CREB isoform represses yolk protein gene expression in the mosquito fat body

In mosquitoes, the steroid 20-hydroxyecdysone (20E) is the main regulator of yolk protein precursor (YPP) gene expression. However, peptide hormones have also been implicated. To investigate involvement of the cAMP-mediated signal-transduction cascade in regulation of mosquito vitellogenic events, we cloned an Aedes aegypti cAMP response element binding protein (AaCREB). The AaCREB contained the domains characteristic to members of the cAMP response element binding protein (CREB) family of transcription factors: a kinase inducible domain region and a bZIP domain responsible for DNA binding and protein dimerization. In the mosquito fat body (site of YPP gene expression), the AaCREB gene was constitutively expressed and produced a transcript of 3.5-4 kb. In vitro fat body organ culture experiments demonstrated that elicitors of the cAMP signal-transduction pathway attenuated 20E-stimulated YPP gene expression. Cell transfection analysis indicated that AaCREB served as a potent repressor of transcription (designated AaCREBr). The role of AaCREBr as a transcriptional repressor supported the electrophoretic mobility shift assay (EMSA) with nuclear extracts from vitellogenic fat bodies. This analysis detected CREB-specific band-shift complexes in nuclear extracts at 24 and 36 h post-blood meal (PBM), when YPP gene expression reaches its peak then terminates. Examination of the regulatory regions of two major YPP genes, vitellogenin (Vg) and vitellogenic carboxypeptidase (VCP), revealed the presence of putative CREB response elements (CREs). These elements competed with the CRE consensus sequence for binding of in vitro-expressed AaCREBr. We propose that AaCREBr functions as a repressor of YPP gene expression at the time of vitellogenesis termination in the fat body.