CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase
CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase
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DOI：
10.1152/ajprenal.00431.2019
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发表时间：
2020-03-01
影响因子：
4.2
通讯作者：
Knepper, Mark A.
中科院分区：
文献类型：
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作者：
Isobe, Kiyoshi;Raghuram, Viswanathan;Knepper, Mark A.
先前的研究表明肌球蛋白轻链激酶（MLCK）参与了肾集合管水通道蛋白2（AQP 2）的调节。为了发现MLCK的信号传导靶点，我们使用CRISPR-Cas9来删除MLCK基因（Mylk）以获得MLCK无效的mpkCCD细胞，并使用稳定同位素标记细胞培养物中的氨基酸进行全面的磷酸化蛋白质组学定量。免疫细胞化学和电子显微镜检查表明，在加工的AQP 2-含有早期内体晚期内体的缺陷。磷酸蛋白质组学实验显示，在1，743个磷酸肽中，在多次重复中定量。107例MLCK缺失导致丰度改变（29例降低，78例增加）。其中一个减少的磷酸肽对应于肌球蛋白调节轻链中的典型靶位点。网络分析表明，目标磷蛋白聚类成不同的结构/功能组：aclomyosin，信号，核膜，基因转录。mRNA加工、能量代谢、中间丝、粘附连接和紧密连接。有显着的重叠之间的衍生MLCK信号网络和先前确定的PKA信号网络。肌动球蛋白类中多种蛋白的存在促使实验表明MLCK缺失抑制了加压素对去乙酰化F-肌动蛋白的正常作用，为AQP 2运输缺陷提供了潜在的解释。核膜中多种蛋白质磷酸化的变化促使测量核大小，显示平均核体积显著增加。我们的结论是MLCK是一个多组分的信号通路的一部分，在细胞质和细胞核，其中包括更多的传统的非肌肉肌球蛋白通过肌球蛋白调节轻链磷酸化的简单调节。
Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates. 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: aclomyosin, signaling, nuclear envelope, gene transcription. mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.