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, Thomas J. Deerinck, Mark H. Ellisman, Susan S. Taylor

Research output: Contribution to journalArticle

131 Citations (Scopus)

Abstract

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 (NO 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 NO 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 NO 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.

Original languageEnglish (US)
Pages (from-to)951-959
Number of pages9
JournalJournal of Cell Biology
Volume145
Issue number5
DOIs
StatePublished - May 31 1999

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Endoplasmic Reticulum
Protein Kinases
Mitochondria
Cyclic AMP-Dependent Protein Kinases
Northern Blotting
Testis
Epitopes
Signal Transduction
Immunohistochemistry
Liver

Keywords

  • AKAP
  • cAMP-dependent protein kinase
  • Endoplasmic reticulum
  • Mitochondria
  • Subcellular localization

ASJC Scopus subject areas

  • Cell Biology

Cite this

NH2-terminal targeting motifs direct dual specificity A-kinase- anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum. / Huang, Lily Jun Shen; Wang, Lin; Ma, Yuliang; Durick, Kyle; Perkins, Guy; Deerinck, Thomas J.; Ellisman, Mark H.; Taylor, Susan S.

In: Journal of Cell Biology, Vol. 145, No. 5, 31.05.1999, p. 951-959.

Research output: Contribution to journalArticle

Huang, Lily Jun Shen ; Wang, Lin ; Ma, Yuliang ; Durick, Kyle ; Perkins, Guy ; Deerinck, Thomas J. ; Ellisman, Mark H. ; Taylor, Susan S. / NH2-terminal targeting motifs direct dual specificity A-kinase- anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum. In: Journal of Cell Biology. 1999 ; Vol. 145, No. 5. pp. 951-959.
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T1 - NH2-terminal targeting motifs direct dual specificity A-kinase- anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum

AU - Huang, Lily Jun Shen

AU - Wang, Lin

AU - Ma, Yuliang

AU - Durick, Kyle

AU - Perkins, Guy

AU - Deerinck, Thomas J.

AU - Ellisman, Mark H.

AU - Taylor, Susan S.

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AB - 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 (NO 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 NO 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 NO 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.

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