Mutations in the Activation Function-2 Core Domain of Steroidogenic Factor-1 Dominantly Suppresses PKA-dependent Transactivation of the Bovine CYP17 Gene

Anne L Jacob,Johan Lund

doi:10.1074/jbc.273.22.13391

Abstract

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.

Highlights

Naling via G-protein coupled cell surface receptors, these hormones increase intracellular cAMP levels and activate cAMP-dependent protein kinase (PKA).1 As a consequence, steroidogenesis is stimulated through a rapid mobilization and movement of the substrate cholesterol to the enzymes, which convert it to biologically active hormones
In the bovine CYP17 gene, which encodes the cytochrome P-450 17␣hydroxylase, an Steroidogenic factor-1 (SF-1) site is present within cAMP-responsive sequence 2 (CRS2) [13], and mutations that interfere with SF-1 binding correlate with decreases in cAMP-stimulated transcription of a linked reporter gene [9]
Several lines of evidence suggest that the nuclear receptor SF-1 may be directly involved in cAMP-dependent transcriptional regulation of steroid hydroxylase genes

Summary

Introduction

Naling via G-protein coupled cell surface receptors, these hormones increase intracellular cAMP levels and activate cAMP-dependent protein kinase (PKA).1 As a consequence, steroidogenesis is stimulated through a rapid mobilization and movement of the substrate cholesterol to the enzymes, which convert it to biologically active hormones. Such mutant SF-1 proteins are able to dominantly suppress the activity of wild-type SF-1 in adrenocortical tumor cells transfected with PKA-C. Western Blot Analysis—To determine expression levels, Cos-1 cells were transfected with plasmids encoding wild-type SF-1 and AF-2 mutants.

Results

Conclusion