Aging, Dauer, and Stature Phenotypes Are Conferred by Structure-Directed Missense Mutations in the Endogenous...

Phosphatidylinositol 3-kinase (PI3K) integrates insulin/IGF signaling (IIS) and Ras inputs to control lifespan, metabolism and growth. Yet the organismal consequences of selective structural perturbations remain poorly understood. Using structure-guided CRISPR/Cas9-dependent genome editing, we dissected functions of AGE-1, the sole Class IA PI3K catalytic subunit in Caenorhabditis elegans. An endogenously tagged AGE-1, containing a long flexible linker, epitope and fluorescent tag, retained full activity, enabling visualization of native protein dynamics in vivo. A likely constitutively activating E630K substitution, modeled on oncogenic p110α alleles, markedly shortened lifespan and enhanced Ras-dependent induction of primary vulval precursor cell (VPC) fate, confirming evolutionary conservation of PI3K activation mechanisms that directly modulate longevity and development. Structural modeling further guided mutation of AGE-1 residues predicted to mediate Ras binding. Surprisingly, a putative AGE-1 variant defective in Ras association, together with a complementary Ras effector-binding mutation, produced enlarged animals with reduced dauer formation. These phenotypes reveal a previously unrecognized Ras>PI3K signaling axis that restrains somatic growth and promotes entry into diapause, counter to canonical IIS models. Together, these structure-informed alleles show that discrete PI3K structural perturbations can differentially uncouple lifespan, growth, and developmental outcomes in vivo. By combining structural modeling with genome editing in a tractable aging model, this work establishes a framework for dissecting conserved signaling enzymes at single-residue resolution and uncovers unexpected organismal roles for PI3K structure in coordinating growth and longevity.