Nima-related kinases (NEKs) are highly conserved among eukaryotes and have been reported to regulate cell cycle processes along with ciliogenesis and DNA repair. Nevertheless, relatively little is known about NEK kinase targets or NEK pathway components. Whereas the human genome encodes for 11 NEK kinases, C. elegans encodes for only 4, including NEKL-2/NEK8 and NEKL-3/NEK6/7. We have reported a requirement for NEKL-2 and NEKL-3 in C. elegans molting and have shown that these kinases influence intracellular trafficking in epidermal cells. Furthermore, NEKL-2 and NEKL-3 appear to function in complementary pathways and within distinct molecular complexes containing several conserved ankyrin-repeat proteins (MLT-2/ANKS6, MLT-3/ANKS3, MLT-4/Inversin). To gain insight into the molecular and cellular functions of NEKL-MLT network, we carried out genetic screens to identify suppressors of the synthetic lethal molting-defective phenotype of
nekl-2;
nekl-3 double mutants. For these screens, we used weak aphenotypic alleles generated by CRISPR (
nekl-2) and the million-mutation project (
nekl-3). Our screens were therefore designed to simultaneously identify both
nekl-2 and
nekl-3 pathway suppressors, thus increasing efficiency. Moreover, by using very weak nekl alleles, our screens should have high sensitivity and thus capture a wide range of suppressor mutations. We also incorporated a novel counter-selection method, using the
peel-1 toxin, to reduce the amount of labor. Finally, a unique subtraction method was carried out in conjunction with whole genome sequencing to help narrow down candidate causal mutations within the suppressor strains. Among the identified suppressors were strong LOF alleles of
fcho-1, and
apm-2/dpy-23.
fcho-1 encodes the C. elegans ortholog of FCHO1, an F-bar protein that regulates the conformation and activity of AP2, a multimeric adapter complex involved in clathrin-mediated endocytosis. Strikingly,
apm-2 encodes the
mu2 subunit of AP2. Further studies have shown that RNAi of the alpha (
apa-2) or sigma2 (
aps-2) subunits of AP2 also suppress
nekl-2;
nekl-3 double-mutant lethality and that
fcho-1(RNAi) suppresses molting defects in other members of the NEKL-MLT network. Our data suggest that the NEKL-MLT network may regulate the activity of the AP2 complex by balancing the actions of FCHO-1. Consistent with this, clathrin puncta, which are mislocalized in
nekl-2;
nekl-3 mutants, are restored to a more wild-type morphology in
nekl-2;
fcho-1;
nekl-3 triple mutants. Interestingly, proteomic studies identified several AP2 subunits in complex with human NEK7, suggesting that NEK family members may regulate clathrin-mediated endocytosis and AP2 in a wide range of species. These results, together with other data, strongly suggest that the regulation of trafficking and endocytosis is a conserved but previously overlooked function of NEK kinases.