CRE DNA binding proteins bind to the AP-1 target sequence and suppress AP-1 transcriptional activity in mouse keratinocytes

Previously, we have shown that nuclear extracts from cultured mouse keratin ocytes induced to differentiate by increasing the levels of extra-cellular calcium contain Fra-1, Fra-2, Jun B, Jun D and c-Jun proteins that bind to the AP-1 DNA binding sequence. Despite this DNA binding activity, AP-1 repo rter activity was suppressed in these cells. Here, we have detected the CRE B family proteins CREB and CREM alpha as additional participants in the AP- 1 DNA binding complex in differentiating keratinocytes, AP-1 and CRE DNA bi nding activity correlated with the induction of CREB, CREM alpha and ATF-1 and CREB phosphorylation at ser(133) (ser(133) phospho-CREB) in the transit ion from basal to differentiating keratinocytes, but the activity of a CRE reporter remained unchanged. In contrast, the CRE reporter was activated in the presence of the dominant-negative (DN) CREB mutants, KCREB and A-CREB, proteins that dimerize with CREB family members and block their ability to bind to DNA. The increase in CRE reporter activity in the presence of thes e mutants suggests that CRE-mediated transcriptional activity is suppressed in keratinocytes through protein-protein interactions involving a factor t hat dimerizes with the CREB leucine zipper. In experiments where the A-CREB mutant was co-transfected with an AP-1 reporter construct, transcriptional activity was also increased indicating that a CREB family member binds AP- 1 sites and represses AP-1 transcriptional activity as well. Exogenous expr ession of the transcriptional repressor CREM alpha down-regulated both CRE and AP-1 reporters in keratinocytes suggesting that this factor may contrib ute to the suppression of AP-1 transcriptional activity observed in differe ntiating keratinocytes.