Mutations in the activation function-2 core domain of steroidogenic factor-1 dominantly suppresses PKA-dependent transactivation of the bovine CYP17 gene.
Steroidogenic factor-1 (SF-1) is a nuclear receptor that is essential for the proper development and function of steroid hormone-producing cells. The activation function-2 (AF-2) domain in SF-1 is a short alpha-helix in the C terminus that is conserved with respect to other nuclear receptors and is important for transactivation of target genes. In order to investigate the possible role of the AF-2 domain of SF-1 in cAMP-dependent transcriptional regulation of the bovine steroid hydroxylase gene CYP17, mutations were introduced and the effects were characterized. The mutant SF-1 proteins were expressed at comparable levels in nonsteroidogenic Cos-1 cells that lack SF-1, and their abilities to bind an SF-1 site from the CYP17 gene were not affected. Transient transfections of wild-type and mutant SF-1 in Cos-1 cells showed that the capacity to transactivate a reporter gene under the control of the SF-1 site from CYP17 was reduced by the mutations in the AF-2 domain of SF-1. A point mutation in the AF-2 region, E454A, resulted in a relative reporter gene activity that was 21% of that observed with wild-type SF-1. Co-transfections of adrenocortical Y-1 cells, which express endogenous SF-1, with the catalytic subunit of cAMP-dependent protein kinase (PKA-C) and the SF-1-dependent reporter gene showed on average a 16-fold increase in activity in the presence of PKA-C. Introduction of the AF-2 mutants of SF-1 into Y-1 cells completely abolished the PKA-C-mediated stimulation of the reporter gene. The transdominant negative effect of the mutant SF-1 proteins suggests that the AF-2 domain is essential for the activation of SF-1 by the cAMP-dependent protein kinase-dependent signaling pathway.
Jacob
A L
AL
Department of Anatomy and Cell Biology, University of Bergen, N-5009 Bergen, Norway.
Lund
J
J
D000818:Animals; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D051379:Mice; D009154:Mutation; D018160:Receptors, Cytoplasmic and Nuclear; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C515058:steroidogenic factor 1, mouse; D000242:Cyclic AMP; D013254:Steroid 17-alpha-Hydroxylase; D017868:Cyclic AMP-Dependent Protein Kinases