Control of clathrin-mediated endocytosis by NIMA family kinases

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. Author summary In order to function properly, cells must continually import materials from the outside. This process, termed endocytosis, is necessary for the uptake of nutrients and for interpreting signals coming from the external environment or from within the body. These signals are critical during animal development but also affect many types of cell behaviors throughout life. In our current work, we show that several highly conserved proteins in the nematode Caenorhabditis elegans, NEKL-2 and NEKL-3, regulate endocytosis. The human counterparts of NEKL-2 and NEKL-3 have been implicated in cardiovascular and renal diseases as well as many types of cancers. However, their specific functions within cells is incompletely understood and very little is known about their role in endocytosis or how this role might impact disease processes. Here we use several complementary approaches to characterize the specific functions of C. elegans NEKL-2 and NEKL-3 in endocytosis and show that their human counterparts likely have very similar functions. This work paves the way to a better understanding of fundamental biological processes and to determining the cellular functions of proteins connected to human diseases.