CAMP RESPONSE ELEMENT-BINDING PROTEIN (CREB) INTERACTS WITH TRANSCRIPTION FACTOR-IIB AND FACTOR-IID

cAMP response element-binding protein (CREB) participates in both cons titutive and cAMP-induced transcription of cAMP-responsive genes. CREB -mediated constitutive transcription requires only CREB-binding sites and a minimal promoter region (containing the TATA through start seque nces), indicating that CREB interacts directly with components of the general transcription machinery. In this study, a coimmunoprecipitatio n assay was used to test for interaction of CREB with the general tran scription factors (TF) TFIIB and TFIID and the core component of TFIID , TATA-binding protein (TBP). Human TFIIB and TBP, tagged with distinc t epitopes (eTFIIB and eTBP), were expressed in and purified from Esch erichia coil, and holo-eTFIID, containing eTBP, was obtained from the HeLa cell line LTR alpha 3. S-35-Labeled CREB, synthesized in vitro an d incubated with eTFIIB, was coimmunoprecipitated with antibody recogn izing eTFIIB, indicating that CREB spe cifically binds to TFIIB. S-35- CREB was coimmunoprecipitated with antibody against eTBP, but only whe n incubated with the holo eTFIID complex, not with eTBP alone. TFIIB i nteracted with TBP, but CREB was not coprecipitated with the eTBP anti body when incubated with eTBP plus TFIIB, so CREB did not form a stabl e ternary complex with TFIIB and TBP. Conversely, depletion of TFIIB f rom the holo-TFIID preparation did not diminish the level of interacti on between CBEB and TFIID. Thus, CREB interacts independently with TFI IB and TFIID, but not directly with TBP. A protein kinase A phosphoryl ation site mutant of CREB and wild-type CREB exhibited equivalent inte raction with TFIIB, indicating that this phosphorylation is not requir ed. Consistent with the role of CREB in promoting constitutive or basa l transcription, the constitutive activation domain of CREB was suffic ient for interaction with both TFIIB and TFIID.