Novel function of phosphoinositide 3-kinase in T cell Ca²⁺ signaling: A phosphatidylinositol 3,4,5-trisphosphate-mediated Ca²⁺ entry mechanism

This study presents evidence that phosphoinositide (PI) 3-kinase is involved in T cell Ca²⁺ signaling via a phosphatidylinositol 3,4,5- trisphosphate PI(3,4,5)P₃-sensitive Ca²⁺ entry pathway. First, exogenous PI(3,4,5)P₃ at concentrations close to its physiological levels induces Ca²⁺ influx in T cells, whereas PI(3,4)P(v) PI(4,5)P₂, and PI(3)P have no effect on [Ca²⁺](i). This Ca²⁺ entry mechanism is cell type-specific as B cells and a number of cell lines examined do not respond to PI(3,4,5)P₃ stimulation. Second, inhibition of PI 3-kinase by wortmannin and by overexpression of the dominant negative inhibitor Δp85 suppresses anti-CD3- induced Ca²⁺ response, which could be reversed by subsequent exposure to PI(3,4,5)P₃. Third, PI(3,4,5)P₃ is capable of stimulating Ca²⁺ efflux from Ca²⁺-loaded plasma membrane vesicles prepared from Jurkat T cells, suggesting that PI(3,4,5)P₃ interacts with a Ca²⁺ entry system directly or via a membrane-bound protein. Fourth, although D-myo-inositol 1,3,4,5- tetrakisphosphate (Ins(1,3,4,5)P₄) mimics PI(3,4,5)P₃ in many aspects of biochemical functions such as membrane binding and Ca²⁺ transport, we raise evidence that Ins(1,3,4,5)P₄ does not play a role in anti-CD3- or PI(3,4,5)P₃-mediated Ca²⁺ entry. This PI(3,4,5)P₃-stimulated Ca²⁺ influx connotes physiological significance, considering the pivotal role of PI 3-kinase in the regulation of T cell function. Given that PI 3-kinase and phospholipase C-γ form multifunctional complexes downstream of many receptor signaling pathways, we hypothesize that PI(3,4,5)P₃-induced Ca²⁺ entry acts concertedly with Ins(1,4,5)P₃-induced Ca²⁺ release in initiating T cell Ca²⁺ signaling. By using a biotinylated analog of PI(3,4,5)P₃ as the affinity probe, we have detected several putative PI(3,4,5)P₃-binding proteins in T cell plasma membranes.