Differential roles of p39Mos–Xp42Mpk1 cascade proteins on Raf1 phosphorylation and spindle morphogenesis in Xenopus oocytes

AbstractFully-grown G2-arrested Xenopus oocytes resume meiosis upon hormonal stimulation. Resumption of meiosis is characterized by germinal vesicle breakdown, chromosome condensation, and organization of a bipolar spindle. These cytological events are accompanied by activation of MPF and the p39Mos–MEK1–Xp42Mpk1–p90Rsk pathways. The latter cascade is activated upon p39Mos accumulation. Using U0126, a MEK1 inhibitor, and p39Mos antisense morpholino and phosphorothioate oligonucleotides, we have investigated the role of the members of the p39Mos–MEK1–Xp42Mpk1–p90Rsk in spindle morphogenesis. First, we have observed at a molecular level that prevention of p39Mos accumulation always led to MEK1 phosphorylation defects, even when meiosis was stimulated through the insulin Ras-dependent pathway. Moreover, we have observed that Raf1 phosphorylation that occurs during meiosis resumption was dependent upon the activity of MEK1 or Xp42Mpk1 but not p90Rsk. Second, inhibition of either p39Mos accumulation or MEK1 inhibition led to the formation of a cytoplasmic aster-like structure that was associated with condensed chromosomes. Spindle morphogenesis rescue experiments using constitutively active Rsk and purified murine Mos protein suggested that p39Mos or p90Rsk alone failed to promote meiotic spindle organization. Our results indicate that activation of the p39Mos–MEK1–Xp42Mpk1–p90Rsk pathway is required for bipolar organization of the meiotic spindle at the cortex