Rap2B-Dependent Stimulation of Phospholipase C-ε by Epidermal Growth Factor Receptor Mediated by c-Src Phosphorylation of RasGRP3

Abstract

Receptor tyrosine kinase regulation of phospholipase C-ε (PLC-ε), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca2+ signaling by the EGF receptor, which activated both PLC-γ1 and PLC-ε, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-ε, and Rap2B-dependent translocation of PLC-ε to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca2+ and expression of lipase-inactive PLC-γ1 but not of PLC-ε. Expression of RasGRP3, a Ca2+/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca2+ signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-γ1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-ε.

Serotonin stimulates GnRH secretion through the c-Src-PLC γ1 pathway in GT1–7 hypothalamic cells

“…In this study, we determined that the Src-mediated activation of PLC g1 and calcium are instrumental in serotonin-mediated GnRH hormone release. Src kinase is considered to be a common mediator of both G protein-coupled receptors and tyrosine kinase-coupled receptors (Haendeler et al 2003, Stope et al 2004. It has also been reported that Src cascades exploit the Gq-PLC pathway for their activation in the process of calcium-dependent pancreatic exocytosis (Tsunoda et al 2004), thereby indicating that Src operates downstream of PLC g1.…”

“…c-Src is involved in serotonin-mediated PLC g1 phosphorylation PLC has been reported to function as a substrate for members of the Src-kinase family (Haendeler et al 2003, Stope et al 2004. It has also been demonstrated that c-Src plays a crucial role in serotonin-mediated calcium responses (Giusti et al 1999, Tolloczko et al 2002, Chang et al 2004, thereby raising the possibility that c-Src may play some role in serotonin signaling.…”

Multiple roles of phosphoinositide-specific phospholipase C isozymes

“…Kataoka et al demonstrated that a PDGF receptor mutant deficient in PLC-γ1 docking site retains its ability to activate PLC-ε (198). However, Schmidt et al suggested that PLC-γ1 can be an upstream regulator of PLC-ε and may exert the effect via activation of RasGRP3 and Rap2B (199).…”

Structure, functional regulation and signaling properties of Rap2B

“…Therefore, Rap2B may regulate a variety of signaling pathways, in addition to cell spreading through its interaction with MAP4K4, MINK, TNIK, PARG1 and RPIP9 (92,93). By contrast, PLC-ε, which is activated by and interacts with Rap2B, facilitates cell growth through activating the Ras-Raf-MAPK/ERK pathway, which increases intracellular Ca +2 levels and activates protein kinase C (75). Therefore, Rap2B may promote the development of tumors through its interaction with PLC-ε.…”

“…The findings from the study by Lopez et al (33) demonstrate that PLC-ε is regulated by the heterotrimeric G protein Gα, and activates the small G protein Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Stope et al (75) suggested that epidermal growth factor receptor triggers the activation of Rap2B via PLCα1 activation and tyrosine phosphorylation of the GEF RasGRP3 by the proto-oncogene tyrosine-protein kinase c-Src, which results in the stimulation of PLC-ε. In 2002, Evellin et al (76) demonstrated that PLC-ε appeared to be stimulated by GPCRs through the formation of cAMP and activation of Rap2B.…”