TRANSCRIPTIONAL REGULATION OF THE JUNB PROMOTER IN MATURE B-LYMPHOCYTES - ACTIVATION THROUGH A CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-LIKE BINDING-SITE

The experiments presented herein were designed to understand the molec ular mechanism(s) by which membrane Ig (mIg)-dependent signals are int egrated at the level of the junB promoter to induce gene transcription . Functional studies using chloramphenicol acetyltransferase reporter gene constructs that contained deleted 5' flanking region junB sequenc es identified a region located between -194 and -87 that contains an E ts binding site and a putative cAMP response element binding site (CRE -like). Point mutagenesis of the CRE-like site blocked junB promoter a ctivation in response to mIg cross-linking in mature Bal17 B cells. Nu clear extract binding activity to a synthetic oligonucleotide containi ng the junB CRE-like site was detected in unstimulated B cells and was increased in response to mIg cross-linking. Binding activity was comp eted with unlabeled oligonucleotides that contained the junB CRE-like site or the somatostatin CRE consensus motif; the latter observation s uggests that members of the activating transcription factor/CRE bindin g protein (CREB) family may mediate mIg-dependent junB transcription. Consistent with this interpretation, recombinant CREB and activating t ranscription factor proteins bound the junB CRE-like site, but did not interact with a mutant CRE-like site. Expression of a dominant negati ve CREB protein blocked mIg-mediated transcription from a junB CRE-lik e site-chloramphenicol acetyltransferase reporter gene. CRE-like nucle oprotein complexes from Bal17 B cells contained constitutively bound C REB-1, which was phosphorylated on serine 133 in response to mIg cross -linking. Activating transcription factor-1 protein was also constitut ively expressed in CRE-like nucleoprotein complexes. Collectively, the se results suggest that components of the protein kinase A signaling p athway are recruited by mIg to induce junB transcription.