RESUMO
The conserved C. elegans protein kinases NEKL-2 and NEKL-3 regulate multiple steps of membrane trafficking and are required for larval molting. Through a forward genetic screen we identified a loss-of-function mutation in catp-1 as a suppressor of molting defects in synthetically lethal nekl-2; nekl-3 double mutants. catp-1 is predicted to encode a membrane- associated P4-type ATPase involved in Na + -K + exchange. Moreover, a mutation predicted to abolish CATP-1 ion-pump activity also suppressed nekl-2; nekl-3 mutants. Endogenously tagged CATP-1 was primarily expressed in epidermal (hypodermal) cells within punctate structures located at or near the apical plasma membrane. Through whole genome sequencing, we identified two additional nekl-2; nekl-3 suppressor strains containing coding-altering mutations in catp-1 but found that neither mutation, when introduced into nekl-2; nekl-3 mutants using CRISPR methods, was sufficient to elicit robust suppression of molting defects. Our data also suggested that the two catp-1 isoforms, catp-1a and catp-1b , may in some contexts be functionally redundant. On the basis of previously published studies, we tested the hypothesis that loss of catp-1 may suppress nekl -associated defects by inducing partial entry into the dauer pathway. Contrary to expectations, however, we failed to obtain evidence that loss of catp-1 suppresses nekl-2; nekl-3 defects through a dauer-associated mechanism or that loss of catp-1 leads to entry into the pre-dauer L2d stage. As such, loss of catp-1 may suppress nekl- associated molting and membrane trafficking defects by altering electrochemical gradients within membrane-bound compartments.
RESUMO
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.