Abstract
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 ACTH-stimulated 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.
Highlights
MARCH 14, 2008 VOLUME 283 NUMBER 11 ous genes with dependence on NADH concentration and the energy and redox state of the cell
Conditions favoring CtBP heterodimer- erodimers prevails in response to ACTH/cAMP and allows ization remain for up to 120 min, this does not main- induction of CYP17 transcription (Fig. 9)
We sought to identify a mechanism of PKAinduced adrenal cortex transcription that corresponds with the induced formation of SF-1-coactivator complexes [20]
Summary
Reagents—Dibutryl 3Ј-5Ј-cyclic adenosine monophosphate (Bt2cAMP) was obtained from Sigma. ␣-Amanitin, H-89, and the catalytic subunit of PKA were obtained from EMD Biosciences, Inc. (La Jolla, CA). 5,6-Carboxy-2Ј,7Ј-dichloro-dihydrofluoroscein diacetate was purchased from Invitrogen. Coimmunoprecipitation and Western Blotting—H295R cells were cultured in 100-mm dishes, and cytoplasmic and nuclear fractions were obtained using the NE-PER kit (Pierce), or whole cell extracts were prepared in radioimmune precipitation assay buffer. For kinetics by coimmunoprecipitation (coIP), 100-g nuclear extracts or for other experiments, an equal fraction or total sample of whole cell lysates were precleared for 45 min at 4 °C and incubated overnight with protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA) and 4 g of anti-CtBP1 (BD Biosciences), 3 g of anti-VP16 or 2 g of GCN5 (Santa Cruz), 2 g of anti-FLAG (Sigma), or 2 g of anti-SF-1 (Millipore). The beads were washed two times in radioimmune precipitation assay buffer with protease inhibitors (EMD) and twice in phosphate-buffered saline, boiled in SDS-PAGE buffer, and separated in 10% gels before transfer to polyvinylidene difluoride membrane (Millipore) and Western blotting with anti-CtBP2. ROX (Bio-Rad), as previously published [20], with primers amplifying the region of the CYP17 promoter from position Ϫ104 to ϩ43: 5Ј-GGC TGG GCT CCA GGA
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