Analysis of an Activator:Coactivator Complex Reveals an Essential Role for Secondary Structure in Transcriptional Activation

Parker, David B.; Jhala, Ulupi S.; Radhakrishnan, Ishwar; Yaffe, Michael B.; Reyes, Christine; Shulman, Andrew I.; Cantley, Lewis C.; Wright, Peter E.; Montminy, Marc

doi:10.1016/s1097-2765(00)80279-8

Cited by 146 publications

(131 citation statements)

References 27 publications

Supporting

Mentioning

123

Contrasting

Unclassified

Order By: Relevance

“…Indeed, the structure of the KID⅐KIX complex supports an inhibitory role for this phosphorylated residue in regulating complex formation (14). But recent studies with antisera specific for phosphorylated Ser-142 and Ser-143 indicate that these sites are not phosphorylated to a significant extent in the cell (M.M.…”

Section: Discussionmentioning

confidence: 99%

“…phoserine-133 promotes this interaction by means of ion-pair and hydrogen-bond interactions with residues in KIX; the Ser-133 phosphate also stabilizes the formation of an amphipathic helix in KID that associates with a shallow hydrophobic groove in KIX (13). Notably, CREB Ser-142 projects into this hydrophobic groove in KIX, and secondary phosphorylation at Ser-142 in vitro blocks complex formation, suggesting a potential mechanism by which various signals may elicit different transcriptional responses (14).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB

Mayr

Canettieri

Montminy

2001

Proc. Natl. Acad. Sci. U.S.A.

171

121

View full text Add to dashboard Cite

A denosine 3Ј,5Ј-cyclic monophosphate (cAMP) stimulates the expression of numerous genes through the protein kinase A-mediated phosphorylation of the cAMP-responsive element-binding protein (CREB) at Ser-133 (1). Ser-133 phosphorylation, in turn, promotes assembly of the transcriptional apparatus through recruitment of the coactivator paralogs CREB-binding protein (CBP) and P300 (2, 3). In addition to its role as a cAMP-responsive activator, CREB appears to function importantly in growth factor-and stress-inducible gene expression (4). Coincident with this wide profile of inducibility, CREB is a substrate for a variety of cellular kinases including pp90rsk (5), protein kinase C (PKC), Akt (6), mitogen-and stressactivated protein kinase 1 (MSK-1) (7), mitogen-activated protein kinase-activated protein 2 (MAPKAP-2) kinase (8), and calcium͞calmodulin-dependent protein kinases II (CaM KII) (9) and IV (10). Overexpression of CREB kinases in transfected cells promotes target gene expression through CREB in some but not all cases. Ser-133 phosphorylation of CREB by CaM KII, for example, fails to promote target gene activation because of secondary CaM KII-dependent phosphorylation of CREB at Ser-142 (9). Whether and to what extent Ser-142 in CREB is phosphorylated in response to external stimuli has not been determined, however.Differences in CREB activity after treatment with cAMP vs. non-cAMP signals are apparent at the promoter level. For example, a single consensus cAMP-responsive element (CRE) is sufficient for target gene activation through CREB in response to cAMP and calcium signals (10, 11). But cellular gene activation in response to nerve growth factor (NGF) requires additional promoter-bound factors that synergize with CREB in a phosphoserine-133-dependent manner (12). In this regard, activation of the c-fos promoter by NGF requires the CRE plus a serum-responsive element that recognizes the ternary complex factor elk-1 and the serum response factor (12).The solution structure of the CREB⅐CBP complex, using relevant interaction domains referred to as KID and KIX, respectively, reveals that Ser-133 phosphorylation of CREB is both necessary and sufficient for complex formation (13). PhosThis paper was submitted directly (Track II) to the PNAS office.Abbreviations: CREB, cAMP-responsive element-binding protein; CBP, CREB-binding protein; CaM KII, calcium͞calmodulin-dependent protein kinase II; CRE, cAMP-responsive element; NGF, nerve growth factor; FRET, fluorescence resonance energy transfer; Fsk, forskolin; TPA, phorbol 12-tetradecanoate 13-acetate; EGF, epidermal growth factor; NLS, nuclear localization signal; GST, glutathione S-transferase; RT-PCR, reverse transcription PCR; GADPH, glyceraldehyde-3-phosphate dehydrogenase; ICER, inducible cAMP early repressor; EYFP, enhanced yellow fluorescent protein; ECFP, enhanced cyan fluorescent protein.*To whom reprint requests should be addressed. E-mail: montminy@salk.edu.The publication costs of this article were defrayed in part by page charge payment. This artic...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB

Mayr

Canettieri

Montminy

2001

Proc. Natl. Acad. Sci. U.S.A.

171

121

View full text Add to dashboard Cite

show abstract

“…S4B). Fluorescein is tagged at the N terminus of the pKID peptide, a construct that has been used in previous studies (35)(36)(37) to show allosteric effects consistent with results from isothermal calorimetry (ITC) assays using unlabeled pKID (11). One rapid association phase was observed upon mixing pKID with varying concentrations of KIX complexes and the observed association rates exhibit a linear dependence on the KIX complex concentration (Fig.…”

Section: Identification Of Kix Residues Affected In Positive and Negamentioning

confidence: 99%

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

Wang

Lodge

Fierke³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/ p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in k off . Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both k on and k off , suggesting stabilization of the binary complex.IDP | protein-protein interaction P rotein-protein interactions (PPIs) underscore all cellular processes, and the mechanistic dissection of PPI networks is thus a high priority (1-4). A particular challenge is defining the mechanism of PPI formation between intrinsically disordered proteins (IDPs) where allosteric changes play a substantive role (5-7). Allosteric communication between binding sites is vital for the proper function of feedback and regulatory circuits in the cell (8, 9). For example, the conformationally dynamic kinase-inducible domain interacting (KIX) domain of the master transcriptional coactivator CREB binding protein (CBP) undergoes a structural shift and stabilization upon binding to a cognate ligand such as the intrinsically disordered transcriptional activation domain (TAD) of mixed-lineage leukemia protein (MLL) (Fig. 1A) (10). The interaction of a second ligand, phosphorylated kinase-inducible domain (pKID) of CREB, is enhanced (up to twofold) by the presence of MLL and several elegant studies have documented allosteric communication between the two binding sites (11-15). However, the relatively modest affinities (micromolar) of the native complexes and the aggregation propensities and the promiscuous binding profiles of the transcriptional activation domains have hampered kinetic dissection of this and other complexes (16,17).The coactivator CBP exists across metazoans (18)(19)(20) and is a transcription hub that interacts with numerous transcriptional activators, using several discrete domains (21,22). The KIX domain within CBP is a 90-residue motif ...

show abstract

“…To address this question, ITC experiments monitoring KIX binding in the presence and absence of MLL19 were performed with a peptide encompassing c-Myb residues 291-315 (Myb25) that includes the minimal trans-activation domain (3). As shown in Fig.…”

Section: Binding Of Mll To Kix Measured By Itc-mentioning

confidence: 99%

“…CBP domains such as KIX, CH1, and CH3 recognize a diverse range of protein sequences that in some cases appear to share a general structural motif in the bound state. For example, the KIX domain binds an amphipathic helix formed by either the phosphorylated kinaseinducible domain (pKID) of CREB or the transactivation domain of c-Myb (3,4). Although both of these ligands bind to the shallow hydrophobic groove formed between KIX helices ␣ 1 and ␣ 3 , the high affinity of CREB for CBP is mediated by critical interactions involving the pKID phosphoryl group and is therefore inducible, whereas the c-Myb activation domain binds constitutively with somewhat lower affinity (5,6).…”

mentioning

confidence: 99%

Cooperativity in Transcription Factor Binding to the Coactivator CREB-binding Protein (CBP)

Goto¹,

Zor²,

Martinez-Yamout

et al. 2002

Journal of Biological Chemistry

171

184

View full text Add to dashboard Cite

Transcriptional activation is mediated by large protein complexes assembled on target gene promoter regions. These complexes contain activators and coactivators of transcription as well as elements of the basal transcription machinery (1). The specificity, timing, and degree of transcriptional activation depend not only on which proteins form this complex but also on how they interact with each other. At the simplest level, direct interactions involved in complex formation provide a mechanism for recruiting specific functionalities to a promoter sequence. However, in some cases, binding events also give rise to allosteric effects on binding or enzymatic activity, providing another means by which transcriptional activity can be modulated.A transcriptional coactivator that may be allosterically regulated by simultaneous interactions with more than one target protein is the cAMP-response element (CREB) 1 -binding protein (CBP), as well as its paralog p300. In addition to its catalytic histone acetyltransferase domain, CBP is composed of several protein-binding modules that serve to bridge genespecific transcription factors with components of the basal transcription complex (2). CBP domains such as KIX, CH1, and CH3 recognize a diverse range of protein sequences that in some cases appear to share a general structural motif in the bound state. For example, the KIX domain binds an amphipathic helix formed by either the phosphorylated kinaseinducible domain (pKID) of CREB or the transactivation domain of c-Myb (3, 4). Although both of these ligands bind to the shallow hydrophobic groove formed between KIX helices ␣ 1 and ␣ 3 , the high affinity of CREB for CBP is mediated by critical interactions involving the pKID phosphoryl group and is therefore inducible, whereas the c-Myb activation domain binds constitutively with somewhat lower affinity (5, 6).It has been proposed that CBP-mediated transcriptional activation can be enhanced by cooperative interactions between CBP and more than one DNA-bound transcriptional activator (7-9). Since the nuclear concentration of CBP is thought to be limiting (10 -12), the presence of multiple protein-binding domains on CBP, together with the high affinity of CBP for the activators involved, should sequester a larger proportion of CBP to specific promoter sites and thereby provide a mechanism for transcriptional synergy by multiple activators. There are a number of cases where synergistic increases in transcription have been shown to occur when multiple CBP-binding activators bind to the same gene promoter. Examples include cooperative transcriptional activation by CREB and the serum response factor (13, 14) as well as enhancement of c-Myb transcriptional activation by core binding factor (15, 16), CCAAT/

show abstract

Analysis of an Activator:Coactivator Complex Reveals an Essential Role for Secondary Structure in Transcriptional Activation

Cited by 146 publications

References 27 publications

Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB

Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

Cooperativity in Transcription Factor Binding to the Coactivator CREB-binding Protein (CBP)

Contact Info

Product

Resources

About