Atypical PI3Ks coordinate chemotaxis, signaling dynamics, and multicellular development in Dictyostelium

Abstract Phosphoinositide 3-kinase (PI3K) signaling regulates protrusion, polarity, membrane uptake, and multicellular development in Dictyostelium discoideum , but these functions have been interpreted largely through canonical Class I PI3Ks and PI(3,4,5)P₃ production. This framework does not fully explain how PI3K-dependent pathways attenuate Ras activity, organize PI(3,4)P₂-associated polarity states, support cAMP relay, or coordinate development. Here, we identify three atypical PI3K-family enzymes—PikF, PikG, and PikH—as functionally distinct regulators of these processes. PikF constrains Ras–phosphoinositide–actin signaling; pikF⁻ cells show prolonged cAMP-stimulated Ras activation, extended PIP₃ recruitment, delayed PI(3,4)P₂ biosensor recovery, elevated peripheral actin activity, impaired chemotactic precision, and delayed abnormal development. PikG acts through a distinct relay-associated pathway: pikG⁻ cells fail to generate endogenous cAMP oscillations, display disrupted ACA polarity, deposit spatially disorganized ACA-positive vesicle trails, and fail to aggregate. PikH, in contrast, supports efficient phagocytic uptake with little effect on acute chemotactic signaling. Kinase-dead rescue experiments show that conserved catalytic lysines are required for PikF- and PikG-dependent development and PikH-dependent uptake. Together, our results reveal that atypical PI3Ks diversify the Dictyostelium PI3K signaling toolkit, separating protrusive signal attenuation, cAMP relay organization, membrane uptake, and multicellular development into distinct kinase-dependent modules.