Expression of a kinase anchor protein 75 depletes type II cAMP-dependent protein kinases from the cytoplasm and sequesters the kinases in a particulate pool.
A kinase anchor proteins (AKAPs) have a C-terminal binding site for the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (PKAII beta) and an N-terminal domain that mediates the targeting and attachment of the anchor protein to intracellular structures. In vitro biochemical studies and recent in situ immunocytochemical analysis (Glantz, S. B., Amat, J. A., and Rubin, C. S. (1992) Mol. Biol. Cell 3, 1215-1228) suggest that AKAPs anchor PKAII beta at specific sites in the dendritic cytoskeleton of neurons. This arrangement would place PKAII beta in proximity with its substrates and create "target sites" for cAMP actions. The foregoing model predicts that (a) RII subunits are freely accessible to AKAPs, (b) PKAII holoenzymes, as well as RII subunits, are anchored, and (c) changes in the level of AKAP can alter the intracellular distribution of type II PKAs. We have addressed these previously untested propositions by overexpressing bovine AKAP75 in a human cell line (HEK293). Non-transfected cells express a low level of endogenous AKAP79, and approximately 90% of RII alpha and RII beta subunits are isolated in the cell cytosol. In contrast, stably transfected cells, which express a 10-fold excess of AKAP75, sequester > 90% of their RII subunits in a particulate pool. Catalytic subunits are also transferred to this pool. AKAP75 accumulates in a cell compartment with biochemical properties characteristic of cytoskeleton. Thus, AKAPs have access to and avidly bind cytoplasmic type II PKAs. Moreover, an increase in AKAP content can alter the particulate/cytoplasmic distribution of PKAII beta and PKAII alpha.
Ndubuka
C
C
Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461.
Li
Y
Y
Rubin
C S
CS
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D002352:Carrier Proteins; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007668:Kidney; D046911:Macromolecular Substances; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D007527:Isoenzymes; D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases