Fig. 5. Mos facilitates activation of MPF in the first meiosis, independently of the MAPK cascade activation. (A) Mos increases the amount of injected GST-cyclin B1 oocytes undergoing GVBD. Oocytes incubated in U0126 were injected with either GST-cyclin B1 (25 µg/ml) alone (B1+U0126), or both GST-cyclin B1 and MBP-Mos (B1+Mos+U0126) in presence or absence of cycloheximide (CHX). Oocytes were scored for GVBD 5 hours after injection. The average of several experiments with different frogs is shown. Oocytes microinjected with MBP-Mos alone (Mos), at the same concentration (20 µg/ml), in absence of U0126 did not undergo GVBD. (B) Oocytes injected with GST-cyclin B1 and MBP-Mos underwent GVBD without activating MAPK. Oocytes microinjected as in A were collected at different times, and analyzed by western blot for diphosphorylated MAPK. Oocytes injected with MBP-Mos alone (Mos) were collected at the end of the experiment. Non-injected immature (stage VI) and progesterone treated mature (control M) oocytes are shown as control. Groups of three oocytes were homogenized and the equivalent of one oocyte was loaded per lane. The band present in all lanes that migrates more slowly than active MAPK corresponds to unspecific staining. (C) Mos facilitates Tyrosine dephosphorylation of Cdc2 in GST-cyclin B1-Cdc2 complexes. Groups of five oocytes treated as in A were collected 1 hour after injection and submitted to GST pull-down assay. The elution of the beads was analyzed by immunoblot with anti-Cdc2 antibodies. The arrow indicates the accumulation of the dephosphorylated form of Cdc2 kinase. Stage VI, one immature oocyte; control M, one progesterone-induced mature oocyte.
