Distinct cAMP response element-binding protein (CREB) domains stimulate different steps in a concerted mechanism of transcription activation Jeong-a Kim, Jingfang Lu, and Patrick G. Quinn* Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, PA 17033 Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved August 9, 2000 (received for review January 21, 2000) Hormones and neurotransmitters rapidly change patterns of gene could promote any (7, 9, 10) or all (11) of the steps in expression in target cells by activating protein kinases that phos- transcription initiation in response to cAMP. However, inter- phorylate and modify the activity of CREB and other transcription pretation of these studies is limited by the possible complica- factors. Although CREB was initially characterized as mediating the tion that PKA phosphorylates and changes the activity of other response to cAMP, CREB phosphorylation and activation are stim- proteins in the nuclear extracts. ulated by diverse extracellular signals and protein kinases in The CREB protein contains two distinct activation domains, a essentially all cells and tissues. CREB stimulates transcription constitutive activation domain (CAD) and a kinase-inducible do- through a constitutive activation domain (CAD), which interacts main (KID) that can act independently to facilitate either consti- with the promoter recognition factor TFIID, and through a kinase- tutive or kinase-inducible transcription activation (13, 14). The inducible domain (KID), when Ser-133 is phosphorylated. The CAD in CREB interacts with the hTAF130͞dTAF110 subunit of present study provides new insight into the mechanism of activa- TFIID, which may facilitate recruitment of a polymerase complex tion by showing that each of the CREB domains contributes to assembly (15, 16) (E. Felinski, J.K., L.J., and P.G.Q., unpublished transcription initiation by stimulating sequential steps in the tran- work). We mapped the interaction between the TATA-binding scription reaction. The CAD effectively assembled a polymerase protein (TBP)-associated factor (TAF) and CREB to hydrophobic complex, as evidenced by constitutive activation in vivo and stim- residues in the CREB CAD (15). Subsequent work showed that ulation of single-round transcription in vitro. In contrast, phos- these mutations also abolish (i) interaction between the proteins in phorylation of the KID in CREB stimulated isomerization of the vitro,(ii) recruitment of a polymerase-containing complex, and (iii) polymerase complex, as determined by abortive initiation, and transcription activation in transfection studies, thereby providing a promoter clearance and͞or reinitiation, as measured by multiple genetic link between interaction, recruitment, and transcription rounds of transcription. Our results provide evidence for a new activation (E. Felinski and P.G.Q., unpublished work). An analo- model for CREB-mediated induction through a concerted mecha- gous mechanism is used to stimulate transcription activation by nism involving establishment of a polymerase complex by the CAD, other constitutively active factors (17–20) and ligand-inducible followed by stimulation of isomerization, promoter clearance, nuclear receptors (21, 22). and͞or reinitiation by phosphorylated KID to enhance target gene Phosphorylation of CREB on Ser-133 has been demon- transcription. strated to promote association with CREB-binding protein (CBP). Bacterially expressed CREB binds CBP in a strictly mportant features of kinase-inducible gene regulation are that phosphorylation-dependent manner and overexpression of Imany of the target genes exhibit basal expression and the CBP augmented induction of a somatostatin reporter by response to kinase activation is extremely rapid, occurring within CREB in F9 cells (23). In that study, CBP also bound TFIIB, minutes (1, 2). CREB was first described as a transcription factor suggesting that it enhances activation through recruitment of mediating induction by extracellular signals activating adenylate essential components of the polymerase complex. In another cyclase and protein kinase A (PKA), which phosphorylates study, CBP binding to CREB was reported in the absence of Ser-133 in CREB and enhances its transcriptional activity (3). CREB phosphorylation (24). More recently, phosphorylated Subsequently, several other signaling pathways were demon- CREB was shown to interact with RNA polymerase complexes strated to activate protein kinases that phosphorylate Ser-133 in HeLa cell nuclear extracts, an effect that could be blocked and enhance transcription of cAMP response element (CRE)- with a CBP peptide that bound CREB or antibody against containing genes in a variety of cells and tissues (calmodulin RNA helicase A (25, 26). It was proposed that phosphorylated kinase, mitogen-activated protein kinase͞p90rsk, protein kinase CREB activates transcription through interaction with CBP C, p38, and protein kinase B (PKB͞Akt) (4, 5). and recruitment of RNA polymerase II complexes. However, For any gene to be transcribed, there must be recruitment the assays used in that study did not assess the contribution of of a polymerase complex, isomerization to expose the template CREB phosphorylation to discrete steps in transcription ini- strand, and promoter clearance of the polymerase to transcribe tiation. Phosphorylated CREB would have to interact with the the body of the gene (6). Previous studies, based on the binding polymerase complex to have an effect on transcription, of heterogeneous activating transcription factor (ATF)͞CREB whether at the recruitment step or at later steps, e.g., isomer- proteins to CRE sites, have produced conflicting results ization, promoter clearance, or reinitiation. (7–12). Early studies using footprinting demonstrated that the presence of an ATF͞CRE site in a promoter produced an extended footprint, caused by a complex containing RNA This paper was submitted directly (Track II) to the PNAS office. polymerase II and TFIIB, TFIID, and TFIIE (7, 8). We Abbreviations: CAD, constitutive activation domain; KID, kinase-inducible domain; CRG, CREB-Gal4; PKA, cAMP-activatable protein kinase A; CRE, cAMP response element; CBP, previously showed that stimulation of in vitro transcription by CREB-binding protein; 5XGT, minimal promoter controlled by five Gal4 sites; G4-DBD, Gal4 cAMP-activatable PKA required a CRE site in the template DNA binding domain. and could be inhibited by a PKA-specific inhibitor peptide or *To whom reprint requests should be addressed. E-mail: [email protected]. by the addition of phosphatase but PKA did not affect binding The publication costs of this article were defrayed in part by page charge payment. This of CREB (12). Additional in vitro transcription studies by article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. others suggested that factors associated with ATF͞CRE sites §1734 solely to indicate this fact. 11292–11296 ͉ PNAS ͉ October 10, 2000 ͉ vol. 97 ͉ no. 21 Downloaded by guest on September 26, 2021 Fig. 1. Distinct activities of the CAD and KID in CREB in phosphorylation-dependent transcription activation in vivo and in vitro.(a) CAD, KID, and DNA-binding domain each contain amino acids 1–8 of CREB plus the indicated CREB amino acid sequence fused to amino acids 4–94 of the Gal4 DNA-binding domain. (b) JEG3 cells were cotransfected with the 5XGT-Luciferase reporter plasmid plus expression plasmids for CREB-Gal4 proteins Ϯ the catalytic subunit of PKA. The data represent the means Ϯ SEM of five independent experiments. (c) A representative experiment shows the effects of PKA titration on in vitro transcription mediated by CRG or S133A. (d) Western blots of purified CREB-GAL4 proteins, incubated ϯ 1 unit of PKA, were performed with anti-CREB (Upper) or anti-P-CREB (Lower) antisera. In the present study, we used assays for discrete steps in tran- specific activity in a mobility shift assay. Where indicated, 0.5 scription initiation to determine the separate and combined con- unit of PKA͞0.5 ␮M okadaic acid were added to phosphorylate tributions of the CREB-activation domains, CAD and KID, to P-KID and P-CRG, after which 0.5 unit of PKA inhibitor was regulation of these different steps in transcription activation. Either added to inhibit further PKA activity. Phosphorylation of CRG or both of the CREB-activation domains were linked to the Gal4 proteins by PKA was demonstrated by Western blotting with a DNA-binding domain in fusion proteins that were used to assess CREB phospho-Ser-133-specific antibody. Rat liver nuclear transcription activation in vivo and in vitro. We directly examined extract preparation and in vitro transcription were performed as the contributions of these activation domains to recruitment, described (12), with the following modifications: in vitro reac- isomerization, or promoter clearance͞reinitiation with single- tions contained 50 ␮g of nuclear extract͞150 fmol of template round transcription, abortive initiation, or multiple-round in vitro (750 fmol of Gal4 sites)͞750 fmol of CREB-Gal4 protein. transcription assays, respectively. We show that the CAD in CREB Template, nuclear extract, and CREB-Gal4 proteins were pre- mediated assembly of a polymerase complex, a process that was incubated for 10 min to allow formation of a preinitiation unaffected by phosphorylation of KID. In contrast, the KID in complex before the addition of nucleotides. In vitro transcription CREB, but not