Huang L J, Wang L, Ma Y, Durick K, Perkins G, Deerinck T J, Ellisman M H, Taylor S S
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0654, USA.
J Cell Biol. 1999 May 31;145(5):951-9. doi: 10.1083/jcb.145.5.951.
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.
由特定靶向基序指导的亚细胞定位是调节信号转导途径的一个新兴主题。对于环磷酸腺苷依赖性蛋白激酶（PKA）而言，这主要是通过其与A激酶锚定蛋白（AKAPs）的结合来实现的。双重特异性AKAP1（D-AKAP1）可与I型和II型调节亚基结合，并具有两个氨基末端（N0和N1）和两个羧基末端（C1和C2）剪接变体（《生物化学杂志》272:8057）。在此我们报告，D-AKAP1的剪接变体以组织特异性方式表达，氨基末端基序作为开关将D-AKAP1定位在不同位点。Northern印迹显示，N1剪接主要在肝脏中表达，而C1剪接在睾丸中占主导。C2剪接呈现出普遍的表达模式。对氨基末端或羧基末端带有表位标签的D-AKAP1（N0）表达构建体进行显微注射，基于免疫细胞化学显示它们定位于线粒体。删除N0（1-30）消除了线粒体靶向，而N0（1-30）-绿色荧光蛋白定位于线粒体。因此，N0的1-30位残基对于线粒体靶向是必需且足够 的。添加N1的33个残基可将D-AKAP1靶向内质网，与绿色荧光蛋白融合的1-63位残基对于内质网靶向是必需且足够的。N1的14-33位残基对于靶向内质网尤为重要；然而，单独与绿色荧光蛋白融合的1-33位残基呈现弥散分布。因此，N1（14-33）具有两种功能：（a）它抑制位于N0的1-30位残基内的线粒体靶向基序，（b）它暴露了一个内质网靶向基序，该基序至少部分包含在N0（1-30）基序内。这代表了一个差异靶向AKAP的首个实例，并为PKA信号网络增加了另一层复杂性。
