Department of Molecular Biology, College of Agriculture and Natural Resources, University of Wyoming, Laramie, Wyoming, United States of America.
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey, United States of America.
PLoS Genet. 2020 Feb 18;16(2):e1008633. doi: 10.1371/journal.pgen.1008633. eCollection 2020 Feb.
Endocytosis, the process by which cells internalize plasma membrane and associated cargo, is regulated extensively by posttranslational modifications. Previous studies suggested the potential involvement of scores of protein kinases in endocytic control, of which only a few have been validated in vivo. Here we show that the conserved NIMA-related kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (the NEKLs) control clathrin-mediated endocytosis in C. elegans. Loss of NEKL-2 or NEKL-3 activities leads to penetrant larval molting defects and to the abnormal localization of trafficking markers in arrested larvae. Using an auxin-based degron system, we also find that depletion of NEKLs in adult-stage C. elegans leads to gross clathrin mislocalization and to a dramatic reduction in clathrin mobility at the apical membrane. Using a non-biased genetic screen to identify suppressors of nekl molting defects, we identified several components and regulators of AP2, the major clathrin adapter complex acting at the plasma membrane. Strikingly, reduced AP2 activity rescues both nekl mutant molting defects as well as associated trafficking phenotypes, whereas increased levels of active AP2 exacerbate nekl defects. Moreover, in a unique example of mutual suppression, NEKL inhibition alleviates defects associated with reduced AP2 activity, attesting to the tight link between NEKL and AP2 functions. We also show that NEKLs are required for the clustering and internalization of membrane cargo required for molting. Notably, we find that human NEKs can rescue molting and trafficking defects in nekl mutant worms, suggesting that the control of intracellular trafficking is an evolutionarily conserved function of NEK family kinases.
内吞作用是细胞内化质膜和相关货物的过程，广泛受到翻译后修饰的调控。先前的研究表明，大量蛋白激酶可能参与了内吞作用的调控，但其中只有少数几种在体内得到了验证。在这里，我们表明保守的 NIMA 相关激酶 NEKL-2/NEK8/9 和 NEKL-3/NEK6/7（NEKLs）控制秀丽隐杆线虫的网格蛋白介导的内吞作用。NEKL-2 或 NEKL-3 活性的丧失导致明显的幼虫蜕皮缺陷和运输标记物在被阻断的幼虫中的异常定位。使用基于植物生长素的去稳定系统，我们还发现，在成年阶段的秀丽隐杆线虫中耗尽 NEKLs 会导致网格蛋白严重定位错误，并显著降低质膜顶端的网格蛋白流动性。使用无偏遗传筛选来鉴定 NEKL 蜕皮缺陷的抑制子，我们鉴定了几种 AP2 的组成部分和调节剂，AP2 是作用于质膜的主要网格蛋白衔接复合物。引人注目的是，降低 AP2 活性可挽救 nekl 突变体的蜕皮缺陷以及相关的运输表型，而增加活性 AP2 的水平则会加剧 nekl 缺陷。此外，在一个独特的相互抑制的例子中，NEKL 抑制减轻了与降低的 AP2 活性相关的缺陷，证明了 NEKL 和 AP2 功能之间的紧密联系。我们还表明，NEKLs 对于蜕皮所需的膜货物的聚类和内化是必需的。值得注意的是，我们发现人类 NEKs 可以挽救 nekl 突变体蠕虫中的蜕皮和运输缺陷，这表明细胞内运输的控制是 NEK 家族激酶的一个进化保守的功能。
