Tyrosine Phosphorylation of the vav Proto-oncogene Product Links FcεRI to the Rac1-JNK Pathway

Abstract

Activation of high affinity IgE Fc receptors (Fc⑀RI) in basophils and mast cells induces the rapid release of histamine and other inflammatory mediators from secretory granules, and initiates a cascade of signal transduction events leading to enhanced production and secretion of various biologically active cytokines (1). One of the earliest events induced upon Fc⑀RI aggregation is the activation of the nonreceptor tyrosine kinases Lyn and Syk, and the tyrosine phosphorylation of cytoplasmic molecules, including phospholipase C-␥ (2). Phosphorylated phospholipase C-␥ hydrolyses phosphatidylinositol 4,5-bisphosphate and liberates inositol 1,4,5-trisphosphate and diacylglycerol, which mobilizes Ca 2ϩ from intracellular and extracellular sources and activates protein kinase C (3), respectively. Whereas these second-messenger generating systems appear to be sufficient for the Fc⑀RI-mediated secretory response (4), how signals initiated by Fc⑀RI aggregation at the plasma membrane are transmitted to the nucleus thereby controlling cytokine gene expression is much less understood.Recently, it has been shown that stimulation of Fc⑀RI in mast cell lines, such as RBL-2H3 cells, leads to the activation of members of the mitogen-activated protein kinase (MAPK) 1 superfamily of serine-threonine kinases. The function of these enzymes is to convert extracellular stimuli to intracellular signals which, in turn, participate in gene expression regulation. In particular, engagement of Fc⑀RI receptors in mast cell lines has been shown to result in the activation of MAPK and JNK (5, 6). In this regard, recently available evidence suggests that engagement of Fc⑀RI with antigen leads to the increased tyrosine phosphorylation of Shc and the association of Shc with Grb2, thus resulting in the recruitment of Sos and the stimulation of the Ras-MAPK pathway. Furthermore, Shc phosphorylation and MAPK activation was shown to be diminished upon overexpression of a dominant negative mutant of Syk, thus suggesting a central role for this kinase in the biochemical route communicating Fc⑀RI to MAPK (5). In contrast, how Fc⑀RI stimulation activates JNK is still unknown.In this study, we thought to dissect the signaling pathway(s) linking Fc⑀RI to MAPK and JNK by reconstructing their respective biochemical routes upon ectopic expression of signaling molecules in COS-7 cells. Using this experimental approach, we provide evidence that whereas Syk and Shc connect Fc⑀RI to the Ras-MAPK pathway, signaling from Fc⑀RI to JNK involves the tyrosine phosphorylation by Syk of a hematopoietic specific guanine-nucleotide exchange factor, Vav, the exchange of GDP for GTP-bound to Rac1, and the consequent stimulation of a kinase cascade leading to JNK activation. MATERIALS AND METHODS RBL-2H3Cell Stimulation-RBL-2H3 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). Before cross-linking of IgE, cells were incubated overnight in DMEM containing 0.1% FBS. Sensitization with anti-trinitrophenyl (TNP) IgE ascites fluid (1:5,000) at 37°C for 2 h a...

The ras-related GTPase rac1 regulates a proliferative pathway selectively utilized by G-protein coupled receptors

“…Control experiments have veri®ed that there is no cross-reactivity of either ligand with either receptor (data not shown) B lymphoma cells, while retransfection of the deleted genes restores responsiveness (Wan et al, 1996;. A direct connection between the IgE Fc (Fcepsilon) receptor and sequential activation of Syk, Vav, rac1 and the JNK pathway has recently been demonstrated (Teramoto et al, 1997). Potential e ectors operating immediately downstream from rac1 include the p21 activated kinase (PAK) which is stimulated upon binding GTP-loaded rac (Manser et al, 1994).…”

“…Potential e ectors operating immediately downstream from rac1 include the p21 activated kinase (PAK) which is stimulated upon binding GTP-loaded rac (Manser et al, 1994). However numerous other e ectors may also respond to rac1 (Tapon and Hall, 1997) and other targets besides PAK may connect rac1 to JNK activation (Gerwins et al, 1997;Teramoto et al, 1996;Burbelo et al, 1995).…”

Rac and Phosphatidylinositol 3-Kinase Regulate the Protein Kinase B in FcεRI Signaling in RBL 2H3 Mast Cells

“…Inhibition by genistein indicate that protein tyrosine kinases participate in activation of Rac. In mast cells, the guanine nucleotide exchange factor Vav is involved in Rac activation (58). Vav was suggested to participate in early signaling events downstream of the Src tyrosine kinase Syk, which causes the tyrosine phosphorylation of Vav, inducing its translocation to the plasma membrane and the activation of Rho GTPases (59).…”

Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential

“…Among them, perhaps the best understood mechanisms are those controlling the conformation and/or localization of the DH-PH domains or their accessibility to bind and stimulate nucleotide exchange on Rho GTPases. This process is best illustrated by Vav1, whose activity is tightly controlled by tyrosine phosphorylation (Crespo et al, 1997;Teramoto et al, 1997). For example, Vav1 is phosphorylated in Tyr174 by Src family tyrosine kinases after cytokine or adhesion receptor activation, and this phosphorylation leads to its activation (Bustelo, 2000).…”