A DOMINANT-NEGATIVE INHIBITOR OF CREB REVEALS THAT IT IS A GENERAL MEDIATOR OF STIMULUS-DEPENDENT TRANSCRIPTION OF C-FOS

Several studies have characterized the upstream regulatory region of c -fos, and identified cis-acting elements termed the cyclic AMP (cAMP) response elements (CREs) that are critical for c-fos transcription in response to a variety of extracellular stimuli, Although several trans cription factors can bind to CREs in vitro, the identity of the transc ription factor(s) that activates the c-fos promoter via the CRE in viv o remains unclear. To help identify the trans-acting factors that regu late stimulus-dependent transcription of c-fos via the CREs, dominant- negative (D-N) inhibitor proteins that function by preventing DNA bind ing of B-ZIP proteins in a dimerization domain-dependent fashion were developed, A D-N inhibitor of CREB, termed A-CREB, was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper domain, The acidic extension of A-CREB interac ts with the basic region of CREB forming a coiled-coil extension of th e leucine zipper and thus prevents the basic region of wild-type CREB from binding to DNA, Other D-N inhibitors generated in a similar manne r with the dimerization domains of Fos, Jun, C/EBP, ATF-2, or VBP did not block CREB DNA binding activity, nor did they inhibit transcriptio nal activation of a minimal promoter containing a single CRE in PC12 c ells, A-CREB inhibited activation of CRE-mediated transcription evoked by three distinct stimuli: forskolin, which increases intracellular c AMP; membrane depolarization, which promotes Ca2+ influx; and nerve gr owth factor (NGF), A-CREB completely inhibited cAMP-mediated, but only partially inhibited Ca2+ and NGF-mediated, transcription of a reporte r gene containing 750 bp of the native c-fos promoter, Moreover, gluta mate induction of c-fos expression in primary cortical neurons was dep endent on CREB, In contrast, induction of c-fos transcription by UV li ght was not inhibited by A-CREB, Lastly, A-CREB attenuated NGF inducti on of morphological differentiation in PC12 cells, These results sugge st that CREB or its closely related family members are general mediato rs of stimulus-dependent transcription of c-fos and are required for a t least some of the long-term actions of NGF.