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-dependent protein kinase is dictated by the interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Using an interaction cloning strategy with RII alpha as a probe, we have isolated cDNAs encoding a novel 761-amino acid protein (named AKAP 95) that contains both RII- and DNA-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 immunofluorescence using purified anti-AKAP 95 antibodies revealed a distinct nuclear staining in a variety of cell types. Direct overlay of fluorescein isothiocyanate-labeled RII alpha onto fixed rat embryo fibroblasts showed that high-affinity binding sites for RII exist in the nucleus and that these sites are blocked by an anchoring inhibitor peptide. Furthermore, AKAP 95 was detected in preparations of RII that were purified from cellular extracts using cAMP-agarose. The results suggest that AKAP 95 could play a role in targeting type II cAMP-dependent protein kinase for cAMP-responsive nuclear events.
Coghlan
V M
VM
Volum Institute of Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
Langeberg
L K
LK
Fernandez
A
A
Lamb
N J
NJ
Scott
J D
JD
D054758:A Kinase Anchor Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D016335:Zinc Fingers
D054758:A Kinase Anchor Proteins; C086194:Akap8 protein, rat; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C517822:Prkar2a protein, rat; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases