BIOLOGY

Identification of a starfish egg PLC-g



Identification of a starfish egg PLC-g

Introduction

At fertilization, eggs undergo a cytoplasmic free Ca2+ rise, which is necessary for stimulating embryogenesis. In starfish eggs, studies using inhibitors designed against vertebrate proteins have shown that this Ca2+ rise requires an egg Src family kinase (SFK) that directly or indirectly activates phospholipase C-gamma (PLC-gamma) to produce IP3, which triggers Ca2+ release from the egg's endoplasmic reticulum (ER) [reviewed in Semin. Cell Dev. Biol. 12 (2001) 45]. To examine in more detail the endogenous factors in starfish eggs that are required for Ca2+ release at fertilization, an oocyte cDNA encoding PLC-gamma was isolated from the starfish Asterina miniata.

This cDNA, designated AmPLC-gamma, encodes a protein with 49% identity to mammalian PLC-gamma1. A 58-kDa Src family kinase interacted with recombinant AmPLC-gamma Src homology 2 (SH2) domains in a specific, fertilization-responsive manner. Immunoprecipitations of sea urchin egg PLC-gamma using an affinity-purified antibody directed against AmPLC-gamma revealed fertilization-dependent phosphorylation of PLC-gamma. Injecting starfish eggs with the tandem SH2 domains of AmPLC-gamma (which inhibits PLC-gamma activation) specifically inhibited Ca2+ release at fertilization. These results indicate that an endogenous starfish egg PLC-gamma interacts with an egg SFK and mediates Ca2+ release at fertilization via a PLC-gamma SH2 domain-mediated mechanism.

Discussion

At fertilization, the sperm initiates a propagated rise of Ca2+ in the egg, which is of central importance in activating the egg to begin development (Jaffe, 1985; Whitaker and Steinhardt, 1985; Kline, 1988). In echinoderm as well as vertebrate eggs, the rise in Ca2+ results, at least in large part, from Ca2+ release from the endoplasmic reticulum in response to a rise in inositol trisphosphate (IP31; Whitaker and Irvine, 1984; Ciapa and Whitaker, 1986; Miyazaki et al., 1992; Mohri et al., 1995; Jaffe, 1996). However, it has not been established how the IP3 is generated at fertilization.

IP3 is produced from phosphatidylinositol bisphosphate (PIP2), by the action of a phospholipase C (PLC; Rhee and Choi, 1992). This family of enzymes includes ß, d, and ? isoforms. PLCß is activated by G proteins, while PLC? is activated by tyrosine kinases. The regulation of PLCd is poorly understood, although the enzymatic activity of all 3 PLC isoforms can be stimulated by an increase in Ca2+ (Park et al., 1992; Wahl et al., 1992; Banno et al., 1994). Very likely the generation of IP3 at fertilization results from the activation of one of these isoforms of phospholipase C.

Both PLCß and PLC? pathways are present in eggs. Expression in frog, mammalian, and starfish eggs of exogenous receptors known to release Ca2+ by a G protein/PLCß pathway, such as serotonin 2c or muscarinic m1 receptors, allows Ca2+ release in eggs when the corresponding agonists are applied (Kline et al., 1988; Williams et al., 1992; Shilling et al., 1994). This indicates that functional PLCß and corresponding G proteins are present. Likewise, expression in frog and starfish eggs of exogenous receptors known to release Ca2+ by a tyrosine kinase/PLC? pathway, such as receptors for EGF or PDGF, allows Ca2+ release in response to ...