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Tr-kit-induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase
Claudio Sette1, Maria Paola Paronetto, Marco Barchi
1Department of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata, Rome, Italy. claudio.sette@uniroma2.it
Abstract:
Microinjection in mouse eggs of tr-kit, a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa, causes resumption of meiosis through activation of phospholipase Cgamma1 (PLCgamma1) and Ca(2+) mobilization from intracellular stores. We show that the Src-like kinase Fyn phosphorylates Tyr161 in tr-kit and that this residue is essential for tr-kit function. Fyn is localized in the cortex region underneath the plasma membrane in mouse oocytes. Using several approaches, we demonstrate that Fyn associates with tr-kit and that the interaction requires Tyr161. The interaction between tr-kit and Fyn triggers activation of the kinase as monitored by both autophosphorylation and phosphorylation of PLCgamma1. Co-injection of tr-kit with the SH2 domain of Fyn, or pre-treatment with a Fyn inhibitor, impairs oocyte activation, suggesting that activation of Fyn by tr-kit also occurs in vivo. Finally, microinjection of constitutively active Fyn triggers oocyte activation downstream of tr-kit but still requires PLC activity. We suggest that the mechanism by which tr-kit triggers resumption of meiosis of mouse eggs requires a functional interaction with Fyn and phosphorylation of PLCgamma1.
Insights
The Src-like kinase Fyn phosphorylates tr-kit, a truncated c-kit protein, initiating mouse egg activation. This interaction is crucial for resuming meiosis via phospholipase Cgamma1 activation and calcium release.
Area of Science:
- Reproductive biology
- Molecular cell biology
- Biochemistry
Background:
- Mouse oocyte activation involves signaling pathways initiated by sperm.
- The truncated c-kit receptor (tr-kit) in sperm is implicated in initiating meiosis resumption.
- Phospholipase Cgamma1 (PLCgamma1) and calcium (Ca2+) mobilization are key events in oocyte activation.
Purpose of the Study:
- To elucidate the role of the Src-like kinase Fyn in tr-kit-mediated mouse egg activation.
- To determine the specific interaction between Fyn and tr-kit and its functional consequences.
- To investigate the signaling cascade involving Fyn, tr-kit, and PLCgamma1.
Main Methods:
- Microinjection of tr-kit and Fyn-related components into mouse eggs.
- Western blotting to detect protein phosphorylation (autophosphorylation and PLCgamma1 phosphorylation).
- Use of Fyn inhibitors and Fyn SH2 domain to assess functional requirements.
Main Results:
- Fyn phosphorylates Tyr161 on tr-kit, a residue essential for its function.
- Fyn associates with tr-kit, and this interaction activates Fyn kinase activity.
- Inhibition of Fyn or disruption of the Fyn-tr-kit interaction impairs oocyte activation.
- Activated Fyn can trigger oocyte activation downstream of tr-kit, but PLC activity remains necessary.
Conclusions:
- The interaction between tr-kit and Fyn is essential for initiating mouse egg activation.
- Fyn-mediated phosphorylation of tr-kit and subsequent PLCgamma1 activation are critical steps in the signaling pathway.
- This study reveals a novel mechanism for sperm-egg interaction involving Fyn and tr-kit.