Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95
Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95
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DOI：
10.1006/excr.1997.3855
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发表时间：
1998-02-01
影响因子：
3.7
通讯作者：
Taskén, K
中科院分区：
文献类型：
--
作者：
Eide, T;Coghlan, V;Taskén, K
II 型环 AMP 依赖性蛋白激酶 (PKA) 通过 RII 亚基与 A 激酶锚定蛋白 (AKAP) 的相互作用定向到不同的亚细胞位点。鉴定并测序了编码 AKAP95 的全长人类克隆，并揭示了与大鼠 AKAP95 89% 同源的 692 个氨基酸开放阅读框（V. M. Coghlan、L. K. Langeberg、A. Fernandez、N.J. Lamb 和 J.D. Scott (1994) J. Biol. Chem. 269, 7658-7665）。使用体细胞杂交和 PCR 将编码 AKAP95 的基因定位到人染色体 19p13.1-q12。覆盖人 AKAP95 氨基酸 414-692 的片段在大肠杆菌中表达，并显示与 RII α 结合。与覆盖 AKAP Ht31 的 RII 结合结构域的肽竞争，消除了 RII α 与 AKAP95 的结合。对静止的人 Hs-68 成纤维细胞的免疫荧光研究显示 AKAP95 位于核内，而 RII α 被排除在细胞核之外。相反，在有丝分裂期间，AKAP95 染色发生显着变化，并且似乎被排除在浓缩染色质之外并位于中期板之外。此外，AKAP95 和 RII α 的亚细胞定位在中期重叠，但在后期开始分离，并在末期 AKAP95 重新进入细胞核时再次分离。最后，RII α 与来自有丝分裂停滞的 HeLa 细胞（而非间期 HeLa 细胞）的 AKAP95 共免疫沉淀，证明这两种分子在有丝分裂过程中存在物理关联。结果显示 AKAP95 在有丝分裂期间明显重新分布，表明 AKAP95 和 RII α 之间的相互作用可能具有细胞周期依赖性。 (C) 1998 年学术出版社。
The cyclic AMP-dependent protein kinase (PKA) type II is directed to different subcellular loci through interaction of the RII subunits with A-kinase anchoring proteins (AKAPs). A full-length human clone encoding AKAP95 was identified and sequenced, and revealed a 692-amino acid open reading frame that was 89% homologous to the rat AKAP95 (V. M. Coghlan, L. K. Langeberg, A. Fernandez, N.J. Lamb, and J.D. Scott (1994) J. Biol. Chem. 269, 7658-7665). The gene encoding AKAP95 was mapped to human chromosome 19p13.1-q12 using somatic cell hybrids and PCR A fragment covering amino acids 414-692 of human AKAP95 was expressed in Escherichia coli and shown to bind RII alpha. Competition with a peptide covering the RII-binding domain of AKAP Ht31 abolished RII alpha binding to AKAP95. Immunofluorescence studies in quiescent human Hs-68 fibroblasts showed a nuclear localization of AKAP95, whereas RII alpha was excluded from the nucleus. In contrast, during mitosis AKAP95 staining was markedly changed and appeared to be excluded from the condensed chromatin and localized outside the metaphase plate. Furthermore, the subcellular localizations of AKAP95 and RII alpha overlapped in metaphase but started to segregate in anaphase and were again separated as AKAP95 reentered the nucleus in telophase. Finally, RII alpha was coimmunoprecipitated with AKAP95 from HeLa cells arrested in mitosis, but not from interphase HeLa cells, demonstrating a physical association between these two molecules during mitosis. The results show a distinct redistribution of AKAP95 during mitosis, suggesting that the interaction between AKAP95 and RII alpha may be cell cycle-dependent. (C) 1998 Academic Press.