Hsu A L, Ching T T, Sen G, Wang D S, Bondada S, Authi K S, Chen C S
Division of Pharmaceutical Sciences, College of Pharmacy and Department of Microbiology and Immunology, Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 40536, USA.
J Biol Chem. 2000 May 26;275(21):16242-50. doi: 10.1074/jbc.M002077200.
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 Ca(2+). This Ca(2+) entry mechanism is cell type-specific as B cells and a number of cell lines examined do not respond to PI(3,4,5)P(3) stimulation. Second, inhibition of PI 3-kinase by wortmannin and by overexpression of the dominant negative inhibitor Deltap85 suppresses anti-CD3-induced Ca(2+) response, which could be reversed by subsequent exposure to PI(3,4,5)P(3). Third, PI(3,4,5)P(3) is capable of stimulating Ca(2+) efflux from Ca(2+)-loaded plasma membrane vesicles prepared from Jurkat T cells, suggesting that PI(3,4,5)P(3) interacts with a Ca(2+) entry system directly or via a membrane-bound protein. Fourth, although D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) mimics PI(3,4,5)P(3) in many aspects of biochemical functions such as membrane binding and Ca(2+) transport, we raise evidence that Ins(1,3,4,5)P(4) does not play a role in anti-CD3- or PI(3,4,5)P(3)-mediated Ca(2+) 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-gamma 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.
本研究表明，磷酸肌醇（PI）3激酶通过磷脂酰肌醇3,4,5-三磷酸PI(3,4,5)P(3)敏感的Ca(2+)内流途径参与T细胞Ca(2+)信号传导。首先，浓度接近其生理水平的外源性PI(3,4,5)P(3)可诱导T细胞中的Ca(2+)内流，而PI(3,4)P(2)、PI(4,5)P(2)和PI(3)P对[Ca(2+)]i无影响。这种Ca(2+)内流机制具有细胞类型特异性，因为所检测的B细胞和许多细胞系对PI(3,4,5)P(3)刺激无反应。其次，渥曼青霉素抑制PI 3激酶以及显性负性抑制剂Deltap85的过表达均抑制抗CD3诱导的Ca(2+)反应，随后暴露于PI(3,4,5)P(3)可使其逆转。第三，PI(3,4,5)P(3)能够刺激从Jurkat T细胞制备的Ca(2+)负载的质膜囊泡中的Ca(2+)外流，这表明PI(3,4,5)P(3)直接或通过膜结合蛋白与Ca(2+)内流系统相互作用。第四，尽管D-肌醇1,3,4,5-四磷酸（Ins(1,3,4,5)P(4)）在膜结合和Ca(2+)转运等许多生化功能方面模拟PI(3,4,5)P(3)，但我们提出证据表明Ins(1,3,4,5)P(4)在抗CD3或PI(3,4,5)P(3)介导的Ca(2+)内流中不起作用。考虑到PI 3激酶在T细胞功能调节中的关键作用，这种PI(3,4,5)P(3)刺激的Ca(2+)内流具有生理意义。鉴于PI 3激酶和磷脂酶C-γ在许多受体信号通路下游形成多功能复合物，我们假设PI(3,4,5)P(3)诱导的Ca(2+)内流与Ins(1,4,5)P(3)诱导的Ca(2+)释放协同作用，启动T细胞Ca(2+)信号传导。通过使用PI(3,4,5)P(3)的生物素化类似物作为亲和探针，我们在T细胞质膜中检测到了几种推定的PI(3,4,5)P(3)结合蛋白。
