p42MAPK-mediated phosphorylation of xEIAP/XLX in Xenopus cytostatic factor-arrested egg extracts.
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.


CONCLUSIONS
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.
Tsuchiya
Yuichi
Y
Department 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>
Yamashita
Shigeru
S
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D002457:Cell Extracts; D024202:Electrophoretic Mobility Shift Assay; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017231:Proto-Oncogene Proteins c-mos / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism
D002457:Cell Extracts; C519842:EIAP protein, Xenopus; D010750:Phosphoproteins; D029867:Xenopus Proteins; D010768:Phosphoserine; D017231:Proto-Oncogene Proteins c-mos; D019950:Mitogen-Activated Protein Kinase 1