Ezrin is a cyclic AMP-dependent protein kinase anchoring protein
Ezrin is a cyclic AMP-dependent protein kinase anchoring protein
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DOI：
10.1093/emboj/16.1.35
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发表时间：
1997-01-02
期刊：
影响因子：
11.4
通讯作者：
Goldenring, JR
中科院分区：
文献类型：
--
作者：
Dransfield, DT;Bradford, AJ;Goldenring, JR
cAMP 依赖性蛋白激酶（A 激酶）锚定蛋白 (AKAP) 负责 II 型 A 激酶的亚细胞隔离。此前，我们鉴定了胃壁细胞中富含的 78 kDa AKAP。我们现在已经使用 II 型 A 激酶调节亚基 (R(II)) 亲和层析从胃底粘膜上清液中纯化出同质的 78 kDa AKAP。纯化的 78 kDa AKAP 可被抗埃兹蛋白（小管肌动蛋白相关蛋白）的单克隆抗体识别。 Sf9 细胞或细菌中产生的重组埃兹蛋白也与 R(II) 结合。重组根素和模蛋白（埃兹蛋白相关蛋白）也在印迹叠加中结合 R(II)。对埃兹蛋白重组截短的分析将 R(II) 结合位点定位到氨基酸 373 和 439 之间的区域。该区域包含一个氨基酸两亲性 α 螺旋推定 R(II) 结合区域。含有两亲性螺旋区域 (ezrin(409-438)) 的合成肽可阻断 R(II) 与埃兹蛋白的结合，但氨基酸 421 处亮氨酸替换为脯氨酸的肽无法抑制 R(II) 结合。在小鼠胃底粘膜中，R(II) 免疫反应性从静息壁细胞中的主要胞质位置重新分布到胃泌素刺激的动物粘膜中的小管模式。这些结果表明，埃兹蛋白是胃壁细胞中的主要 AKAP，并且可能起到将 II 型 A 激酶束缚到分泌小管附近的区域的作用。
cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (R(II)) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound R(II). Recombinant radixin and moesin, ezrin-related proteins, also bound R(II) in blot overlay. Analysis of recombinant truncations of ezrin mapped the R(II) binding site to a region between amino acids 373 and 439. This region contained a lit-aminoacid amphipathic alpha-helical putative R(II) binding region. A synthetic peptide containing the amphipathic helical region (ezrin(409-438)) blocked R(II) binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit R(II) binding. In mouse fundic mucosa, R(II) immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.