Siddiqui R A, English D
Experimental Cell Research Program, The Methodist Research Institute, MPC 1417, 1701 N. Senate Ave., Indianapolis, IN 46202, USA.
Biochim Biophys Acta. 2000 Jan 3;1483(1):161-73. doi: 10.1016/s1388-1981(99)00172-9.
Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.
磷脂酰肌醇3'-激酶（PI 3'-激酶）在肝细胞、内皮细胞和肿瘤细胞向激活细胞酪氨酸激酶的激动剂迁移过程中发挥重要作用。我们检测了磷脂酸（PA）诱导的嗜中性白细胞的PI 3'-激酶依赖性趋化反应，以阐明该酶潜在影响细胞迁移的机制。免疫沉淀的蛋白质印迹分析表明，PA诱导了在PA刺激的细胞中共免疫沉淀的三种参与功能激活的不同蛋白质的酪氨酸磷酸化。这些蛋白质被鉴定为lyn、syk和PI 3'-激酶的85 kDa调节亚基。PI 3'-激酶抑制剂渥曼青霉素和LY294002抑制了对PA而非其他几种嗜中性粒细胞激动剂的趋化反应。趋化抑制是由钙动员的上游抑制引起的。用乙二醇双（β-氨基乙基醚）N，N，N'，N'-四乙酸（EGTA）螯合细胞外钙不影响PA诱导的趋化性，而用1，2-双（2-氨基苯氧基）乙烷-N，N，N'，N'-四乙酸（BAPTA）螯合细胞内钙减弱了这种反应。因此，在没有钙内流情况下从细胞内储存库动员的钙所引起的细胞内Ca(2+)水平变化调节PA诱导的趋化性。此外，PI 3'-激酶抑制减弱了激动剂依赖性的肌醇1,4,5-三磷酸（IP(3)）生成，表明PI 3'-激酶通过介导PA刺激细胞中磷脂酶C（PLC）的激活对细胞内钙动员发挥作用。此外，PI 3'-激酶抑制剂LY294002也抑制了PA刺激细胞中syk的磷酸化。因此，我们提出局限于质膜内小叶的PI 3'-激酶产物在syk激活、钙动员和趋化迁移诱导中发挥作用。
