Figure 4. Analysis of SmSak activity in Xenopus oocytes.

A. Constitutively active SmSak induces germinal vesicle breakdown (GVBD) and meiosis progression in Xenopus oocytes. Different samples of oocytes (n = 15) were microinjected with cRNA encoding SmSak, SmSak^DK, SmSakT₁₇₅D, SmSakT₁₇₅D^DK or SmSakT₁₇₅A. Percentages of oocytes exhibiting GVBD 18 h after microinjection of cRNA are indicated. Non-injected oocytes were used as control. Western blot analysis of oocyte extracts was performed as described in materials and methods to detect the gel-shift of endogenous phosphorylated Plx1 and Cdc25C proteins (indicated by arrows). The expression of the different forms of V5-tagged recombinant SmSak was confirmed by the use of anti-V5 antibodies. B. SmSak mutants do not alter the GVBD induced by progesterone. The experiment was performed as in A, except that PG was added to the different oocyte groups two hours after microinjection of cRNA preparations. Gel-shifts of Plx1 and Cdc25C are observed in all oocyte groups associated with high levels of GVBD. C. Endogenous Plx1 is required for the induction of GVBD by SmSakT₁₇₅D. Oocytes were injected or not with 5 ng anti-Plx1 antibodies one hour before the injection of 10 or 60 ng SmSak₁₇₅D cRNA. Meiosis resumption was monitored as in A and B. SmSakT₁₇₅D induces GVBD (as already shown in A) following the injection of 60 ng cRNA but has no more effect in Plx1-depleted oocytes. In control oocytes, it was confirmed that anti-Plk1 antibodies blocked completely the GVBD induced by PG. D. Comparative analysis of the capacity of SmSak and SmPlk1 to induce GVBD in different conditions. Normal versus Plx1-depleted oocytes were incubated with or without (w/o) PG following the expression of SmPlk1, SmPlkT₁₈₂D, SmSak or SmSakT₁₇₅D. PG stimulates GVBD in the oocytes expressing each of the parasite kinases, provided that Plx1 is functional in oocytes. In Plx1-depleted oocytes, only SmPlk1 proteins are efficient. In the absence of PG, only constitutively active kinases are efficient, provided that Plx1 is functional. In Plx1-depleted oocytes, only SmPlkT₁₈₂D can induce GVBD. These results were correlated with our previous data [23] and those presented in A, B and C. All percentages of GVBD represent the mean of three independent experiments. In A, B and C, blots are representative for the three experiments.