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 ph ospholipid phosphatidylinositol 3,5-bisphosphate (PtdIns3,5P(2)) was identi fied in yeast, fibroblasts, SV40-transformed kidney (COS-7) cells, and plat elets. The discovery of this novel phospholipid has increased the complexit y of the metabolism relating to the generation of biologically active inosi tol-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 ce lls with wortmannin or treatment with hypertonic medium (high concentration sorbitol) led to the opposite effect. Herein we demonstrate that interleuk in-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 o f 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 obta ined when the pre-B cell line BaF/3 ex pressing the high affinity IL-2 rece ptor was used. Using a constitutively active regulatory subunit of type I p hosphatidylinositol 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-sti mulated type I phosphatidylinositol 3-kinase activation, we demonstrate tha t 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 PtdI ns3,5P(2) in both T- and B-lymphocytes and indicate its positive regulation by the mitogen IL-2.