Recruitment of an RNA Polymerase II Complex Is Mediated by the Constitutive Activation Domain in CREB, Independently of CREB Phosphorylation

Abstract

The cAMP response element binding protein (CREB) is a bifunctional transcription activator, exerting its effects through a constitutive activation domain (CAD) and a distinct kinase inducible domain (KID), which requires phosphorylation of Ser-133 for activity. Both CAD and phospho-KID have been proposed to recruit polymerase complexes, but this has not been directly tested. Here, we show that the entire CREB activation domain or the CAD enhanced recruitment of a complex containing TFIID, TFIIB, and RNA polymerase II to a linked promoter. The nuclear extracts used mediated protein kinase A (PKA)-inducible transcription, but phosphorylation of CRG (both of the CREB activation domains fused to the Gal4 DNA binding domain) or KID-G4 did not mediate recruitment of a complex, and mutation of the PKA site in CRG abolished transcription induction by PKA but had no effect upon recruitment. The CREB-binding protein (CBP) was not detected in the recruited complex. Our results support a model for transcription activation in which the interaction between the CREB CAD and hTAFII130 of TFIID promotes the recruitment of a polymerase complex to the promoter.The cAMP response element (CRE) mediates both constitutive and cAMP-induced transcription activation of many genes in a variety of cell types (8,30,40,43,50,52). The CRE-binding protein, CREB, a member of the basic leucine zipper family of transcription factors, binds constitutively to the CRE in the promoter of the target gene (51) and can activate constitutive transcription in the absence of hormonal stimuli (3,29,50). Extracellular stimuli that activate protein kinases can lead to phosphorylation of CREB on Ser-133, e.g., by cAMP-activated protein kinase A (PKA), resulting in a further enhancement of transcriptional activation (3,16,50). Mutation of the Ser-133 PKA phosphorylation site in CREB to an alanine abolishes kinase-inducible activation (17, 50) but not constitutive activation (3, 29, 50). We and others have shown that these constitutive and kinase-inducible activities map to two separate and independently acting transcription activation domains: a constitutive activation domain (CAD) responsible for activating constitutive transcription and a kinase-inducible domain (KID) that mediates activation in response to cAMP-activated PKA (3, 29, 50) and several other kinases (11,15,26,63,68). However, the exact mechanism of action of these domains in stimulating constitutive and kinaseinducible transcription has not been defined.Transcription of a class II gene by RNA polymerase II requires the assembly of general transcription factors and coactivators around the transcription start site in the gene's promoter (reviewed in references 22 and 44). The general transcription factors (TFIID, TFIIA, TFIIB, TFIIF-pol II, TFIIE, and TFIIH) were initially identified as the basic nuclear components required to reconstitute in vitro transcription by RNA polymerase II (9, 54-56, 66, 67). These general factors are required for accurate and optimal positioning of RNA polymerase II at ...

Protein Kinase A Phosphorylates Hepatocyte Nuclear Factor-6 and Stimulates Glucose-6-phosphatase Catalytic Subunit Gene Transcription

“…It may be possible to prove that PKA-dependent phosphorylation increases the transactivation potential of HNF-6 by analyzing the effect of mutating the three PKA phosphorylation sites in the context of an HNF-6 molecule in which the basal activation domains (37,44) have been mutated but only if these same domains are not also required for the PKA response. These observations are somewhat related to those recently reported by Quinn and co-workers (46,47), who investigated the relative contributions of different domains in CREB to transcription initiation. Of particular note is the observation that the constitutive activation domain in CREB mediates recruitment of the polymerase complex, whereas the kinase-inducible domain mediates later PKA-stimulated steps in transcription initiation.…”

Chromatin-Dependent Cooperativity between Constitutive and Inducible Activation Domains in CREB

“…In contrast to these studies, Felinski et al have recently proposed that transcriptional induction via CREB proceeds exclusively via the Q2 domain without a requirement for CBP-containing complexes (8). Rather, they propose that phospho (Ser133) CREB stimulates CREB activity via an allosteric mechanism that potentiates Q2 activity.…”

“…The Q2 domain has been shown to stimulate transcription by recruiting TFIID activity to the promoter via a direct interaction with TAF II 130 (8,10,26). To map the domain of TAF II 130 that interacts with CREB, we performed GST pulldown assays.…”

“…Rather, they propose that phospho (Ser133) CREB stimulates CREB activity via an allosteric mechanism that potentiates Q2 activity. The use of naked DNA templates to evaluate CREB activity and TFIID recruitment in these studies (8) could mask potential effects of CBP/p300 on CREB-dependent transcription, however, prompting us to evaluate CREB-dependent tran-scription on a chromatin template. Our studies confirm the importance of CBP/p300 in this process and provide new insights into the mechanism by which these HATs mediate cooperativity between constitutive and inducible domains in CREB.…”

Regulation of the human SOX9 promoter by Sp1 and CREB

“…Interestingly, CREB can stimulate both basal transcription and inducible transcription through its bipartite transcriptional activation domain [34,38,39]. The domain contains a constitutive activation domain that can interact with components of the general transcriptional machinery and a kinase-inducible domain that when phosphorylated at a serine residue promotes the recruitment of transcriptional co-activators such as CREB-binding protein and p300 [38][39][40][41][42][43]. A recent study of CREB binding has shown that the protein has the ability to bind its cognate site in responsive gene promotes either as a monomer or as a homodimer or a heterodimer with other transcription factors such as NFIL3 [44].…”