Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle.
Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle.
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DOI：
10.1073/pnas.2204618119
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发表时间：
2022-08-23
影响因子：
11.1
通讯作者：
Meredith, Stephen C.
中科院分区：
文献类型：
--
作者：
Srivastava, Atul K.;Venkata, Bharat Somireddy;Sweat, Yan Y.;Rizzo, Heather R.;Jean-Francois, Lea;Zuo, Li;Kurgan, Kathleen W.;Moore, Patrick;Shashikanth, Nitesh;Smok, Izabela;Sachleben, Joseph R.;Turner, Jerrold R.;Meredith, Stephen C.
紧密连接形成可选择性渗透的密封层，限制了细胞旁的流量。紧密连接通透性增加与肠道疾病有关。先前的研究表明，酪蛋白激酶2(CK2)在紧密连接蛋白occludin的细胞质尾部的一个非结构化区域内磷酸化S408。此外，CK2抑制和随后的S408去磷酸化降低了体内外细胞旁的通透性，并在体内减轻了实验性免疫介导的肠道疾病。在这里，我们证明了S408的磷酸化增强了非结构区域和远端α-螺旋束之间的分子内相互作用，从而限制了与紧密连接蛋白闭锁带1(ZO-1)的结合。相反，S408去磷酸化限制了分子内的相互作用，在体外促进了occludin与ZO-1的结合，并在体内增强了occludin在紧密连接处的锚定。Occludin是一种跨越四膜的紧密连接蛋白。长的C末端细胞质结构域代表了近一半的occludin序列，包括一个由三个α-螺旋组成的远端束，介导与其他紧密连接成分的相互作用。位于α-螺旋束附近的一个短小的非结构化区域是一个磷酸化热点，其中S408的磷酸化起到了分子开关的作用，改变了紧密连接蛋白的相互作用和屏障功能。在这里，我们使用核磁共振来定义S408磷酸化对非结构区域和α-螺旋束之间分子内相互作用的影响。S408假磷酸化影响三个α-螺旋之间的铰链位置的构象。进一步的顺磁松弛增强和微尺度热电泳法研究表明，非结构区域与α螺旋束相互作用。这些非结构结构域之间的相互作用被S408磷酸化增强，并允许非结构区域阻碍结合部位，从而降低了阻滞素尾部与闭锁小带1(ZO-1)的亲和力。相反，S408去磷酸化减弱了分子内的相互作用，暴露了结合部位，并增加了occludin与ZO-1的结合亲和力。与ZO-1结合增加一致，转基因小鼠的活体成像和光漂白后荧光恢复(FRAP)分析表明，与野生型EGFP-occludin相比，增强型绿色荧光蛋白(EGFP)标记的非磷酸化occludin的紧密连接锚定增加。总体而言，这些数据定义了S408磷酸化修饰阻塞素尾部构象以调节紧密连接蛋白相互作用和细胞旁通透性的机制。
Tight junctions form selectively permeable seals that limit paracellular flux. Increased tight junction permeability has been associated with intestinal disease. Previous studies indicate that casein kinase 2 (CK2) phosphorylates S408 within an unstructured region of the cytoplasmic tail of the tight junction protein occludin. Moreover, CK2 inhibition and consequent S408 dephosphorylation reduce paracellular permeability in vitro and in vivo and attenuate experimental immune-mediated intestinal disease in vivo. Here, we show that S408 phosphorylation enhances intramolecular interactions between the unstructured region and the distal α-helical bundle to limit binding to the tight junction protein zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation limits intramolecular interactions, promotes occludin binding to ZO-1 in vitro, and enhances occludin anchoring at the tight junction in vivo. Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.
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影响因子：
7.7
作者：
Murakami T;Frey T;Lin C;Antonetti DA
通讯作者：
Antonetti DA
影响因子：
14.8
作者：
Fawzi, Nicolas L.;Ying, Jinfa;Torchia, Dennis A.;Clore, G. Marius
通讯作者：
Clore, G. Marius
DOI：
10.1083/jcb.141.7.1539
发表时间：
1998-06-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Furuse M;Fujita K;Hiiragi T;Fujimoto K;Tsukita S
通讯作者：
Tsukita S
影响因子：
3.3
作者：
Chen, YH;Lu, Q;Jeansonne, B
通讯作者：
Jeansonne, B
occludin S408磷酸化调节紧密的连接蛋白相互作用和屏障功能。
DOI：
10.1083/jcb.201010065
发表时间：
2011-05-02
期刊：
The Journal of cell biology
影响因子：
--
作者：
Raleigh DR;Boe DM;Yu D;Weber CR;Marchiando AM;Bradford EM;Wang Y;Wu L;Schneeberger EE;Shen L;Turner JR
通讯作者：
Turner JR