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cAMP stimulates diacylglycerol kinase‐θ phosphorylation in adrenocortical cells (605.18)
Author(s) -
Cai Kai,
Sewer Marion
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.605.18
Subject(s) - diacylglycerol kinase , phosphorylation , phosphatidic acid , kinase , biology , signal transduction , microbiology and biotechnology , transcription factor , protein kinase a , steroidogenic factor 1 , nuclear receptor , chemistry , biochemistry , protein kinase c , phospholipid , membrane , gene
Diacylglycerol kinase theta (DGKθ) is an intercellular lipid kinase that coordinates nuclear phospholipid metabolism and cortisol production in the adrenal cortex by generating phosphatidic acid (PA), an agonist of the nuclear receptor steroidogenic factor 1 (SF1). We previously found that cAMP signaling induces the transcription of DGKθ and increases nuclear DGK activity. Moreover, mass spectrometric analysis of DGKθ isolated from H295R human adrenocortical cells has revealed that DGKθ is a phosphoprotein. However, the functional significance of DGKθ phosphorylation is unclear. Additionally, the mechanism by which cAMP signaling regulates DGKθ activity is not known. We show that dibutyryl cAMP increases the phosphorylation of DGKθ in H295R human adrenocortical cells. Alanine substitution at Ser‐354 reduces cAMP‐stimulated phosphorylation impairs the ability of DGKθ to activate SF1‐dependent transcription of CYP17. Cycloheximide sensitivity assays revealed that the S354A mutant is more stable than the wild type. Notably, we also identify Ser‐241 as a second site that is phosphorylated, where the S241A mutant abrogates DGKθ‐stimulated activation of a CYP17 reporter gene. Since Ser‐241 lies near to two proline residues, Pro‐244 and Pro‐245, that have been identified as key for optimal catalytic activity, we postulate that mutation of these prolines also alters the ability of DGKθ to be phosphorylated at this site. Indeed, P244L and P245L impair SF1‐dependent CYP17 luciferase activity. In summary, we identify DGKθ as an effector in the cAMP signaling pathway and a target of protein kinase A. Grant Funding Source : Supported by NIH DK084178.