Cysteine 70 of ankyrin-G is S-palmitoylated and is required for function of ankyrin-G in membrane domain assembly.
Cysteine 70 of ankyrin-G is S-palmitoylated and is required for function of ankyrin-G in membrane domain assembly.
复制标题
Ankyrin-G的半胱氨酸70已被S-膜酰化化，并且是膜结构域组装中Ankyrin-G的功能所必需的。
DOI：
10.1074/jbc.m112.417501
复制
发表时间：
2012-12-21
期刊：
影响因子：
--
通讯作者：
Bennett V
中科院分区：
文献类型：
--
作者：
He M;Jenkins P;Bennett V
背景：锚蛋白-G靶向多种细胞类型中的特化膜结构域。结果：锚蛋白-G在位于锚蛋白重复螺线管第一环的保守半胱氨酸70处被S-棕榈酰化。结论：半胱氨酸70是膜结构域组装过程中锚蛋白G发挥功能所必需的。意义：这一发现为锚蛋白如何在膜结构域的形成和维持中发挥功能提供了新的见解。锚蛋白-G（AnkG）协调不同的膜结构域，包括上皮细胞侧膜和神经元轴突起始段的蛋白质组成。然而，AnkG本身如何定位于这些膜结构域尚不清楚。我们报道了在低钙条件下生长的Madin-Darby犬肾（MDCK）细胞中，尽管这些细胞缺乏顶-底极性，并且显示出AnkG伴侣、E-钙粘蛋白和β2-血影蛋白的质膜结合丧失，但AnkG仍然存在于质膜上。随后，我们证明使用诱变和质谱分析，AnkG是S-棕榈酰化完全在Cys-70，这是位于第一个锚蛋白重复的一个环，并在脊椎动物锚蛋白家族中是保守的。此外，C70 A突变废除了在低钙条件下生长的MDCK细胞中190-kDa AnkG的膜结合。C70 A 190-kDa AnkG不能恢复内源性AnkG耗尽的MDCK细胞中上皮细胞侧膜的生物发生。此外，C70 A 270-kDa AnkG未能聚集在AnkG耗尽培养的海马神经元的轴突起始段，并且未能募集神经成束蛋白以及电压门控钠通道。C70 A突变的这些作用与其S-棕榈酰化的证据相结合，与棕榈酰化对AnkG在上皮侧膜和神经元轴突起始段的膜结构域生物发生中的靶向和功能的要求一致。
Background: Ankyrin-G targets to specialized membrane domains in multiple cell types. Results: Ankyrin-G is S-palmitoylated at a conserved cysteine 70 located in the first loop of the ankyrin repeat solenoid. Conclusion: Cysteine 70 is required for function of ankyrin-G in membrane domain assembly. Significance: This finding provides new insights into how the ankyrin proteins exert their functions in formation and maintenance of membrane domains. Ankyrin-G (AnkG) coordinates protein composition of diverse membrane domains, including epithelial lateral membranes and neuronal axon initial segments. However, how AnkG itself localizes to these membrane domains is not understood. We report that AnkG remains on the plasma membrane in Madin-Darby canine kidney (MDCK) cells grown in low calcium, although these cells lack apical-basal polarity and exhibit loss of plasma membrane association of AnkG partners, E-cadherin and β2-spectrin. We subsequently demonstrate using mutagenesis and mass spectrometry that AnkG is S-palmitoylated exclusively at Cys-70, which is located in a loop of the first ankyrin repeat and is conserved in the vertebrate ankyrin family. Moreover, C70A mutation abolishes membrane association of 190-kDa AnkG in MDCK cells grown in low calcium. C70A 190-kDa AnkG fails to restore biogenesis of epithelial lateral membranes in MDCK cells depleted of endogenous AnkG. In addition, C70A 270-kDa AnkG fails to cluster at the axon initial segment of AnkG-depleted cultured hippocampal neurons and fails to recruit neurofascin as well as voltage-gated sodium channels. These effects of C70A mutation combined with evidence for its S-palmitoylation are consistent with a requirement of palmitoylation for targeting and function of AnkG in membrane domain biogenesis at epithelial lateral membranes and neuronal axon initial segments.