CLONING AND CHARACTERIZATION OF AKAP-95, A NUCLEAR-PROTEIN THAT ASSOCIATES WITH THE REGULATORY SUBUNIT OF TYPE-II CAMP-DEPENDENT PROTEIN-KINASE

The subcellular location of the type II cAMP-depend- ent protein kinas e is dictated by the interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Using an interaction cloning strateg y with RII alpha as a probe, we have isolated cDNAs encoding a novel 7 61-amino acid protein (named AKAP 95) that con- tains both RII- and DN A-binding domains. Deletion analysis and peptide studies revealed that the RII-binding domain of AKAP 95 is located between residues 642 and 659 and includes a predicted amphipathic helix. Zinc overlay and DNA binding studies suggest that the DNA-binding domain is composed of two CC/HH-type zinc fingers between residues 464 and 486 and residues 553 and 576. The AKAP was detected in a nuclear matrix fraction, and immu nofluorescence using purified anti-AKAP 95 antibodies revealed a disti nct nuclear staining in a variety of cell types. Direct overlay of flu orescein isothiocyanate-labeled RII alpha onto fixed rat embryo fibrob lasts showed that high-affinity binding sites for RII exist in the nuc leus and that these sites are blocked by an anchoring inhibitor peptid e. Furthermore, AKAP 95 was detected in preparations of RII that were purified from cellular extracts using cAMP-agarose. The results sugges t that AKAP 95 could play a role in targeting type II cAMP-dependent p rotein kinase for cAMP-responsive nuclear events.