Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79.
Postsynaptic densities (PSD) are a network of proteins located on the internal surface of excitatory synapses just inside the postsynaptic membrane. Enzymes associated with the PSD are optimally positioned to respond to signals transduced across the postsynaptic membrane resulting from excitatory synaptic transmission or neurotransmitter release. We present evidence suggesting that type II cAMP-dependent protein kinase (PKA) is anchored to the PSD through interaction of its regulatory subunit (RII) with an A-Kinase Anchor Protein (AKAPs). A cDNA for the human RII-anchoring protein, AKAP 79, was isolated by screening an expression library with radiolabeled RII. This cDNA (2621 base pairs) encodes a protein of 427 amino acids with 76% identity to bovine brain AKAP 75 and 93% identity to a carboxyl-terminal RII-binding fragment of murine brain AKAP 150. A bacterially expressed 92-amino acid fragment, AKAP 79 (335-427) was able to bind RII alpha. Disruption of secondary structure by site-directed mutagenesis at selected residues within a putative acidic amphipathic helix located between residues 392 and 408 prevented RII binding. Immunological studies demonstrate that AKAP 79 is predominantly expressed in the cerebral cortex and is a component of fractions enriched for postsynaptic densities. AKAP antisera strongly cross-react with a 150-kDa protein in murine PSD believed to be AKAP 150. Co-localization of the type II PKA in purified PSD fractions was confirmed immunologically by detection of RII and enzymologically by measuring cAMP-stimulated phosphorylation of the heptapeptide substrate Kemptide. Approximately 30% of the PSD kinase activity was specifically inhibited by PKI 5-24 peptide, a highly specific inhibitor of PKA. We propose that AKAP 79 and AKAP 150 function to anchor the type II PKA to the PSD, presumably for a role in the regulation of postsynaptic events.
Carr
D W
DW
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
Stofko-Hahn
R E
RE
Fraser
I D
ID
Cone
R D
RD
Scott
J D
JD
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D016548:Prosencephalon / Q000378:metabolism*; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013574:Synaptosomes / Q000378:metabolism*; D013961:Thyroid Gland / Q000502:physiology
D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D048868:Adaptor Proteins, Signal Transducing; C415703:Akap5 protein, rat; D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D004247:DNA; D011494:Protein Kinases