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.[1]References
- Mutations in the activation function-2 core domain of steroidogenic factor-1 dominantly suppresses PKA-dependent transactivation of the bovine CYP17 gene. Jacob, A.L., Lund, J. J. Biol. Chem. (1998) [Pubmed]
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