Loss of Copine D Leads to Ras Activation in Dictyostelium discoideum

Copines are a family of calcium-dependent phospholipid-binding proteins found in most eukaryotes. The expression of multiple copine genes is dysregulated in various types of human cancers. Despite this, a common mechanistic function for copines remains unknown. We are studying copines in Dictyostelium discoideum, which has six copine genes (cpnA-cpnF). Cells lacking cpnA or cpnC (cpnA- and cpnC-, respectively) exhibit many phenotypes, including defects in development, chemotaxis, adhesion, and contractile vacuole (CV) function. To further characterize the function of copines, this study tested the hypothesis that CpnD is responsible for cellular functions distinct from CpnA and CpnC. In this study, we obtained two cpnD mutants that were generated via restriction enzyme-mediated integration (REMI) mutagenesis; one in the first exon (cpnD(i291)), and one in the second exon (cpnD(i459)) of the endogenous cpnD gene. Throughout our experiments, we found that cpnD mutants had increased cellular proliferation in both axenic and bacterial cultures. Additionally, we found that cpnD mutants exhibited precocious development and had significantly larger fruiting bodies than the parental cell line. We further investigated the morphology of cpnD mutants and found that they were significantly larger than parental cells and exhibited decreased cell-substrate adhesion. cpnD mutants also had increased activated Ras compared to the parental cell line, along with significantly smaller CVs, a phenotype that was rescued after PI3K inhibition. Finally, we found that GFP-tagged CpnD localizes to the leading edge of both randomly migrating cells and in cells responding to folate. This study is the first to describe copine proteins as having a regulatory function in Ras activation and downstream signaling effects. Additionally, this study further supports our hypothesis that copines act as nonredundant cellular proteins in Dictyostelium to regulate numerous processes.