Distinct activation domains within cAMP response element-binding protein (CREB) mediate basal and cAMP-stimulated transcription

Abstract

Activation of protein kinase A (PKA) by cAMP results in phosphorylation of cAMP response element-binding protein (CREB) and induction of specific gene expression. However, whether CREB participates directly in basal (PKA-independent) transcription is still an open question, and existing studies conflict over the identification of putative basal activation domains. In the present study, the activation domain of CREB, whether fused to the GAL4 DNA binding domain (CRG) or in native CREB, stimulated basal activity of the minimal tk promoter, but not the minimal SV40 early promoter. Cotransfection with PKI, a specific inhibitor of PKA, blocked PKA-induced expression of both promoters, but did not block CRG-mediated basal expression of the tk promoter. In addition, both CRG and a PKA phosphorylation site mutant provided comparable stimulation of basal tk promoter activity. Examination of a series of CREB deletion mutants mapped basal activity to interacting domains, located on either side of the previously identified PKA activation domain (amino acids 98-142). This PKA-independent activity mapped primarily to a bipartite COOH-terminal basal activation domain (amino acids 165-252). Its major component bears no obvious homology to previously identified activation domains, whereas a minor component is glutamine-rich. This COOH-terminal domain acts independently and provides the majority of basal activation but requires an NH2-terminal domain (amino acids 41-86) to provide full basal activity. A repressor domain (amino acids 142-165), deletion of which enhanced both basal and PKA-activated transcription, was also identified. This work establishes that CREB contains distinct basal and PKA-activated domains, that they operate independently for both loss of function and gain of function, and that they work on different promoters in different cell types.