The identification of phosphatidylinositol 3,5-bisphosphate in T-lymphocytes and its regulation by interleukin-2

In recent times 3-phosphoinositides have emerged as important regulators of cell metabolism, survival, and proliferation. During the last year, the phospholipid phosphatidylinositol 3,5-bisphosphate (PtdIns3,5P(2)) was identified in yeast, fibroblasts, SV40-transformed kidney (COS-7) cells, and platelets. The discovery of this novel phospholipid has increased the complexity of the metabolism relating to the generation of biologically active inositol-containing lipids. We describe here the identification of PtdIns3,5P(2) in the CTLL-8 mouse T-lymphocyte cell line using two in vivo radiolabeling protocols. Treatment of the cells with UV radiation led to an increase in the cellular content of PtdIns3,5P(2). In contrast, preincubation of the cells with wortmannin or treatment with hypertonic medium (high concentration sorbitol) led to the opposite effect. Herein we demonstrate that interleukin-2 (IL-2), the growth factor required for CTLL-2 cell proliferation, was able to increase the level of PtdIns3,5P(2) with similar kinetics to that of the formation of phosphatidylinositol: 3,4-bisphosphate (PtdIns3,4P(2)). An increase in this novel 3-phosphorylated lipid in response to IL-2 seems to be a general property of this cytokine because a similar result was obtained when the pre-B cell line BaF/3 ex pressing the high affinity IL-2 receptor was used. Using a constitutively active regulatory subunit of type I phosphatidylinositol 3-kinase and cells expressing a deletion of the serine-rich domain of the IL-2 receptor beta chain, which is required for IL-2-stimulated type I phosphatidylinositol 3-kinase activation, we demonstrate that IL-2-induced generation of PtdIns3,5P(2) is related to the activation of this enzyme. The results show for the first time the identification of PtdIns3,5P(2) in both T- and B-lymphocytes and indicate its positive regulation by the mitogen IL-2.