PIKI-1, a class II PI 3-kinase, functions in endocytic trafficking

Abstract

Cellular membrane trafficking, including endocytosis and exocytosis, is a complex process coordinated by trafficking-associated proteins, cargo molecules, the cytoskeleton, and membrane lipids. The NIMA-related kinases NEKL-2 (human NEK8/9) and NEKL-3 (human NEK6/7) are conserved regulators of membrane trafficking in Caenorhabditis elegans that are required for the completion of molting. Using a genetic approach, we isolated reduction-of-function mutations in piki-1 that suppress nekl-associated molting defects. piki-1 encodes the sole predicted C. elegans Class II PI 3-kinase (PI3K), a relatively understudied class of lipid modifiers that contribute to the production of PI 3-phosphate (PI(3)P) and PI 3,4-bisphosphate (PI(3,4)P2). Using genetically encoded lipid sensors, we found that PIKI-1 was responsible for the production of PI(3,4)P2 in the C. elegans epidermis but played only a minor role in contributing to PI(3)P levels. Consistent with this, both PI(3,4)P2 and PIKI-1 partially colocalized to early endosomes, and reduction of PIKI-1 affected the size and protein composition of early endosomal compartments marked by RAB-5, EEA-1, and SNX-1. Reduced PIKI-1 also led to increased tubulation of endosomal compartments associated with recycling or the degradation of cellular debris. In contrast to studies using mammalian cell culture, PIKI-1 was largely dispensable for clathrin-mediated endocytosis in the worm epidermis, a polarized epithelium. Notably, reduction of PIKI-1 function mitigated defects in early endosomes associated with the depletion of NEKL-2. We propose that reduction of PIKI-1 function may suppress nekl molting defects by partially restoring endocytic trafficking function within a subset of compartments, including the early endosome. We also show that inhibition of HIPR-1, an ortholog of the mammalian PI(3,4)P₂-binding proteins, HIP1 and HIPR1, suppresses nekl molting defects, consistent with a model that loss of PIKI-1 alters the binding of endocytic regulators in a manner that partially compensates for the loss of NEKL-2 activity.