 gene by p53; reciprocally, p53 acting through CBP sequences potentiates activation of a Gal4-responsive target gene by a Gal4(1-147)-CBP(1678-2441) fusion protein. A double point mutation that destroys the transactivation function of p53 also abolishes its binding to CBP and its synergistic function with CBP. The ability of p53 to interact physically and functionally with a coactivator (CBP) that has histone acetyltransferase activity and with components (TAFs) of the general transcription machinery indicates that it may have different functions in a multistep activation pathway (Gu, 1997a).

The tumor suppressor p53 exerts antiproliferation effects through its ability to function as a sequence-specific DNA-binding transcription factor. p53 can be modified by acetylation both in vivo and in vitro. The site of p53 acetylated by its coactivator, p300, resides in a C-terminal domain known to be critical for the regulation of p53 DNA binding. The N-terminal activation domain of p53 physically interacts with this C-terminal domain of CBP. The acetylation of p53, which takes place on the most C-terminal amino acids of p53, can dramatically stimulate p53's sequence-specific DNA-binding activity, possibly as a result of an acetylation-induced conformational change. These observations clearly indicate a novel pathway for p53 activation and, importantly, provide an example of an acetylation-mediated change in the function of a nonhistone regulatory protein. These results have significant implications regarding the molecular mechanisms of various acetyltransferase-containing transcriptional coactivators whose primary targets have been presumed to be histones (Gu, 1997b).

The products of the p53 and CBP/p300 genes have been individually implicated in the control of cell growth and the regulation of transcription. p53 is known to act both as a positive and negative regulator of gene expression. p300/CBP is contained in a specific DNA-bound protein complex together with p53. The region of p300 that interacts with p53 extends from residues 1514 to 1737; these are C-terminal sequences that encompass sequences upstream and are partially included in the third cysteine/histidine-rich domain. In both wild-type and mutant conformation p53 forms a specific protein complex with p300. However, in only its wild-type conformation, p53 inhibits (in a p300-dependent manner) a promoter containing the DNA-binding sequences for the transcription factor AP1. This promoter does not contain a p53-binding site. Thus p53 inhibits the p300-mediated activation of a promoter containing the DNA-binding sites for the transcription factor AP1. p300 stimulates the transcriptional activity of p53 on p53-regulated promoters, and it enhances the responsiveness to ionizing radiation, a physiological upstream modulator of p53 function. A dominant negative form of p300 prevents transcriptional activation by p53; it counteracts p53-mediated G1 arrest and apoptosis. The data implicate p300 as an important component of p53-signaling, thus providing new insight into the mechanisms of cellular proliferation (Avantaggiati, 1997).

The p53 tumor suppressor gene product interacts with the p300 transcriptional coactivator that regulates the transactivation of p53-inducible genes. The adenovirus E1A protein has been shown to bind to p300 and inhibit its function. E1A inhibits transactivation by p53 and also promotes p53 accumulation by a p300-dependent mechanism. Murine double minute 2 (Mdm2) is a transcriptional target of p53 that binds to p53 and inhibits its transcriptional activity. E1A inhibits mdm2 transactivation without affecting the expression of p21(WAF1) or Bax, thus resulting in high levels of p53 accumulation and apoptosis. Ectopic expression of p300 restores Mdm2 levels and inhibits p53-dependent apoptosis, as does ectopic expression of Mdm2. Thus, p300 is required for mdm2 induction by p53 and the subsequent Mdm2 directed inhibition of p53 stabilization. Inhibition of p300 by E1A results in stabilization of p53 and causes apoptosis. E1B 19K or Bcl-2 expression in E1A-transformed cells abrogates p53-dependent apoptosis by restoring mdm2 transactivation by p53. Hence, p300 regulation of mdm2 expression controls apoptotic activity of p53, and 19K or Bcl-2 bypass E1A inhibition of p300 transactivation of Mdm2 (Thomas, 1998).

The p53 tumor suppressor protein is a sequence-specific transcription factor that modulates the response of cells to DNA damage. Recent studies suggest that full transcriptional activity of p53 requires the coactivators CREB binding protein (CBP)/p300 and PCAF. These coactivators interact with each other, and both possess intrinsic histone acetyltransferase activity. Furthermore, p300 acetylates p53 to activate its sequence-specific DNA binding activity in vitro. PCAF also acetylates p53 in vitro at a lysine residue distinct from that acetylated by p300, thereby increasing the ability of p53 to bind to its cognate DNA site. Antibodies were generated to acetylated p53 peptides at either of the two lysine residues that are targeted by PCAF or p300: these antibodies are highly specific for both acetylation and the particular site. Using these antibodies, acetylation of these sites in vivo has been detected, and interestingly, acetylation at both sites increases in response to DNA-damaging agents. These data indicate that site-specific acetylation of p53 increases under physiological conditions that activate p53 and identify CBP/p300 and PCAF as the probable enzymes that modify p53 in vivo (Liu, 1999).

The nuclear p300/CBP proteins function as coactivators of gene transcription. Using cells deficient in p300 or CBP, it has been shown that p300, and not CBP, is essential for ionizing radiation-induced accumulation of the p53 tumor suppressor and thereby p53-mediated growth arrest. The results demonstrate that deficiency of p300 results in increased degradation of p53. These findings suggest that p300 contributes to the stabilization and transactivation function of p53 in the cellular response to DNA damage (Yuan, 1999).

In response to DNA damage, p53 undergoes post-translational modifications (including acetylation) that are critical for its transcriptional activity. However, the mechanism by which p53 acetylation is regulated is still unclear. This study describes an essential role for HLA-B-associated transcript 3 (Bat3)/Scythe in controlling the acetylation of p53 required for DNA damage responses. Depletion of Bat3 from human and mouse cells markedly impairs p53-mediated transactivation of its target genes Puma and p21. Although DNA damage-induced phosphorylation, stabilization, and nuclear accumulation of p53 are not significantly affected by Bat3 depletion, p53 acetylation is almost completely abolished. Bat3 forms a complex with p300, and an increased amount of Bat3 enhances the recruitment of p53 to p300 and facilitates subsequent p53 acetylation. In contrast, Bat3-depleted cells show reduced p53-p300 complex formation and decreased p53 acetylation. Furthermore, consistent with in vitro findings, thymocytes from Bat3-deficient mice exhibit reduced induction of puma and p21, and are resistant to DNA damage-induced apoptosis in vivo. These data indicate that Bat3 is a novel and essential regulator of p53-mediated responses to genotoxic stress, and that Bat3 controls DNA damage-induced acetylation of p53 (Sasaki, 2007).

CBP interactions with Smads and Stats

TGF-beta and activin induce the phosphorylation and activation of Smad2 and Smad3, but how these proteins stimulate gene transcription is poorly understood. TGF-beta receptor phosphorylation of Smad3 promotes its interaction with the paralogous coactivators CBP and p300, whereas CBP/p300 binding to nonphosphorylated Smad3 or its oligomerization partner Smad4 (Drosophila homolog: Medea) is negatively regulated by Smad-intramolecular interactions. Furthermore, p300 and TGF-beta receptor-phosphorylated Smad3 synergistically augment transcriptional activation. Thus, CBP/p300 are important components of activin/TGF-beta signaling and may mediate the antioncogenic functions of Smad2 and Smad4 (Janknecht, 1998).

The transforming growth factor-beta (TGF-beta) superfamily of growth factors and cytokines has been implicated in a variety of physiological and developmental processes within the cardiovascular system. Smad proteins are a recently described family of intracellular signaling proteins that transduce signals in response to TGF-beta superfamily ligands. It has been demonstrated by both a mammalian two-hybrid and a biochemical approach that human Smad2 and Smad4, two essential Smad proteins involved in mediating TGF-beta transcriptional responses in endothelial and other cell types, can functionally interact with the transcriptional coactivator CREB binding protein (CBP). This interaction is specific in that it requires ligand (TGF-beta) activation and is mediated by the transcriptional activation domains of the Smad proteins. A closely related, but distinct endothelial-expressed Smad protein, Smad7, which does not activate transcription in endothelial cells, does not interact with CBP. Furthermore, Smad2,4-CBP interactions involve the COOH terminus of CBP, a region that interacts with other regulated transcription factors such as certain signal transduction and transcription proteins and nuclear receptors. Smad-CBP interactions are required for Smad-dependent TGF-beta-induced transcriptional responses in endothelial cells, as evidenced by inhibition with overexpressed 12S E1A protein and reversal of this inhibition with exogenous CBP. This report demonstrates a functional interaction between Smad proteins and an essential component of the mammalian transcriptional apparatus (CBP) and extends insight into how Smad proteins may regulate transcriptional responses in many cell types. Thus, functional Smad-coactivator interactions may be an important locus of signal integration in endothelial cells (Topper, 1998).

Smads regulate the transcription of defined genes in response to TGF-beta receptor activation, although the mechanisms of Smad-mediated transcription are not well understood. The TGF-beta-inducible Smad3 uses the tumor suppressor Smad4/DPC4 and CBP/p300 as transcriptional coactivators, which associate with Smad3 in response to TGF-beta. The association of CBP with Smad3 was localized to the carboxyl terminus of Smad3, which is required for transcriptional activation, and a defined segment in CBP. Mad4 shows ligand-inducible interaction with the two CBP segments in two-hybrid assays in Mv1Lu cells. This is in contrast with the lack of Smad4-CBP interaction in coimmunoprecipitation and yeast two-hybrid experiments, suggesting that this interaction is mediated through the ligand-dependent association of Smad4 with endogenous Smad3, which in turn interacts in a ligand-dependent fashion with CBP. In addition, coexpression of Smad4 in SW480.7 cells increases the interaction of Smad3 with the amino- and carboxy-terminal domains of CBP, whereas coexpression of Smad3 promotes the association of Smad4 and CBP in mammalian two-hybrid assays. These results thus suggest a ternary protein complex, whereby the ligand-dependent interaction of Smad3 with CBP (primarily its carboxy-terminal segment) is stabilized by Smad4. This interpretation is consistent with the participation of all three proteins in a nucleoprotein complex at the promoter. The stabilization by Smad4 may be required for the ability of CBP to efficiently coactivate Smad3. CBP/p300 stimulates both TGF-beta- and Smad-induced transcription in a Smad4/DPC4-dependent fashion. Smad3 transactivation and TGF-beta-induced transcription are inhibited by expressing E1A, which interferes with CBP functions (Feng, 1998).

SMADs are transforming growth factor beta (TGF-beta) receptor substrates and mediators of TGF-beta transcriptional responses. Evidence is provided that the coactivators p300 and CBP interact with Smads 1 through 4. The biological relevance of this interaction is shown in vivo by overexpression of the adenovirus E1A protein and mutant forms of E1A that lack p300-binding sites. Wild-type E1A, but not the mutants, inhibits SMAD-dependent transcriptional responses to TGF-beta. E1A also inhibits the intrinsic transactivating function of the Smad4 MH2 domain. In addition, overexpression of p300 enhances SMAD-dependent transactivation. These results suggest a role for p300/CBP in SMAD-mediated transcriptional activation and provide an explanation for the observed ability of E1A to interfere with TGF-beta action (Pouponnot, 1998).

Transforming growth factor-beta family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressor DPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. A 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), is essential for mediating these signaling activities. The functional activity of the SAD is reported in this study. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. Transcriptional activation by the SAD is p300-dependent; this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex (de Caestecker, 2000).

Members of the transforming growth factor-ß superfamily play critical roles in controlling cell growth and differentiation. Effects of TGF-ß family ligands are mediated by Smad proteins. To understand the mechanism of Smad function, attempts were made to identify novel interactors of Smads by use of a yeast two-hybrid system. A 396-amino acid nuclear protein termed SNIP1 was cloned and shown to harbor a nuclear localization signal (NLS) and a Forkhead-associated (FHA) domain. The FHA domain has been shown to be a modular phosphothreonine recognition motif expressed on a variety of nuclear proteins, and is suggested to play a docking role analogous to that of the modular phosphotyrosine domain recognition site, SH2. The carboxyl terminus of SNIP1 interacts with Smad1 and Smad2 in yeast two-hybrid as well as in mammalian overexpression systems. However, the amino terminus of SNIP1 harbors binding sites for both Smad4 and the coactivator CBP/p300. Interaction between endogenous levels of SNIP1 and Smad4 or CBP/p300 is detected in NMuMg cells as well as in vitro. Overexpression of full-length SNIP1 or its amino terminus is sufficient to inhibit multiple gene responses to TGF-ß and CBP/p300, as well as the formation of a Smad4/p300 complex. Studies in Xenopus laevis further suggest that SNIP1 plays a role in regulating dorsomedial mesoderm formation by the TGF-ß family member nodal. Thus, SNIP1 is a nuclear inhibitor of CBP/p300 and its level of expression in specific cell types has important physiological consequences by setting a threshold for TGF-ß-induced transcriptional activation involving CBP/p300 (Kim, 2000).

A region of Smad4 called the SAD, located in the middle linker region just amino-terminal to the MH2 domain (amino acids 275-322), has been shown to be both necessary and sufficient for the transcriptional-activating activity of Smad4 through its interaction with CBP/p300. It is noteworthy that Smad4 deletion constructs of the MH2 domain inclusive of the SAD motif are unable to bind SNIP1 either in vitro or in vivo. A recent crystallographic structural analysis of the transcriptionally active domain of Smad4, including the SAD (amino acids 276-552), has shown that inclusion of the SAD alters the previously published structure of the inactive MH2 domain by stabilizing a glutamine-rich helical extension from the core. Thus, it is suggested that inclusion of this domain, in the absence of other constraints imposed by the MH1 domain, may restrict interactions of SNIP1 with the MH2 domain (Kim, 2000 and references therein).

Importantly, SNIP1 also interacts constitutively with CBP/p300 through the same amino-terminal domain that mediates its principal interaction with Smad4. On this basis, a model is proposed in which the relative levels of SNIP1 and nuclear Smad4 contribute to setting limits on both the basal activity as well as the maximum level of transcriptional activation of target genes that can be achieved in a cell following ligand activation. In this model, high levels of SNIP1 relative to nuclear Smad4 will restrict the interaction between the activated Smad complex and CBP/p300 with resultant inhibition of the ligand-dependent transcriptional response. However, high levels of Smad4 relative to that of SNIP1 will favor the formation of transcriptionally active Smad4/coactivator complexes and Smad4/SNIP1 complexes, which may initiate proteasomal degradation of SNIP1. In the case of the homeodomain repressor TGIF, which, like SNIP1, suppresses both basal and TGF-ß-activated transcription, a competitive mechanism has also been proposed whereby the relative levels of TGIF and Smad2 determine formation of mutually exclusive Smad2/inhibitor and Smad2/coactivator complexes. Suppression of signaling responses from TGF-ß family ligands by the adenoviral oncoprotein E1A, which interacts with the MH2 domain R-Smads, has also been shown to involve competition for binding of these Smads to the C/H3 domain of p300. These data suggest that SNIP1 acts in a similar fashion to inhibit interaction of Smad4 with the C/H1 domain of p300 (Kim, 2000 and references therein).

SNIP1 mRNA and protein are broadly expressed, and, in the limited number of cell lines examined, do not change with TGF-ß treatment. However, preliminary observations of highly selective patterns of immunohistochemical staining for SNIP1 in tissues suggest that its expression is under stringent control. Activation of BMP or TGF-ß-signaling pathways can lead to degradation of SNIP1 through a process involving antizyme and the proteasome subunit HsN3. Proteolytic degradation has also been suggested to be important in regulation of the suppressor activity of Ski and SnoN, where it has been proposed that a TGF-ß-signal leads to activation of Smad3, which then mediates degradation of SnoN and Ski. Mechanisms such as these suggest that the balance of activated Smad complexes and repressor proteins in the nucleus is critical to regulation of the signal-transduction pathways from TGF-ß family ligands (Kim, 2000 and references therein).

Although the cloning and characterization of SNIP1 was based on its ability to bind to Smad proteins and to inhibit Smad-dependent signaling, its ability to inhibit the transcriptional activating activity of the NF-kappaB transcriptional activator p65 shows that its action is not limited to Smad-dependent signaling pathways. Because NF-kappaB, like Smad4, interacts with p300 through the C/H1 domain, it is speculated that SNIP1 might also suppress transcription dependent on other factors interacting with this same domain of p300 including Stat2 and Stat3, ets-1, p53 and MDM2. However, the inability of SNIP1 to inhibit the p300-dependent activity of Gal4-p53 now suggests that the specificity of SNIP1 may be more narrowly defined in terms of the region of p300 with which it interacts and may possibly also be restricted by other parameters such as direct binding to the transcription factor itself. As such, SNIP1 could both limit the magnitude of a particular cellular response and serve to mediate an additional level of crosstalk between various transcriptional regulators that interact with it to fine tune cellular proliferation, differentiation, and response to injury and stress (Kim, 2000 and references therein).

The cytokines LIF (leukemia inhibitory factor) and BMP2 (bone morphogenetic protein-2) signal through different receptors and transcription factors, namely STATs (signal transducers and activators of transcription) and Smads. LIF and BMP2 act in synergy on primary fetal neural progenitor cells to induce astrocytes. The transcriptional coactivator p300 interacts physically with STAT3 at its amino terminus in a cytokine stimulation-independent manner, and with Smad1 at its carboxyl terminus in a cytokine stimulation-dependent manner. The formation of a complex between STAT3 and Smad1, bridged by p300, is involved in the cooperative signaling of LIF and BMP2 and the subsequent induction of astrocytes from neural progenitors (Nakashima, 1999).

Balancing signals derived from the TGFbeta family are crucial for regulating cell proliferation and differentiation, and in establishing the embryonic axis during development. TGFbeta/BMP signaling leads to the activation and nuclear translocation of Smad proteins, which activate transcription of specific target genes by recruiting P/CAF and p300. The two members of the ZEB family of zinc finger factors (ZEB-1/deltaEF1 and ZEB-2/SIP1) regulate TGFbeta/BMP signaling in opposite ways: ZEB-1/deltaEF1 synergizes with Smad-mediated transcriptional activation, while ZEB-2/SIP1 represses it. These antagonistic effects by the ZEB proteins arise from the differential recruitment of transcriptional coactivators (p300 and P/CAF) and corepressors (CtBP) to the Smads. Thus, while ZEB-1/deltaEF1 binds to p300 and promotes the formation of a p300-Smad transcriptional complex, ZEB-2/SIP1 acts as a repressor by recruiting CtBP. This model of regulation by ZEB proteins also functions in vivo, where they have opposing effects on the regulation of TGFbeta family-dependent genes during Xenopus development (Postigo, 2003).

The family of Smad proteins mediates transforming growth factor-β signaling in cell growth and differentiation. Smad proteins repress or activate TGF-β signaling by interacting with corepressors (e.g., Ski; see Drosophila snoN) or coactivators (e.g., CREB binding protein [CBP]), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads, and whether they can interact with Smad3 at the same binding surface on Smad3. This study investigated the interactions among purified constructs of Smad, Ski and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Ski (aa 16-192) interacts directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g., Smad2 or Smad3, to form a hexamer; Ski (aa 16-192) interacts with an R-Smad/Smad4 heterotrimer to form a pentamer. CBP (aa 1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer, and with an R-Smad/Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP compete with each other for binding to Smad3. Mutational studies reveal that domains of Ski and CBP interact with Smad3 at a portion of the Smad anchor for receptor activation (SARA)-binding surface. These results suggest that Ski negatively regulates TGF-β signaling by replacing CBP in R-Smad complexes. A working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-β pathway (Chen, 2007),

CBP interactions with Cyclin E-Cdk2

The p300 and CREB binding protein (CBP) transcriptional coactivators interact with a variety of transcription factors and regulate their activity. Among the interactions that have been described, the COOH-terminal region of p300 binds to cyclin E-cyclin-dependent kinase 2 (cyclin E-Cdk2) and TFIIB, as well as to the E1A gene products of adenovirus. Inhibition of Cdk activity by Cdk inhibitors, such as p21 or p27, potentiates NF-kappaB activity and provides a mechanism to coordinate cell cycle progression with the transcription of genes expressed during growth arrest. In this report, the specific domains of p300 required for the binding of p300 to cyclin E-Cdk2, TFIIB, and E1A have been examined, as well as the ability of these proteins to interact with p300, either alone or in combination. 12S E1A, an inhibitor of p300-dependent transcription, reduces the binding of TFIIB to p300, but not that of cyclin E-Cdk2, to p300. In contrast, 13S E1A, a pleiotropic transcriptional activator, does not inhibit TFIIB binding to p300, although it enhances the interaction of cyclin E-Cdk2 with p300. Modification of cyclin E-Cdk2 is most likely required for association with p300 since the interaction is observed only with cyclin E-Cdk2 purified from mammalian cells. Domain swap studies show that the cyclin homology domain of TFIIB is involved in interactions with p300, although the homologous region from cyclin E does not mediate this interaction. These findings suggest that p300 or CBP function is regulated by interactions of various proteins with a common coactivator domain (Felzien, 1999).

The observations of cooperative and competitive interactions of cyclin E-Cdk2 and TFIIB with p300 provide mechanistic explanations for several previously described functional activities of these proteins. For instance, expression of the p21 cyclin-dependent kinase inhibitor activates human immunodeficiency virus transcription through NFkappa and p300. This finding suggested that inhibition of cyclin E-Cdk2 complexes activates NF-kappaB through p300 and that active cyclin E-Cdk2 antagonizes this activation. It has also been shown that p21 specifically inhibits cyclin E-Cdk2 complexes associated with p300-Rel A and CBP-Rel A complexes, confirming that one mechanism for p21 activation of NF-kappaB through p300-CBP is by means of its inhibition of associated cyclin E-Cdk2 complexes. While active cyclin E-Cdk2 complexes seem to inhibit p300-CBP function, TFIIB contributes to the activation of transcription by p300-CBP, as demonstrated by its involvement in the recruitment of a CBP-containing RNA polymerase II holoenzyme to the beta interferon enhancer. Thus, the observation reported in this paper that TFIIB and cyclin E-Cdk2 complexes compete for binding to a common region of p300 provides an explanation for the opposing effects of TFIIB and cyclin E-Cdk2 complexes on p300 activity. This example of competitive interactions at the COOH terminus of p300 could be a mechanism that occurs with additional regulatory proteins to control a variety of promoters dependent on p300 (Felzien, 1999 and references).

Although the binding of TFIIB and cyclin E-Cdk2 to p300 is competitive, cyclin E-Cdk2 and TFIIB differ in the biochemical basis for their interaction with p300. The binding of TFIIB involves, in part, its cyclin homology domain, but the corresponding region of cyclin E alone cannot facilitate p300 binding. This result is consistent with the findings that sequences within both the amino- and carboxy-terminal regions of TFIIB are necessary for its interaction with CBP. Intact cyclin E-Cdk2 complexes from nuclear extracts are required for interactions with p300, suggesting that a specific conformation or posttranslational modification of cyclin E-Cdk2 or additional polypeptides are needed to mediate interactions between cyclin E-Cdk2 and p300. Cdk2 function is modulated at specific cell cycle phases by phosphorylation and dephosphorylation at certain threonine and tyrosine residues. The requirement of the assembled cyclin E-Cdk2 complex and an additional role of phosphorylation of critical residues may ensure the formation of cyclin E-Cdk2-p300 complexes at distinct times for proper regulation of certain genes (Felzien, 1999 and references).

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