Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5
Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5
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DOI：
10.1074/jbc.m117.788364
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发表时间：
2017-09-01
影响因子：
4.8
通讯作者：
Tornroth-Horsefield, Susanna
中科院分区：
文献类型：
--
作者：
Roche, Jennifer Virginia;Survery, Sabeen;Tornroth-Horsefield, Susanna
肾水通道水通道蛋白2（AQP 2）和溶酶体运输调节相互作用蛋白LIP 5之间的相互作用将AQP 2靶向多泡体并促进溶酶体降解。这种相互作用是以加压素依赖性方式控制AQP 2顶端膜丰度的过程的一部分，从而允许尿量调节。加压素调节AQP 2 C末端四个位点的磷酸化（Ser（256）、Ser（261）、Ser（264）和Thr（269）），其中Ser（256）是关键的，足以使AQP 2从储存囊泡转运到顶膜。然而，AQP 2磷酸化是否调节AQP 2-LIP 5复合物亲和力尚不清楚。在这里，我们使用远蛋白质印迹分析和微量热泳显示，AQP 2结合LIP 5的磷酸化依赖的方式。我们构建了五个磷酸化模拟突变体（S256 E，S261 E，S264 E，T269 E和S256 E/T269 E）和一个C-末端截短突变体（Delta P242），该突变体缺乏所有磷酸化位点，但保留了先前提出的LIP 5结合位点。CD光谱表明，野生型水通道蛋白2和磷酸模拟突变体具有相似的整体结构，但显示可能产生的C-末端构象变化的熔融温度的差异。非磷酸化的AQP 2与LIP 5结合的亲和力最高，而AQP 2-Delta P242的亲和力低20倍，这是通过微量热泳测定的。AQP 2-S256 E、S261 E、T269 E和S256 E/T269 E均具有降低的亲和力。这种作用对于AQP 2-S256 E最为突出，这与其在顶端膜靶向中的作用非常吻合。AQP 2-S264 E的亲和力与非磷酸化AQP 2相似，可能表明其在外泌体排泄中发挥作用。我们的数据表明，AQP 2磷酸化变构控制其与LIP 5的相互作用，说明如何改变亲和力相互作用的蛋白质形成的基础上，通过翻译后修饰调节AQP 2贩运。
The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. This interaction is part of a process that controls AQP2 apical membrane abundance in a vasopressin-dependent manner, allowing for urine volume adjustment. Vasopressin regulates phosphorylation at four sites within the AQP2 C terminus (Ser(256), Ser(261), Ser(264), and Thr(269)), of which Ser(256) is crucial and sufficient for AQP2 translocation from storage vesicles to the apical membrane. However, whether AQP2 phosphorylation modulates AQP2-LIP5 complex affinity is unknown. Here we used far-Western blot analysis and microscale thermophoresis to show that the AQP2 binds LIP5 in a phosphorylation-dependent manner. We constructed five phospho-mimicking mutants (S256E, S261E, S264E, T269E, and S256E/T269E) and a C-terminal truncation mutant (Delta P242) that lacked all phosphorylation sites but retained a previously suggested LIP5-binding site. CD spectroscopy indicated that wild-type AQP2 and the phospho-mimicking mutants had similar overall structure but displayed differences in melting temperatures possibly arising from C-terminal conformational changes. Non-phosphorylated AQP2 bound LIP5 with the highest affinity, whereas AQP2-Delta P242 had 20-fold lower affinity as determined by microscale thermophoresis. AQP2-S256E, S261E, T269E, and S256E/T269E all had reduced affinity. This effect was most prominent for AQP2-S256E, which fits well with its role in apical membrane targeting. AQP2-S264E had affinity similar to non-phosphorylated AQP2, possibly indicating a role in exosome excretion. Our data suggest that AQP2 phosphorylation allosterically controls its interaction with LIP5, illustrating how altered affinities to interacting proteins form the basis for regulation of AQP2 trafficking by post-translational modifications.