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© The Rockefeller University Press, 0021-9525/1999//951 $5.00
The Journal of Cell Biology, Volume 145, Number 5, , 1999 951-959

Regular Articles

NH2-Terminal Targeting Motifs Direct Dual Specificity A-Kinase–anchoring Protein 1 (D-AKAP1) to Either Mitochondria or Endoplasmic Reticulum



Lily Jun-shen Huang*, Lin Wang*, Yuliang Ma*, Kyle Durick*, Guy Perkins{ddagger}, Thomas J. Deerinck{ddagger}, Mark H. Ellisman{ddagger}, and Susan S. Taylor*

* Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, and the {ddagger} National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California 92093-0654

Subcellular localization directed by specific targeting motifs is an emerging theme for regulating signal transduction pathways. For cAMP-dependent protein kinase (PKA), this is achieved primarily by its association with A-kinase–anchoring proteins (AKAPs). Dual specificity AKAP1, (D-AKAP1) binds to both type I and type II regulatory subunits and has two NH2-terminal (N0 and N1) and two COOH-terminal (C1 and C2) splice variants (Huang et al., 1997. J. Biol. Chem. 272:8057). Here we report that the splice variants of D-AKAP1 are expressed in a tissue-specific manner with the NH2-terminal motifs serving as switches to localize D-AKAP1 at different sites. Northern blots showed that the N1 splice is expressed primarily in liver, while the C1 splice is predominant in testis. The C2 splice shows a general expression pattern. Microinjecting expression constructs of D-AKAP1(N0) epitope-tagged at either the NH2 or the COOH terminus showed their localization to the mitochondria based on immunocytochemistry. Deletion of N0(1-30) abolished mitochondrial targeting while N0(1-30)-GFP localized to mitochondria. Residues 1–30 of N0 are therefore necessary and sufficient for mitochondria targeting. Addition of the 33 residues of N1 targets D-AKAP1 to the ER and residues 1–63 fused to GFP are necessary and sufficient for ER targeting. Residues 14–33 of N1 are especially important for targeting to ER; however, residues 1–33 alone fused to GFP gave a diffuse distribution. N1(14-33) thus serves two functions: (a) it suppresses the mitochondrial-targeting motif located within residues 1–30 of N0 and (b) it exposes an ER-targeting motif that is at least partially contained within the N0(1-30) motif. This represents the first example of a differentially targeted AKAP and adds an additional level of complexity to the PKA signaling network.

Key Words: cAMP-dependent protein kinase • AKAP • mitochondria • endoplasmic reticulum • subcellular localization

Abbreviations used in this paper: AKAP, A-kinase–anchoring protein; C, cAMP-dependent protein kinase catalytic subunit; D-AKAP1, dual specificity AKAP1; GFP, green fluorescent protein; PKA, cAMP-dependent protein kinase; R, cAMP-dependent protein kinase regulatory subunit; RI, type I regulatory subunit of PKA; RII, type II regulatory subunit of PKA; SH2, src homology 2.

This research was supported in part by grants from the American Cancer Society (BE-48L to S.S. Taylor), the National Institutes of Health (1PO1DK54441-01 to S.S. Taylor), and the National Center for Research Resources (NIH RR04050 to M.H. Ellisman). The costs of publication of this article were defrayed in part by the payment of page charges. L.J.-s. Huang is supported by UCSD Cell Molecular and Genetics Training Grant 2T32GM07240-21A1. K. Durick is supported by the Markey Charitable Trust as a Fellow and by NIH Training Grant NCI T32 CA09523.

Dr. Durick's current address is Aurora Biosciences, 11010 Torreyana Road, San Diego, CA 92121.


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