Organelle-specific targeting of protein kinase AII (PKAII). Molecular and in situ characterization of murine A kinase anchor proteins that recruit regulatory subunits of PKAII to the cytoplasmic surface of mitochondria.
Experiments were designed to test the idea that A kinase anchor proteins (AKAPs) tether regulatory subunits (RII) of protein kinase AII (PKAII) isoforms to surfaces of organelles that are bounded by phospholipid bilayers. S-AKAP84, one of three RII-binding proteins encoded by a single-copy murine gene, was studied as a prototypic organelle-associated AKAP. When S-AKAP84 was expressed in HEK293 cells, the anchor protein was targeted to mitochondria and excluded from other cell compartments. The RII tethering site is located in the cytoplasm adjacent to the mitochondrial surface. Endogenous RII subunits are not associated with mitochondria isolated from control cells. Expression of S-AKAP84 in transfected HEK293 cells triggered a redistribution of 15% of total RII to mitochondria. Thus, the tethering region of the organelle-inserted anchor protein is properly oriented and avidly binds RII (PKAII) isoforms in intact cells. Two critical domains in S-AKAP84 were mapped. Residues 1 to 30 govern insertion of the polypeptide into the outer mitochondrial membrane; amino acids 306-325 constitute the RII-binding site. Properties established for S-AKAP84 in vitro and in situ strongly suggest that a physiological function of this protein is to concentrate and immobilize RII (PKAII) isoforms at the cytoplasmic face of a phospholipid bilayer.
Chen
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Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Lin
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Rubin
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D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D018076:DNA, Complementary; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D015388:Organelles / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology*
D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; C486333:Akap1 protein, mouse; D002352:Carrier Proteins; D018076:DNA, Complementary; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D017868:Cyclic AMP-Dependent Protein Kinases