Novel Function of Phosphoinositide 3-Kinase in T Cell Ca2+ Signaling

Abstract

This study presents evidence that phosphoinositide (PI) 3-kinase is involved in T cell Ca 2؉ signaling via a phosphatidylinositol 3,4,5-trisphosphate PI(3,4,5)P 3 -sensitive Ca 2؉ entry pathway. First, exogenous PI(3,4,5)P 3 at concentrations close to its physiological levels induces Ca 2؉ influx in T cells, whereas PI(3,4)P 2 , PI(4,5)P 2 , and PI(3)P have no effect on [ entry. This PI(3,4,5)P 3 -stimulated Ca 2؉ 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-␥ form multifunctional complexes downstream of many receptor signaling pathways, we hypothesize that PI(3,4,5)P 3 -induced Ca 2؉ entry acts concertedly with Ins(1,4,5)P 3 -induced Ca 2؉ release in initiating T cell Ca 2؉ signaling. By using a biotinylated analog of PI(3,4,5)P 3 as the affinity probe, we have detected several putative PI(3,4,5)P 3 -binding proteins in T cell plasma membranes. channel (7, 8). Third, a TCR-operated Ca 2ϩ entry (TROCE) mechanism is activated in response to TCR-CD3 stimulation (2). However, this putative pathway is less well characterized. It is known to be independent of the depletion of intracellular Ca 2ϩ and inhibited by SKF96365, a Ca 2ϩ channel blocker, and phorbol esters (9).In this paper, we present data suggesting a new function of phosphoinositide (PI) 3-kinase in T cell Ca 2ϩ regulation via a PI(3,4,5)P 3 -sensitive Ca 2ϩ entry mechanism. In response to TCR activation, PI 3-kinase and other signaling molecules such as PLC-␥1 are recruited to the plasma membrane to form multifunctional complexes (10 -12). Activation of PI 3-kinase results in a transient accumulation of M levels of PI(3,4,5)P 3 and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ), both absent in quiescent T cells (13). To date, a clear consensus on the mode of action of these lipid messengers in regulating TCR signaling has yet to emerge. Putative downstream effectors for PI(3,4,5)P 3 and PI(3,4)P 2 in receptor-stimulated signaling include Ca 2ϩ -independent PKC isozymes (␦, ⑀, , ), PLC-␥, Akt, and so forth (14). The results of this study suggest that PI(3,4,5)P 3 mediates a novel Ca 2ϩ entry mechanism on plasma membranes. Given the intimate relationship between PI 3-ki-* This work was supported by National Institutes of Health Grants GM53448 (to C.-S. C.) and AI21490 (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.ʈ To whom correspondence should be addressed: ASTeCC Facility, Rm. 323B, University of Kentucky, Lexington, KY 40506-0286. Tel.: 606-257-2300 (ext. 261); Fax: 606-257-2489; E-mail: cchen1@pop. uky.edu. 1 The abbreviations used are: TCR, T cell receptor; PI 3-kinase, phosphoinositide 3-kinase; PLC, phospholipase C; PI(3,4,5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PI(4,...

Phosphoinositide 3-Kinase Facilitates Antigen-stimulated Ca2+ Influx in RBL-2H3 Mast Cells via a Phosphatidylinositol 3,4,5-Trisphosphate-sensitive Ca2+Entry Mechanism

“…It is noteworthy that the proposed involvement of PI3K in the regulation of a non-capacitative Ca 2ϩ influx pathway is reminiscent of our finding of a PI(3,4,5)P 3 -sensitive Ca 2ϩ entry mechanism in platelets (19) and Jurkat T cells (20). Consequently, we hypothesize that this PI (3,4,5 …”

“…The identity and purity of all inositol phosphates and inositol lipids were verified by 1 H and 31 P NMR and high resolution mass spectrometry. Published data from this and other laboratories have shown that PI(3,4,5)P 3 and other inositol lipids are cell-permeant and can readily fuse with cell membranes to exert cellular responses in different types of cells, including platelets (19), NIH3T3 cells (23), adipocytes (24), and Jurkat T cells (20). Fura-2 acetoxymethyl ester (fura-2 AM), fluo-3 acetoxymethyl ester (fluo-3 AM), and 2-aminoethyoxy diphenylborate (2-APB) were purchased from Calbiochem.…”

“…[ 3 H]Inositol Phosphate Turnover Analysis-The examination of phosphoinositol turnover was carried out according to a modification of the procedure described previously (20). In brief, RBL-2H3 cells were incubated with myo- [2-3 H]inositol (10 Ci/10 6 cells/ml) in Eagle's minimum essential medium supplemented with 10% fetal bovine serum.…”

Requirement of Ca2+and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-γ

“…It was reported recently that the phosphatidylinositol 3-kinase (PI3K)͞Akt pathway could be involved also in the IFN-␥-induced phosphorylation of Stat1 S727 in human tumor cell lines or mouse embryonic fibroblasts (15). PI3K has been shown to facilitate antigen-stimulated Ca 2ϩ flux in Jurkat T cells and mast cells (44,45), which could result in activation of Ca 2ϩ -dependent serine kinases. It seems likely that in different cellular contexts, more than one serine kinase pathway can converge on Stat1 during or after its activation through tyrosine phosphorylation on the cell surface.…”

Gs Protein-coupled Receptor Agonists Induce Transactivation of the Epidermal Growth Factor Receptor in T84 Cells

“…One possibility is that PI3K may alter the activity of one or more of the transport proteins that comprise the Cl Ϫ secretory machinery, and in this regard PI3K has been shown to alter ion channel activity in some cell types (48,49). However, previous studies from our laboratory suggest an inhibitory effect of PI3K on intestinal epithelial K ϩ channels (50), an effect that does not explain our current data because inhibition of K ϩ channel function would likely lead to attenuation of Cl Ϫ secretory responses (2).…”