Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

Abstract

We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca](i) and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca](i), PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca](i) release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca](i) release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization.

Keywords: 1,2-dicapryloyl-sn-glycero-3-phosphate; 1,2-dioctanoyl-sn-glycero-3-[phospho-l-serine]; 5-fluoro-2-indolyl des-chlorohalopemide; DAG; ELSD; Exocytosis; FIPI; IP3; LPA; LPC; Membrane fusion; Membrane rafts; PA; PC; PE; PI; PI3; PI345P3; PI34P2; PI35P2; PI4; PI45P2; PI5; PKC; PLC; PLCγ; PLD; PS; Phospholipase Cγ; Phospholipase D; RT-PCR; S1P; [Ca](i); dPA; dPS; evaporative light scattering detector; inositol 1,4,5-trisphosphate; intracellular calcium; lysophosphatidic acid; lysophosphatidylcholine; phosphatidic acid; phosphatidylcholine; phosphatidylethanolamine; phosphatidylinositol; phosphatidylinositol 3,4,5-trisphosphate; phosphatidylinositol 3,4-bisphosphate; phosphatidylinositol 3,5-bisphosphate; phosphatidylinositol 3-phosphate; phosphatidylinositol 4,5-bisphosphate; phosphatidylinositol 4-phosphate; phosphatidylinositol 5-phosphate; phosphatidylserine; phospholipase C; phospholipase C-γ; phospholipase D; protein kinase C; reverse transcriptase polymerase chain reaction; sn 1,2-diacylglycerol; sphingosine-1-phosphate.