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

Subcellular localization directed by specific targeting motifs is an emergi ng theme for regulating signal transduction pathways, For cAMP-dependent pr otein kinase (PKA), this is achieved primarily by its association with A-ki nase-anchoring proteins (AKAPs), Dual specificity AKAP1, (D-AKAP1) binds to both type I and type II regulatory subunits and has two NH2-terminal (NO a nd NI) and two COOH-terminal (C1 and C2) splice variants (Huang et al., 199 7. J. Biol. Chem, 272:8057). Here we report that the splice variants of D-A KAP1 are expressed in a tissue-specific manner with the NH2-terminal motifs serving as switches to localize D-AKAP1 at different sites. North ern blot s showed that the N1 splice is expressed primarily in liver, while the C1 s plice is predominant in testis, The C2 splice shows a general expression pa ttern. Microinjecting expression constructs of D-AKAP1(NO) epitope-tagged a t either the NH2 or the COOH terminus showed their localization to the mito chondria based on immunocytochemistry. Deletion of NO(1-30) abolished mitoc hondrial 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 NI targets D-AKAP1 to the ER and residues 1-63 fused to GFP are necessary and sufficient for ER targeting. Residues 1 4-33 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 th e first example of a differentially targeted AKAP and adds an additional le vel of complexity to the PKA signaling network.