Loss of Copine D Leads to Ras Activation in Dictyostelium discoideum

Copines are a family of calcium-dependent phospholipid-binding proteins conserved across numerous eukaryotes. The expression of multiple copine genes is dysregulated in several human cancers, yet their molecular functions remain poorly understood. We are investigating copine genes (cpnA-cpnF) in the amoeba Dictyostelium discoideum, a well-established model for studying conserved signaling pathways that regulate chemotaxis. During development, individual amoebae release and move toward cAMP. cAMP chemotaxis utilizes Ras/PI3K signaling, a pathway that is highly conserved in mammalian cells and often dysregulated in cancer. Using two cpnD mutants that were generated via restriction enzyme-mediated integration (REMI), we found that cpnD mutants exhibited increased cellular proliferation, precocious development, and larger fruiting bodies. Additionally, we found that cpnD mutants had increased cell spreading, producing a flattened morphology and larger cell area. Because activated Ras has been shown to promote this phenotype, we measured Ras activity and found that cpnD mutants exhibited increased Ras activation. cpnD mutants also formed significantly smaller contractile vacuoles during osmotic stress. Inhibition of PI3K suppressed both the enlarged cell area and reduced contractile vacuole phenotypes, indicating that these defects result from increased Ras/PI3K signaling. Finally, we found that GFP-tagged CpnD transiently localized to the plasma membrane following cAMP stimulation, suggesting CpnD may have a role in cAMP signaling. Together, these findings identify CpnD as a potential negative regulator of Ras signaling and provide the first evidence of copine involvement in the Ras/PI3K pathway, suggesting a conserved mechanism that may help explain how altered copine expression contributes to cancer progression.