Genetic analysis of daf-18/PTEN missense mutants for starvation resistance and developmental regulation during Caenorhabditis elegans L1 arrest

Abstract

Abstract Mutations in the well-known tumor suppressor PTEN are observed in many cancers. PTEN is a dual-specificity phosphatase that harbors lipid and protein-phosphatase activities. The Caenorhabditis elegans PTEN ortholog is daf-18, which has pleiotropic effects on dauer formation, aging, starvation resistance, and development. Function of 3 daf-18 point-mutants, G174E, D137A, and C169S, had previously been investigated using high-copy transgenes in a daf-18 null background. These alleles were generated based on their mammalian counterparts and were treated as though they specifically disrupt lipid or protein-phosphatase activity, or both, respectively. Here, we investigated these alleles using genome editing of endogenous daf-18. We assayed 3 traits relevant to L1 starvation resistance, and we show that each point mutant is essentially as starvation-sensitive as a daf-18 null mutant. Furthermore, we show that G174E and D137A do not complement each other, suggesting overlapping effects on lipid and protein-phosphatase activity. We also show that each allele has strong effects on nucleocytoplasmic localization of DAF-16/FoxO and dauer formation, both of which are regulated by PI3K signaling, similar to a daf-18 null allele. In addition, each allele also disrupts M-cell quiescence during L1 starvation, though D137A has a weaker effect than the other alleles, including the null. Our results confirm that daf-18/PTEN is important for promoting starvation resistance and developmental arrest and that it is a potent regulator of PI3K signaling, and they highlight challenges of using genetic analysis to link specific DAF-18/PTEN enzymatic activities to particular phenotypes.