Evidence for the Involvement of a Src-Related Tyrosine Kinase inXenopusEgg Activation

Abstract

Recently, we have purified a Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), from oocytes of Xenopus laevis and found that the enzyme is activated within 1 min following fertilization [Sato et al. (1996) J. Biol. Chem. 271, 13250-13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996) Dev. Biol. 177, 558-567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner independent or upstream of calcium signaling.

Ca2+ signaling differentiation during oocyte maturation

“…In Xenopus fertilization induces the activation of Src-related protein tyrosine kinases (PTK), which induces PLCg activation through an unknown mechanism (Sato et al, 1999(Sato et al, , 2000Tokmakov et al, 2002). 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).…”

Mammalian Fertilization: From Sperm Factor to Phospholipase Cζ

“…Activation of SFKs occurs soon after fertilization in sea urchin, zebrafish and Xenopus eggs (Kinsey, 1996;Sato et al, 1999;Abassi et al, 2000;Wu and Kinsey, 2000), and recent studies have suggested that activation of SFK(s) is required for sperm-induced [Ca 2+ ] i release in sea urchin, starfish, ascidian, and Xenopus eggs (Giusti et al, 1999Abassi et al, 2000;Runft and Jaffe, 2000;Sato et al, 2000). For instance, inhibition of SFK signaling by injection of the SFK SH2 domain, or by various SFK inhibitors, prevented fertilization-associated activation of PLCc and [Ca 2+ ] i release in starfish and Xenopus eggs, and, consistent with these findings, activation of the egg SFK(s) was sufficient to induce PLCc activation and subsequent [Ca 2+ ] i release in these species Sato et al, 2000Sato et al, , 2003.…”

Focused Proteomics on Egg Membrane Microdomains to Elucidate the Cellular and Molecular Mechanisms of Fertilization in the African Clawed Frog Xenopus laevis

“…In that paper, we showed data about chromatographic fractionation of membrane-associated proteins and in vitro protein kinase assay, by which we could detect an elevation of the activity of xSrc in response to fertilization. Our further studies demonstrated that pharmacological (e.g., use of inhibitors; Sato et al 1999Sato et al , 2000Sato et al , 2001 or molecular biological inhibition of xSrc (i.e., expression of kinase-negative mutant of xSrc; manuscript in preparation) impairs the ability of Xenopus eggs to undergo calcium reactions and egg activation in response to sperm and that Xenopus eggs can be activated in a Src-dependent manner by artifi cial egg activators that interacts with the egg surface (i.e., RGD peptide and cathepsin B; Sato et al 1999 ;Mahbub Hasan et al 2005 ) and by hydrogen peroxide that may directly activate xSrc (Sato et al 2001 ). These results suggest that xSrc acts between gamete interaction/fusion and an increase in intracellular calcium concentration at fertilization ( Fig.…”