Calcium Release at Fertilization in Starfish Eggs Is Mediated by Phospholipase Cγ

Abstract

Although inositol trisphosphate (IP3) functions in releasing Ca2+ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP3. To distinguish between a role for PLCγ, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCβ, which is activated by a G protein, we injected starfish eggs with a PLCγ SH2 domain fusion protein that inhibits activation of PLCγ. In these eggs, Ca2+ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCγSH2 protein is a specific inhibitor of PLCγ in the egg, since it did not inhibit PLCβ activation of Ca2+ release initiated by the serotonin 2c receptor, or activation of Ca2+ release by IP3 injection. Furthermore, injection of a PLCγ SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca2+ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cγ by an SH2 domain-mediated process stimulates the production of IP3 that causes intracellular Ca2+ release.

Ca2+ signaling differentiation during oocyte maturation

“…However, PLCg activation in Xenopus does not involve SH2-domain dependent recognition of phospho-tyrosine residues (Runft et al, 1999), in contrast to starfish for example (Carroll et al, 1997). Therefore, how Srcrelated PTK activate PLCg remains vague at this point.…”

Mammalian Fertilization: From Sperm Factor to Phospholipase Cζ

“…The SH2 domains recognize tyrosine-phosphorylated proteins, including active tyrosine kinases that interact with and activate PLCc isozymes (Margolis et al, 1989;Meisenhelder et al, 1989;Rhee, 2001). PLCc isozymes are thought to operate in ascidian and echinoderm fertilization, as it was shown that injection of excess PLCc SH2 domain, acting in a dominant negative manner, prevented sperm-induced [Ca 2+ ] i release in eggs of these species (Carroll et al, 1997Shearer et al, 1999;Runft and Jaffe, 2000). On the contrary, using a similar approach, it was shown that [Ca 2+ ] i release in Xenopus and mouse fertilization occurred unperturbed in the presence of excess SH2 domains, suggesting that PLCc activation is not important in these species, or that it occurs by a different mechanism Runft et al, 1999).…”

Calcium signalling in early embryos

“…But the inactivation of the NAADP pathway inhibits membrane currents (Churchill et al 2003;Moccia et al 2004), not the fertilization calcium wave (Churchill et al 2003). In starfish, the fertilization wave is prevented by an InsP 3 chelator and by SH2 domains directed against Src and PLCg (Carroll et al 1997;Giusti et al 1999b), but equally NAADP can mobilize calcium even when the InsP 3 pathway is downregulated (Lim et al 2001).…”

“…The Src kinase stimulates PLCg to produce InsP 3 (Giusti et al 1999a(Giusti et al , 2000bShearer et al 1999;Jaffe et al 2001;Runft Linda et al 2004). SH2 domains inhibitory to PLCg and Src-family kinases will block fertilization in sea urchins, starfish, zebrafish and ascidians (Carroll et al 1997(Carroll et al , 1999Runft et al 1999Runft et al , 2002Shearer et al 1999;Rongish & Kinsey 2000;Kinsey William et al 2003), but not in mammals or frogs (Mehlmann et al 1998;Mehlmann & Jaffe 2005). Nonetheless, in frogs, Src-related tyrosine kinase co-immunoprecipitates with PLCg after fertilization and complex formation is blocked by the protein tyrosine kinase inhibitor PP1 (Sato et al 2000).…”