A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs
A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs
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DOI：
10.1016/s0022-2836(02)00479-5
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发表时间：
2002-07-12
影响因子：
5.6
通讯作者：
Feliciello, A
中科院分区：
文献类型：
--
作者：
Cardone, L;de Cristofaro, T;Feliciello, A
A-激酶锚蛋白（AKAP）在底物/效应蛋白附近组装多酶信号传导复合物，从而引导和放大膜产生的信号。S-AKAP 84和AKAP 121是具有相同NH 2末端的选择性剪接产物。这些AKAP结合并靶向蛋白激酶A（PKA）至线粒体外膜。在酵母双杂交筛选中，微管蛋白被鉴定为S-AKAP 84的结合伴侣。大鼠睾丸提取物的免疫沉淀和共沉淀实验证实了微管和S-AKAP 84之间的相互作用。睾丸生殖细胞（GC 2）的原位免疫染色显示，AKAP 121集中在线粒体间期和有丝分裂纺锤体在M期。纯化的微管蛋白直接与S-AKAP 84结合，但不与在残基1-30处缺乏线粒体靶向结构域（MT）的缺失突变体结合。预测MT形成高度疏水的α-螺旋轮，其也可能介导与微管蛋白的相互作用。通过定点突变破坏车轮废除微管蛋白结合，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，但不定位于线粒体。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-AKAP 84/AKAP 121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。(C)2002爱思唯尔科技有限公司版权所有。
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 NH2 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. (C) 2002 Elsevier Science Ltd. All rights reserved.