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p42MAPK-mediated phosphorylation of xEIAP/XLX in Xenopus cytostatic factor-arrested egg extracts.
Tsuchiya Y
,
Yamashita S
.
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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.
Figure 1. Phosphorylation-dependent electrophoretic mobility shift of xEIAP/XLX in CSF-arrested egg extracts. (A) Schematic models of recombinant proteins used in this study. (B) Time-dependent changes of recombinant proteins incubated in CSF-arrested (CSF) and interphase (INT) egg extracts. 35S-radiolabeled 6XHis-FL, MBP-FL, MBP-Î1, and MBP-Î2 were mixed with either CSF-arrested or interphase egg extracts and incubated for indicated time periods (0, 2, and 4 h). After separation by SDS-PAGE, radiolabeled and MBP-fused recombinants were detected by image analyzer and Western blot, respectively. Molecular weights of standard proteins are indicated at the left. The larger (~70 k) and smaller cleavage products (50â55 k) of MBP-fusions are indicated by single and double asterisks, respectively. (C) Phosphorylation-dependent electrophoretic mobility shift of xEIAP/XLX. Endogenous xEIAP/XLX in CSF-arrested egg extracts were retrieved by antibody-coated beads and incubated with (+) or without (-) calf intestine alkaline phosphatase. After separation by SDS-PAGE, xEIAP/XLX was detected by Western blot. Positions of phosphorylated (P-xEIAP) and unphosphorylated (xEIAP) forms of xEIAP/XLX are indicated by arrowheads. The position of IgG heavy chain (IgG HC) is indicated by the line.
Figure 2. Phosphorylation of xEIAP/XLX by p42MAPK in CSF-arrested egg extracts. (A) The effects of kinase inhibitors. MBP-Î1 was incubated in CSF-arrested (CSF) and interphase (INT) egg extracts, respectively, with or without the indicated kinase inhibitors (R, 100 μM roscovitine; U, 100 μM U0126; S, 10 μM staurosporine) for indicated time periods (0 and 2 h). After separation by SDS-PAGE, MBP-Î1 was detected by Western blot. (B) Phosphorylation of xEIAP/XLX by p42MAPK and CSF-arrested egg extracts. MBP-Î1, MBP-Î2, and control substrates Histone H1 and myelin basic protein (Myelin BP) were incubated with Cdc2/Cyclin B2 (Cdc2), p42MAPK (MAPK), CSF-arrested (CSF) egg extracts, or interphase (INT) egg extracts in the presence of [γ-32P] ATP. After separation by SDS-PAGE, incorporated radioactivity was detected by image analyzer.
Figure 3. p42MAPK phosphorylates Ser235/251/254 of xEIAP/XLX. (A) Amino acid sequence of xEIAP/XLX residues 219â269. Indicated three Ser residues are changed to either Ala or Asp. (B) Phosphorylation of WT, Ser235Ala single mutant (1A), Ser251/254Ala double mutant (2A), and Ser235/251/254Ala triple mutant (3A). (C) Phosphorylation of WT, 3A, and Ser235/251/254Asp triple mutant (3D). In (B) and (C), MBP-Î1 variants were incubated with CSF-arrested egg extracts (CSF) or p42MAPK (MAPK) in the presence of [γ-32P] ATP. After separation by SDS-PAGE, incorporated radioactivity was detected by image analyzer. Same amounts of substrates were also stained with Coomassie Brilliant Blue (Input). In (B), the phosphorylation intensities were calculated using NIH ImageJ software and indicated below.
Figure 4. Phosphorylation of Ser235/251/254 affects neither protein stability nor apoptosis-inhibiting activity of xEIAP/XLX. (A) Stability and electrophoretic mobility shift of xEIAP/XLX. The WT/3A/3D forms of 35S-radiolabeled 6XHis-FL, MBP-FL, and MBP-Î1 were incubated in CSF-arrested (CSF) or interphase (INT) egg extracts for indicated time periods (0, 2, and 4 h). After separation by SDS-PAGE, radiolabeled and MBP-fused recombinants were detected by image analyzer and Western blot, respectively. Molecular weights of standard proteins are indicated at the left. The larger (~70 k) and smaller cleavage products (50â55 k) of MBP-fusions are indicated by single and double asterisks, respectively. (B) Apoptosis inhibition by MBP-FL. (C) Apoptosis inhibition by MBP-Î1. In (B) and (C), nuclear fragmentation assays were carried out as previously described [11]. Open circles, MBP (negative control); closed circles, WT; open triangles, 3A; closed triangles, 3D; open squares, MBP-xXIAP (positive control). Data are represented as mean + S.E.M. (N = 5).
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