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Molecular and Cellular Biology, October 2007, p. 6669-6685, Vol. 27, No. 19
0270-7306/07/$08.00+0     doi:10.1128/MCB.00355-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cyclic AMP-Stimulated Interaction between Steroidogenic Factor 1 and Diacylglycerol Kinase Facilitates Induction of CYP17

School of Biology and the Parker H. Petit Institute for Bioengineering & Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332

Received 27 February 2007/ Returned for modification 17 April 2007/ Accepted 20 July 2007

In the human adrenal cortex, adrenocorticotropin (ACTH) activates CYP17 transcription by promoting the binding of the nuclear receptor steroidogenic factor 1 (SF1) (Ad4BP, NR5A1) to the promoter. We recently found that sphingosine is an antagonist for SF1 and inhibits cyclic AMP (cAMP)-dependent CYP17 gene transcription. The aim of the current study was to identify phospholipids that bind to SF1 and to characterize the mechanism by which ACTH/cAMP regulates the biosynthesis of this molecule(s). Using tandem mass spectrometry, we show that in H295R human adrenocortical cells, SF1 is bound to phosphatidic acid (PA). Activation of the ACTH/cAMP signal transduction cascade rapidly increases nuclear diacylglycerol kinase (DGK) activity and PA production. PA stimulates SF1-dependent transcription of CYP17 reporter plasmids, promotes coactivator recruitment, and induces the mRNA expression of CYP17 and several other steroidogenic genes. Inhibition of DGK activity attenuates the binding of SF1 to the CYP17 promoter, and silencing of DGK- expression inhibits cAMP-dependent CYP17 transcription. LXXLL motifs in DGK- mediate a direct interaction of SF1 with the kinase and may facilitate binding of PA to the receptor. We conclude that ACTH/cAMP stimulates PA production in the nucleus of H295R cells and that this increase in PA concentrations facilitates CYP17 induction.

Published ahead of print on 30 July 2007.