A cAMP-regulated RNA-binding protein that interacts with phosphoenolpyruvate carboxykinase (GTP) mRNA.
Cyclic-AMP stabilizes phosphoenolpyruvate carboxykinase (GTP) (PEPCK) mRNA against degradation. To investigate the mechanism of this effect, RNA mobility shift assays were used to determine the interaction of cellular proteins with specific domains from the mRNA. We report here the identification of a protein with an affinity for sequences of PEPCK mRNA with a predicted stem-loop structure. RNA-protein complex formation was significantly reduced if the double-stranded RNA probe was preheated to 90 degrees C. The RNA-binding protein did not bind to the hairpin structure of poly(rI)-poly (rC), indicating some degree of sequence specificity and that the RNA-binding protein is not the interferon-induced double-stranded RNA-activated protein kinase. The binding activity was contained in the cytosolic fraction (100,000 x g) of rat hepatoma FTO-2B cells and was significantly enhanced by high concentrations of KCl. Chromatography on an anion exchanger separated the binding activity from a factor which, upon reconstitution, inhibited the interaction with the RNA probe. Incubation of cells with cAMP resulted in a 3-4-fold decrease in the activity of the RNA-binding protein. An inhibition in complex formation was observed with extracts as early as 60 min after exposure of cells to cAMP. Liver extracts from rats starved for 72 h also had reduced binding activity compared to extracts from fed animals. Cellular extracts treated with alkaline phosphatase exhibited an elevated level of complex formation. An analysis by SDS-polyacrylamide gel electrophoresis of the RNA-protein complex after ultraviolet light cross-linking demonstrated that the RNA-binding protein had a molecular mass of approximately 100 kDa. On the basis of these results, we suggest that liver cells contain a protein whose interaction with PEPCK mRNA is regulated by cAMP-dependent phosphorylation and which may be responsible for the cAMP-mediated control of PEPCK mRNA half-life.
Nachaliel
N
N
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794-8661.
Jain
D
D
Hod
Y
Y
D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D010744:Phosphoric Monoester Hydrolases / Q000494:pharmacology; D012333:RNA, Messenger / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D013217:Starvation; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000242:Cyclic AMP; D010744:Phosphoric Monoester Hydrolases; D010729:Phosphoenolpyruvate Carboxykinase (GTP)