Cardone Luca, de Cristofaro Tiziana, Affaitati Adelina, Garbi Corrado, Ginsberg Michael D, Saviano Michele, Varrone Stelio, Rubin Charles S, Gottesman Max E, Avvedimento Enrico V, Feliciello Antonio
Dipartimento di Biologia e Patologia Molecolare e Cellulare, BioGem Consortium, Instituto di Endocrinologia ed Oncologia Sperimentale CNR, Universitá Federico II, via S. Pansini 5, 80131 Naples, Italy.
J Mol Biol. 2002 Jul 12;320(3):663-75. doi: 10.1016/s0022-2836(02)00479-5.
A-kinase anchor proteins (AKAPs) assemble multi-enzyme signaling complexes in proximity to substrate/effector proteins, thus directing and amplifying membrane-generated signals. S-AKAP84 and AKAP121 are alternative splicing products with identical NH(2) termini. These AKAPs bind and target protein kinase A (PKA) to the outer mitochondrial membrane. Tubulin was identified as a binding partner of S-AKAP84 in a yeast two-hybrid screen. Immunoprecipitation and co-sedimentation experiments in rat testis extracts confirmed the interaction between microtubules and S-AKAP84. In situ immunostaining of testicular germ cells (GC2) shows that AKAP121 concentrates on mitochondria in interphase and on mitotic spindles during M phase. Purified tubulin binds directly to S-AKAP84 but not to a deletion mutant lacking the mitochondrial targeting domain (MT) at residues 1-30. The MT is predicted to form a highly hydrophobic alpha-helical wheel that might also mediate interaction with tubulin. Disruption of the wheel by site-directed mutagenesis abolished tubulin binding and reduced mitochondrial attachment of an MT-GFP fusion protein. Some MT mutants retain tubulin binding but do not localize to mitochondria. Thus, the tubulin-binding motif lies within the mitochondrial attachment motif. Our findings indicate that S-AKAP84/AKAP121 use overlapping targeting motifs to localize signaling enzymes to mitochondrial and cytoskeletal compartments.
A激酶锚定蛋白（AKAPs）在底物/效应蛋白附近组装多酶信号复合物，从而引导和放大膜产生的信号。S-AKAP84和AKAP121是具有相同NH₂末端的可变剪接产物。这些AKAPs将蛋白激酶A（PKA）结合并靶向到线粒体外膜。在酵母双杂交筛选中，微管蛋白被鉴定为S-AKAP84的结合伴侣。大鼠睾丸提取物中的免疫沉淀和共沉降实验证实了微管与S-AKAP84之间的相互作用。睾丸生殖细胞（GC2）的原位免疫染色显示，AKAP121在间期集中在线粒体上，在M期集中在有丝分裂纺锤体上。纯化的微管蛋白直接与S-AKAP84结合，但不与在第1至30位残基处缺乏线粒体靶向结构域（MT）的缺失突变体结合。预测MT会形成一个高度疏水的α螺旋轮，该螺旋轮也可能介导与微管蛋白的相互作用。通过定点诱变破坏该螺旋轮会消除微管蛋白结合，并减少MT-GFP融合蛋白的线粒体附着。一些MT突变体保留微管蛋白结合，但不定位到线粒体。因此，微管蛋白结合基序位于线粒体附着基序内。我们的研究结果表明，S-AKAP84/AKAP121使用重叠的靶向基序将信号酶定位到线粒体和细胞骨架区室。
