Identification and Characterization of a Novel Transcriptional Activation Domain in the CREB-binding Protein

Sandra Bisotto,Stephanie Minorgan,Robert P Rehfuss

doi:10.1074/jbc.271.30.17746

Sandra Bisotto, Stephanie Minorgan + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.30.17746

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Abstract

The CREB-binding protein (CBP) plays a central role in the regulation of gene expression by several different second messenger pathways including serum growth factors, cAMP and phorbol esters. CBP specifically binds to the phosphorylated forms of CREB and c-Jun and is thought to activate transcription through a C-terminal activation domain. In this report, we demonstrate that the C terminus of CBP is dispensable for its ability to stimulate phospho-CREB activity, and, further, that the deletion of this domain produces highly active, mutant forms of CBP. The novel N-terminal activation identified by this deletional analysis consists of the first 714 amino acids of CBP and is sufficient for high levels of transcriptional activity. This domain is also capable of stimulating the activity of a second cAMP-regulated factor, ATF-1. Surprisingly, ATF-1 activity is not significantly stimulated by full-length CBP suggesting that the C-terminal domain of CBP may also serve to regulate ATF-1/CBP activity. Additionally, the demonstration that one of our hyperactive CBP mutants is able to activate a nonphosphorylatable mutant of CREB (M1 CREB) provides the first evidence that CBP may play a role in regulating the basal transcriptional activity of CREB.

Highlights

Induced changes in the levels of intracellular cyclic AMP regulate the expression of many cellular and viral genes through a common promoter element termed the CRE1 [1]
The TFIIB Binding Site of CREB-binding protein (CBP) Is Dispensable for Activity—To determine if the ability of CBP to act as a CREB coactivator was dependent on the presence of the TFIIB binding site, we examined the transcriptional activity of a mutant form of CBP which lacks this region
Sequences which comprise the TFIIB binding site were removed by constructing an in-frame deletion of the region (Fig. 1A) and the ability of the resulting mutant (CBP ⌬E) to potentiate the activity of CREB was determined by measuring its ability to stimulate CREB-dependent transcriptional activity in F9 cells

Summary

Introduction

Induced changes in the levels of intracellular cyclic AMP (cAMP) regulate the expression of many cellular and viral genes through a common promoter element termed the CRE1 [1]. This interaction is required for PKA-dependent transcriptional activation as disruption of the complex by microinjection of either specific peptide fragments of the phospho-CREB binding domain or anti-CBP blocking antibodies is able to block stimulated expression of a CRE-dependent reporter gene in fibroblast cells [10, 11].

Results

Conclusion