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 keratinocytes 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 reporter activity was suppressed in these cells. Here, we have detected the CREB family proteins CREB and CREMalpha as additional participants in the AP-1 DNA binding complex in differentiating keratinocytes. AP-1 and CRE DNA binding activity correlated with the induction of CREB, CREMalpha and ATF-1 and CREB phosphorylation at ser133 (ser133 phospho-CREB) in the transition 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 these mutants suggests that CRE-mediated transcriptional activity is suppressed in keratinocytes through protein-protein interactions involving a factor that 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 expression of the transcriptional repressor CREMalpha down-regulated both CRE and AP-1 reporters in keratinocytes suggesting that this factor may contribute to the suppression of AP-1 transcriptional activity observed in differentiating keratinocytes.
Rutberg
S E
SE
Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Adams
T L
TL
Olive
M
M
Alexander
N
N
Vinson
C
C
Yuspa
S H
SH
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000502:physiology; D002454:Cell Differentiation; D051786:Cyclic AMP Response Element Modulator; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D019281:Dimerization; D015603:Keratinocytes / Q000378:metabolism*; D016350:Leucine Zippers; D046911:Macromolecular Substances; D051379:Mice; D010766:Phosphorylation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D012097:Repressor Proteins; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; D018808:Transcription Factor AP-1; D051786:Cyclic AMP Response Element Modulator; D004247:DNA; D002118:Calcium