Barker S A, Caldwell K K, Hall A, Martinez A M, Pfeiffer J R, Oliver J M, Wilson B S
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Mol Biol Cell. 1995 Sep;6(9):1145-58. doi: 10.1091/mbc.6.9.1145.
We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.
我们研究了磷脂酰肌醇3激酶（PI 3激酶）抑制剂渥曼青霉素对RBL-2H3肥大细胞模型中抗原介导信号传导的影响。在RBL-2H3细胞中，高亲和力IgE受体（FcεR1）的交联激活至少两种细胞质蛋白酪氨酸激酶Lyn和Syk，并刺激分泌、膜 ruffling、铺展、胞饮作用以及与细胞 - 底物粘附增加相关的肌动蛋白斑块形成。此外，FcεR1交联激活PI 3激酶。先前已表明渥曼青霉素会导致PI 3激酶活性的剂量依赖性抑制，并且还抑制抗原刺激的脱颗粒作用。我们报告渥曼青霉素也显著抑制抗原诱导的肌醇（1,4,5）P3合成。与其他细胞系统中暗示磷脂酰肌醇（3,4,5）P3参与ruffling的证据一致，用渥曼青霉素预处理RBL-2H3细胞可抑制FcεR1交联引起的膜ruffling和液体胞饮作用。然而，渥曼青霉素不抑制抗原诱导的肌动蛋白聚合、受体内化，或在贴壁细胞中抗原刺激后发生的肌动蛋白依赖性铺展和粘附斑块形成过程。渥曼青霉素也不能抑制FcεR1偶联的酪氨酸激酶Lyn或Syk中的任何一种，或通过体外激酶测定法测量的丝裂原活化蛋白激酶的激活。令人惊讶的是，使用针对PI 3激酶p85亚基的抗血清从FcεR1激活的细胞制备的免疫沉淀物中存在大量体外丝氨酸/苏氨酸激酶活性。这种活性可通过用渥曼青霉素预处理细胞或通过直接向激酶测定中添加渥曼青霉素来抑制，这表明PI 3激酶本身能够作为一种蛋白激酶起作用。我们得出结论，FcεR1交联激活PI 3激酶的脂质和蛋白激酶活性，并且用渥曼青霉素抑制这些活性会导致FcεR1介导的信号反应的一个子集的选择性阻断。
