In the human adrenal cortex, the peptide hormone adrenocorticotropin (ACTH) directs cortisol and adrenal androgen biosynthesis by activating a cAMP/cAMP-dependent protein kinase (PKA) pathway. Carboxyl-terminal binding protein 1 (CtBP1) is a corepressor that regulates transcription of the CYP17 gene by periodically interacting with steroidogenic factor-1 in response to ACTH signaling. Given that CtBP1 function is regulated by NADH binding, we hypothesized that ACTHstimulated changes in cellular pyridine nucleotide concentrations modulate the ability of CtBP1 to repress CYP17 transcription. Further, we postulated that PKA evokes changes in the phosphorylation status of CtBP1 that control the ability of the protein to bind to steroidogenic factor-1 and the coactivator GCN5 (general control nonderepressed 5) and repress CYP17 gene expression. We show that ACTH alters pyridine nucleotide redox state and identify amino acid residues in CtBP1 that are targeted by PKA and PAK6. Both ACTH/cAMP signaling and NADH/NAD ؉ ratio stimulate nuclear-cytoplasmic oscillation of both CtBP proteins. We provide evidence that PKA 1) induces metabolic changes in the adrenal cortex and 2) phosphorylates CtBP proteins, particularly CtBP1 at T144, resulting in CtBP protein partnering and ACTH-dependent CYP17 transcription.
CtBP12 was originally identified as a repressor of the adenoviral early antigen 1A oncogenic transcription activator (1) but has subsequently been shown to have 2-hydroxy acid dehydrogenase activity and a conserved lactate dehydrogenase structure (2, 3), although the endogenous substrates targeted by this activity are probably different (4). Perhaps via a preference for binding of reduced pyridine nucleotide (5, 6) or innate dehydrogenase activity (7), CtBP1 and 2 are able to regulate numerous genes with dependence on NADH concentration and thus the energy and redox state of the cell. Intriguingly, CtBP1 transcriptionally represses the SirT1 histone deacetylase when NADH is elevated (8), possibly regulating SirT1 energy-restricted longevity in some species. E-cadherin is repressed by CtBP (9 -11) during hypoxia-induced tumor metastasis, where hypoxia significantly increases cellular NADH (9). Of note, there is a report that hypoxia stimulates adrenal output of corticosterone secretion and steroidogenic acute regulatory protein and peripheral benzodiazepine receptor in rat pups (12).Although CtBP represses transcription of nuclear receptor target genes (13-19), CtBP1 has only been shown to interact with SF-1 (20). Bartholin et al. (13) showed that the functional CtBP interaction motif of a retinoid X receptor-interacting corepressor is required for acute repression of a retinoic acidinducible reporter and retinoic acid-induced teratogenesis. CtBP1 and 2 reversibly interact with and reinforce the corepressor function of RIP140 (140-kDa receptor interacting protein) (14,15,17,18), which in a ligand-dependent manner interacts with nuclear receptors (21, 22), including SF-1 (23), via one or more of several NR boxes (LXX(L/...