Cyclic Adenosine 3′,5′-Monophosphate (cAMP) Enhances cAMP-Responsive Element Binding (CREB) Protein Phosphorylation and Phospho-CREB Interaction with the Mouse Steroidogenic Acute Regulatory Protein Gene Promoter

• Published in: Endocrinology (Philadelphia) Vol. 146; no. 3; pp. 1348 - 1356
• Main Authors: Clem, Brian F., Hudson, Elizabeth A., Clark, Barbara J.
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Endocrine Society 01.03.2005; Oxford University Press
• Subjects: Acetylation; Activating Transcription Factor 1; Activator protein 1; Adenosine; Affinity chromatography; AMP; Animals; Biological and medical sciences; Cell Nucleus - metabolism; Chromatin; Chromatin Immunoprecipitation; Chromatography; CREB-Binding Protein; Cyclic AMP - metabolism; Cyclic AMP response element modulator; Cyclic AMP response element-binding protein; Cyclic AMP Response Element-Binding Protein - metabolism; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA-Binding Proteins - metabolism; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Gene expression; Histones - metabolism; Immunoblotting; Immunoprecipitation; Kinases; Leydig Cells; Male; Mice; Nuclear Proteins - metabolism; Phosphoproteins - genetics; Phosphorylation; Promoter Regions, Genetic; Protein folding; Protein kinase A; Proteins; Signal Transduction; Steroidogenic acute regulatory protein; Time Factors; Trans-Activators - metabolism; Transcription factors; Transcription Factors - metabolism; Tumor cells; Vertebrates: endocrinology; Purine nucleoside; Adenosine; Phosphorylation; Interaction; Rodentia; Cyclic AMP; Steroidogenic acute regulatory protein; Binding protein; Vertebrata; Mammalia; Gene; Mouse; Animal; Transcription factor CREB
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2004-0761

Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5′-TGACGTCA-3′) within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.