Related Experiment Video
Updated: Jul 15, 2026
Xenopus laevis as a Model to Identify Translation Impairment
Published on: September 27, 2015
p42MAPK-mediated phosphorylation of xEIAP/XLX in Xenopus cytostatic factor-arrested egg extracts
Yuichi Tsuchiya1, Shigeru Yamashita
1Department of Biochemistry, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo, Japan. tsuchiya@med.toho-u.ac.jp <tsuchiya@med.toho-u.ac.jp>
Background:
BIR family proteins are evolutionarily conserved anti-apoptotic molecules. One member of Xenopus BIR family proteins, xEIAP/XLX, is a weak apoptosis inhibitor and rapidly degraded in a cell-free apoptotic execution system derived from interphase egg extracts. However, unfertilized eggs are naturally arrested at the metaphase of meiosis II by the concerted activities of Mos-MEK-p42MAPK-p90Rsk kinase cascade (cytostatic factor pathway) and many mitotic kinases. Previous studies suggest that cytostatic factor-arrested egg extracts are more resistant to spontaneous apoptosis than interphase egg extracts in a p42MAPK-dependent manner. We tested whether xEIAP/XLX might be phosphorylated in cytostatic factor-arrested egg extracts, and also examined whether xEIAP/XLX could be functionally regulated by phosphorylation.
Results:
We found that p42MAPK was the major kinase phosphorylating xEIAP/XLX in cytostatic factor-arrested egg extracts, and three Ser residues (Ser 235/251/254) were identified as p42MAPK-mediated phosphorylation sites. We characterized the behaviors of various xEIAP/XLX mutants that could not be phosphorylated by p42MAPK. However, neither protein stability nor anti-apoptotic ability of xEIAP/XLX was significantly altered by the substitution of Ser with either Ala or Asp at these three sites.
Conclusion:
xEIAP/XLX is physiologically phosphorylated by p42MAPK in Xenopus unfertilized eggs. However, this protein may not serve as an essential mediator of p42MAPK-dependent anti-apoptotic activity.
Insights
Xenopus xEIAP/XLX protein is phosphorylated by p42MAPK in unfertilized eggs. However, this phosphorylation does not significantly affect its stability or anti-apoptotic function, suggesting it
Area of Science:
- Cell Biology
- Molecular Biology
- Apoptosis Research
Background:
- BIR family proteins are conserved anti-apoptotic molecules.
- Xenopus xEIAP/XLX, a BIR protein, is a weak apoptosis inhibitor and unstable in interphase egg extracts.
- Unfertilized Xenopus eggs are arrested in meiosis II by the cytostatic factor (CSF) pathway, exhibiting increased apoptosis resistance.
Purpose of the Study:
- To investigate if xEIAP/XLX is phosphorylated in CSF-arrested egg extracts.
- To determine if phosphorylation regulates the function and stability of xEIAP/XLX.
Main Methods:
- Analysis of xEIAP/XLX phosphorylation in CSF-arrested Xenopus egg extracts.
- Identification of p42MAPK phosphorylation sites on xEIAP/XLX.
- Characterization of xEIAP/XLX mutants with altered phosphorylation sites (Ser to Ala or Asp substitutions).
Main Results:
- p42MAPK was identified as the primary kinase phosphorylating xEIAP/XLX in CSF-arrested egg extracts.
- Three serine residues (Ser 235/251/254) were confirmed as p42MAPK phosphorylation sites.
- Mutating these sites did not significantly alter xEIAP/XLX protein stability or its anti-apoptotic activity.
Conclusions:
- xEIAP/XLX undergoes physiological phosphorylation by p42MAPK in Xenopus unfertilized eggs.
- This p42MAPK-mediated phosphorylation does not appear to be a critical mechanism for xEIAP/XLX's anti-apoptotic function in this context.
