A CAMP-REGULATED RNA-BINDING PROTEIN THAT INTERACTS WITH PHOSPHOENOLPYRUVATE CARBOXYKINASE (GTP) MESSENGER-RNA

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 c ellular proteins with specific domains from the mRNA. We report here t he identification of a protein with an affinity for sequences of PEPCK mRNA with a predicted stem-loop structure. RNA-protein complex format ion was significantly reduced if the double-stranded RNA probe was pre heated to 90-degrees-C. The RNA-binding protein did not bind to the ha irpin structure of poly(rI)-poly (rC), indicating some degree of seque nce specificity and that the RNA-binding protein is not the interferon -induced double-stranded RNA-activated protein kinase. The binding act ivity was contained in the cytosolic fraction (100,000 x g) of rat hep atoma FTO-2B cells and was significantly enhanced by high concentratio ns of KCl. Chromatography on an anion exchanger separated the binding activity from a factor which, upon reconstitution, inhibited the inter action 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 inhi bition 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 f ed animals. Cellular extracts treated with alkaline phosphatase exhibi ted an elevated level of complex formation. An analysis by SDS-polyacr ylamide gel electrophoresis of the RNA-protein complex after ultraviol et light cross-linking demonstrated that the RNA-binding protein had a molecular mass of approximately 100 kDa. On the basis of these result s, we suggest that liver cells contain a protein whose interaction wit h PEPCK mRNA is regulated by cAMP-dependent phosphorylation and which may be responsible for the cAMP-mediated control of PEPCK mRNA half-li fe.