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*
- From the Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461
Abstract
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.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant GM22792.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U95145 and U95146.
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↵‡ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, F-229, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2505; Fax: 718-430-8922; E-mail: rubin{at}aecom.yu.edu.
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↵1 The abbreviations used are: PKA, cAMP-dependent protein kinase; R, regulatory subunit of cAMP-dependent protein kinase; AKAP, A kinase anchor protein; S-AKAP84, spermatid A kinase anchor protein with an apparentM r of 84,000; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s).
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↵2 R.-Y. Lin, Q. Chen, and C. S. Rubin, unpublished observations.
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↵3 Q. Chen and C. S. Rubin, unpublished results.
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- Received February 6, 1997.
- Revision received March 14, 1997.
