Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis
Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis
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DOI：
10.1006/dbio.2000.9725
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发表时间：
2000-07-01
影响因子：
2.7
通讯作者：
Taskén, K
中科院分区：
文献类型：
--
作者：
Reinton, N;Collas, P;Taskén, K
采用蛋白激酶A II型重叠筛选、cDNA末端快速扩增和数据库检索相结合的方法，构建了一个9923 bp的重叠群，其中开放阅读框编码一个1901个氨基酸的A激酶锚定蛋白（AKAP），SDS-PAGE电泳迁移率为220 kDa，命名为人AKAP 220（hAKAP 220）。hAKAP 220的氨基酸序列与大鼠AKAP 220在1167个C-末端残基中具有高度相似性，但在N-末端含有727个在报道的大鼠AKAP 220序列中不存在的残基。hAKAP 220 mRNA在人睾丸和分离的人粗线期精母细胞和圆形精子细胞中以高水平表达。hAKAP 220蛋白存在于人类男性生殖细胞和成熟精子中。特异性抗体的免疫荧光标记表明，hAKAP 220定位于减数分裂前粗线期精母细胞的细胞质和减数分裂后生殖细胞的中心体中，而在延长期精母细胞和成熟精子中发现了中段/中心体定位。hAKAP 220蛋白连同PKA I型和II型以及蛋白磷酸酶I的一部分对精子尾部的洗涤剂提取具有抗性，这表明与细胞骨架结构相关。与此相反，S-AKAP 84/D-AKAP 1也存在于中间片段中，在相同的条件下提取。抗hAKAP 220抗血清共免疫沉淀人睾丸裂解物中PKA的I型和II型调节亚基，表明hAKAP 220通过单独的或通过共同的结合基序与两类R亚基相互作用。(C)北京大学出版社.
Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids. The hAKAP220 protein was present in human male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicated that hAKAP220 was localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization was found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I was resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, was extracted under the same conditions, Anti-hAKAP220 antisera coimmunoprecipitated both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s). (C) 2000 Academic Press.