p42MAPK-mediated phosphorylation of xEIAP/XLX in Xenopus cytostatic factor-arrested egg extracts
© Tsuchiya and Yamashita; licensee BioMed Central Ltd. 2007
Received: 06 October 2006
Accepted: 11 April 2007
Published: 11 April 2007
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.
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.
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.
In various animal species including Xenopus, ovulated mature eggs have to survive without the support of surrounding follicle cells until successful fertilization. In contrast with the long life of immature oocytes in ovary, the life of ovulated mature eggs is limited to only a few days. Many reports indicate that aged eggs without fertilization or parthenogenetically activated eggs eventually die by apoptosis [reviewed in [1–3]]. Although the exhaustion of nutrients can contribute to oocyte/egg apoptosis , the mechanism of this machinery is still poorly understood.
The translation of Mos protein kinase begins during oocyte maturation and automatically activates Mos-MEK-ERK (p42MAPK in Xenopus oocyte)-p90Rsk kinase cascade. This is called CSF (cytostatic factor) pathway because its primary role is to arrest the cell cycle until fertilization [reviewed in [5, 6]]. In vertebrates, CSF arrests mature eggs at the metaphase of meiosis II, and many mitotic kinases including Cdc2/cyclin B are also kept active. Recent studies suggest that CSF pathway also regulates apoptosis [7–11], but the exact targets are largely unknown.
Baculovirus IAP repeat (BIR) family proteins are evolutionarily conserved zinc-coordinating proteins, and some members inhibit apoptosis by blocking caspase activities [reviewed in [12, 13]]. We recently identified four BIR family proteins in Xenopus eggs and examined their apoptosis-inhibiting activities using a cell-free system derived from interphase egg extracts . Whereas xXIAP was a physiological apoptosis inhibitor, xEIAP (identical with XLX reported by Holley et al. ) only weakly inhibited apoptosis, and neither xSurvivin1/xBIR1 nor xSurvivin2/SIX showed anti-apoptotic activities. However, both CSF and mitotic kinases are inactive in interphase egg extracts, and we wondered whether BIR family proteins might be functionally regulated by phosphorylation in CSF-arrested egg extracts. We found that p42MAPK directly phosphorylated xEIAP/XLX on three Ser residues in the Ser-rich region between BIR2 and RING finger domains in CSF-arrested egg extracts. The effects of phosphorylation on the stability and anti-apoptotic activity of xEIAP/XLX were also examined.
Results and Discussion
Phosphorylation-dependent electrophoretic mobility shift of xEIAP/XLX in CSF-arrested egg extracts
Phosphorylation of xEIAP/XLX by p42MAPK in CSF-arrested egg extracts
p42MAPK phosphorylates Ser235/251/254 of xEIAP/XLX
Phosphorylation of Ser235/251/254 affects neither protein stability nor apoptosis-inhibiting activity of xEIAP/XLX
Our data indicate that, although xEIAP/XLX is a physiologically phosphorylatable substrate for p42MAPK, it may not be a direct mediator of p42MAPK-dependent anti-apoptotic activity in CSF-arrested egg extracts. One possible role of xEIAP/XLX might be to titrate or ubiquitylate pro-apoptotic molecules, thereby indirectly supporting the anti-apoptotic role of xXIAP [19–23]. Otherwise, xEIAP/XLX could regulate the abundance of xXIAP [24–26] or ubiquitylate apoptotic signal transducers [22, 27–30]. Further studies to address these issues are currently in progress. During the preparation of this manuscript, Greenwood and Gautier also reported that xEIAP/XLX is phosphorylated mainly by MAPK during meiosis .
Preparation of recombinant proteins
Vector construction, bacterial expression and affinity purification of maltose binding protein (MBP)-fused recombinant proteins were previously described . In vitro translation of 35S-radiolabeled 6XHis-tagged recombinant proteins in rabbit reticulocyte lysates using TnT T7 Quick (Promega, Tokyo, Japan) and Pro-Mix (GE Healthcare, Tokyo, Japan) was carried out according to manufacture's instructions. Site-directed mutagenesis was performed using QuikChange (Stratagene, CA, USA) and confirmed by DNA sequencing.
Preparation of Xenopus egg extracts
Preparations of CSF-arrested, interphase, and apoptotic egg extracts were previously described [11, 15–18]. Where indicated, roscovitine (Calbiochem-Merck, Tokyo, Japan), U0126 (Sigma-Aldrich, Tokyo, Japan), and staurosporine (Sigma-Aldrich) were supplied to egg extracts.
Protein stability assay
MBP-fused recombinants were added to egg extracts at 1 μg/ml, whereas rabbit reticulocyte lysates containing radiolabeled recombinants were mixed with egg extracts at 1:9. After incubation, samples were resolved by SDS-PAGE, and remaining MBP-fused and radiolabeled recombinants were detected by Western blot using anti-MBP antiserum (New England Biolabs, MA, USA) and by BAS-5000 image analyzer (Fuji Film, Tokyo, Japan), respectively.
Protein phosphorylation assay
Recombinant activated p42MAPK was purchased from New England Biolabs. Active Cdc2/Cyclin B2 complex was immunoprecipitated from CSF-arrested egg extracts by affinity-purified anti-Xenopus Cyclin B2 antibody immobilized on Affi-Prep Protein A beads (Bio-Rad, Tokyo, Japan). Histone H1 and myelin basic protein were purchased from Roche (Tokyo, Japan) and Sigma-Aldrich, respectively. Kinase assay using purified p42MAPK and Cdc2/Cyclin B2 was performed in 10 μl of Assay Buffer (10 mM HEPES-KOH, pH 7.7, 15 mM MgCl2, 1 mM DTT) containing respective kinase, 1 μg of substrate protein, 100 μM cold ATP, and 37 kBq of [γ-32P] ATP (GE Healthcare). For phosphorylation assay by egg extracts, either CSF-arrested or interphase egg extracts were first diluted 5-fold with MEB-TX (20 mM HEPES-KOH, pH 7.7, 15 mM MgCl2, 80 mM sodium glycerol 2-phosphate, 20 mM EGTA, 1 mM DTT, 0.2 mM phenylmethanesulfonyl fluoride, 0.1% Triton X-100). Diluted extracts were then supplied with 1 μg of substrate protein and 37 kBq of [γ-32P] ATP in 10 μl. After the reaction at 30°C for 30 min, samples were resolved by SDS-PAGE and analyzed with image analyzer.
Dephosphorylation of endogenous xEIAP/XLX
Affinity-purified anti-xEIAP/XLX antibody was first immobilized on Affi-Prep Protein A beads and then mixed with CSF-arrested egg extracts to retrieve endogenous xEIAP/XLX. After extensive washing, the xEIAP/XLX-loaded beads in Assay Buffer were incubated with calf intestine alkaline phosphatase (Roche) overnight at 30°C, followed by resolution by SDS-PAGE and Western blot with the same antibody.
Apoptosis inhibition assay
Recombinant proteins were added to interphase egg extracts at 10 μg/ml, and apoptotic nuclear fragmentation was observed as previously described .
We thank the members of our lab for helpful discussions. This work was supported by Project Research Grant No. 17–23 from Toho University School of Medicine and in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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