Specific nuclear binding of adenosine 3',5'-monophosphate-binding protein complex with subsequent poly(A) RNA synthesis in embryonic chick cartilage.

We used embryonic chick pelvic cartilage as a model to study the mechanism by which cyclic AMP increases RNA synthesis. Isolated nuclei were incubated with [32P]-8-azidoadenosine 3,5'-monophosphate ([32P]N3cAMP) with no resultant specific nuclear binding. However, in the presence of cytosol proteins, nuclear binding of [32P]N3cAMP was demonstrable that was specific, time dependent, and dependent on a heat-labile cytosol factor. The possible biological significance of the nuclear binding of the cyclic AMP-protein complex was identified by incubating isolating nuclei with either cyclic AMP or cytosol cyclic AMP-binding proteins prepared by batch elution DEAE cellulose chromatography (DEAE peak cytosol protein), or both, in the presence of cold nucleotides and [3H]uridine 5'-triphosphate. Poly(A) RNA production occurred only in nuclei incubated with cyclic AMP and the DEAE peak cytosol protein preparation. Actinomycin D inhibited the incorporation of [3H]uridine 5'-monophosphate into poly(A) RNA. The newly synthesized poly(A) RNA had a sedimentation constant of 23S. Characterization of the cytosol cyclic AMP binding proteins using [32P]N3-cAMP with photoaffinity labeling three major cAMP-binding complexes (41,000, 51,000, and 55,000 daltons). The 51,000 and 55,000 dalton cyclic AMP binding proteins were further purified by DNA-cellulose chromatography. In the presence of cyclic AMP they stimulated poly(A) RNA synthesis in isolated nuclei. The 51,000-dalton cyclic AMP-binding protein was the predominant one that bound to the nuclei. While cyclic AMP-dependent protein kinsae activity was present in the cytosol and DEAE peak cytosol proteins, it was not present in the DNA-cellulose-bound, cyclic AMP-binding proteins. We conclude that one possible mechanism by which cyclic AMP increases RNA synthesis is by complexing to a 51,000-dalton cytosol cyclic AMP-binding protein and being subsequently translocated to the nucleus, where it is specifically bound and associated with induction of poly(A) RNA synthesis.

Duke Scholars

Author Warner Miller Burch Jr. Medicine, Endocrinology, Metabolism, and Nutrition