NH2-Terminal targeting motifs direct dual specificity A-kinase-anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum

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 (N0 and N1) and two COOH-terminal (C1 and C2) splice variants (. 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.

Figure 1

Schematic diagram of the various splices and isoforms of D-AKAP1. (a) Schematic diagram of various mouse homologues of D-AKAP1. All four isoforms share a 525–amino acid core, including the R-binding domain. A putative KH domain was identified in isoforms AKAP100 and D-AKAP1c (AKAP121). This domain is shown as the hatched box and the corresponding sequences are underlined. (b) The NH₂ terminal 30 and 63 residues are shown for N0 and N1, respectively. Residues RRCSY represent the putative PKA phosphorylation site identified in N1.

Figure 2

Splice variants of D-AKAP1 are expressed in a tissue-specific manner. The same Northern blot containing immobilized samples of mRNAs of selected adult tissues was probed with ³²P-radiolabeled cDNAs specific for the various splice variants: core (A), N1 (B), C1 (C), and C2 (D). The size of the mRNA was indicated on the right side of the blots. Panel a is taken from Huang et al. (1997) with permission.

Figure 3

Cell-free expression of [HA]-N0 and [HA]-N1. [³⁵S]Methionine-labeled [HA]-N0 and [HA]-N1 were expressed in an in vitro transcription-translation system and visualized by autoradiography (A). These products were also subjected to immunoblot analysis using anti-RPP7 (B) or anti-N1 (C).

Figure 4

The N0 splice localizes to mitochondria. Confocal images of [HA]-N0 localization using anti-HA antibodies (red) costained with a mitochondria fluorescent marker (A) or a mitochondrial targeted GFP marker (B) (both green). Yellow staining indicates overlapped localization. Individual staining patterns were shown in the insets. The blue staining shows immunofluorescent detection of an IgG injection marker (A). [HA]- N0(Δ1-30) shows a diffused cytoplasmic stain (C), while N0- [GFP] exhibits a mitochondrial pattern (D). These results are summarized schematically in E. The first 30 residues of N0 are aligned with the first 15 residues of Hexokinase I. Identical residues are indicated in black boxes, and homologous residues are indicated in gray boxes.

Figure 4

The N0 splice localizes to mitochondria. Confocal images of [HA]-N0 localization using anti-HA antibodies (red) costained with a mitochondria fluorescent marker (A) or a mitochondrial targeted GFP marker (B) (both green). Yellow staining indicates overlapped localization. Individual staining patterns were shown in the insets. The blue staining shows immunofluorescent detection of an IgG injection marker (A). [HA]- N0(Δ1-30) shows a diffused cytoplasmic stain (C), while N0- [GFP] exhibits a mitochondrial pattern (D). These results are summarized schematically in E. The first 30 residues of N0 are aligned with the first 15 residues of Hexokinase I. Identical residues are indicated in black boxes, and homologous residues are indicated in gray boxes.

Figure 5

The N1 splice localizes to endoplasmic reticulum. (A) Confocal image of [HA]-N1 localization with anti-HA antibodies (red) costained with anti-Bip antibodies (green). Yellow staining indicates overlapped localization. Individual staining patterns were shown in the insets. The blue staining shows immunofluorescent detection of an IgG injection marker. (B) N1(Δ1-13) (red) colocalizes with Bip (green). (C) N1(Δ1-24) (red) does not colocalize with Bip (green). The results in A–C are summarized schematically in D. In addition, mutagenesis studies of the putative PKA phosphorylation site (boxed residues) are also summarized (data not shown). The dotted residues indicate the amino acids used to replace the serine residue.

Figure 5

The N1 splice localizes to endoplasmic reticulum. (A) Confocal image of [HA]-N1 localization with anti-HA antibodies (red) costained with anti-Bip antibodies (green). Yellow staining indicates overlapped localization. Individual staining patterns were shown in the insets. The blue staining shows immunofluorescent detection of an IgG injection marker. (B) N1(Δ1-13) (red) colocalizes with Bip (green). (C) N1(Δ1-24) (red) does not colocalize with Bip (green). The results in A–C are summarized schematically in D. In addition, mutagenesis studies of the putative PKA phosphorylation site (boxed residues) are also summarized (data not shown). The dotted residues indicate the amino acids used to replace the serine residue.

Figure 6

The N1 splice does not contain an ER-targeting signal. Confocal images of 10T1/2 cells transfected with (1-63)N1-[GFP] which displayed ER localization (A), and (1-33)N1-[GFP] with a diffuse pattern (B).

Figure 7

Schematic summary of various NH₂-terminal motifs that target D-AKAP1 to either mitochondria or endoplasmic reticulum.