Molting is conserved process in members of Nematoda phylum, in which the apical extracellular matrix of the epidermis, the cuticle, is remodeled through a process of degradation and re-synthesis. Using a genetic approach, we identified two NIMA family ser/thr kinases, NEKL-2 and NEKL-3, as essential for the completion of molting. NEKL-3 is highly similar to mammalian Nek6 and Nek7, which regulate several cell cycle processes involving microtubules. Co-expression of Nek6 and Nek7 can rescue molting defects in
nekl-3 mutants, indicating that these proteins share conserved molecular functions. Unlike Nek6 and Nek7, however, NEKL-3 does not play a role in cell cycle events. Rather, NEKL-3 acts specifically in the hypodermis to promote cuticle shedding, possibly through the regulation of microtubules, vesicle trafficking, or both. NEKL-2 is most similar to mammalian Nek8, which may control microtubule organization during ciliogenesis. NEKL-2 functions in the hypodermis where it is required for clathrin-mediated endocytosis. Inhibition of
nekl-2 leads to decreased internalization of LRP-1, an essential molting factor and receptor for low-density lipoproteins. Decreased endocytosis of LRP-1 in
nekl-2 mutants may lead to decreased cholesterol intake and the reduced synthesis of sterols required for molting. Notably, although NEKL-2 and NEKl-3 are both expressed in a punctate pattern close to the apical surface of the
hyp7 epidermal syncytium, their expression patterns do not overlap, suggesting that they carry out distinct functions. Consistent with this, unlike
nekl-2, loss of
nekl-3 does not perturb endocytosis. Genetic analyses indicate that
nekl-2 and
nekl-3 carry out partially redundant functions that promote normal molting. In addition, our data, together with studies of their mammalian orthologs, indicate that NEKL-2 and NEKL-3 associate with ankyrin repeat proteins, which may function as scaffolds for NEKL-2 and NEKL-3 signaling. Loss of several conserved ankyrin-repeat proteins, including MLT-4/Inversin, lead to specific defects in molting similar to
nekl-2 and
nekl-3 mutants. We have also identified several suppressors of a hypomorphic allele of
nekl-3,
sv3, which include several kinases and phosphatases that are likely to function in the NEKL-3 pathway. In addition,
nekl-3 defects are suppressed by
cdc-42 depletion, suggesting that CDC-42 could be a target for negative regulation by NEKL-3 and that NEKL-3 may therefore control the polarity of epidermal cytoskeletal components during molting cycles. Taken together, our studies have identified several unique roles for NIMA family kinases that we expect are conserved in other eukaryotes, including mammals, but which have been largely overlooked.