Activation of Wee1 by p42 MAPK In Vitro and in CyclingXenopusEgg Extracts

Abstract

Xenopus oocytes and eggs provide a dramatic example of how the consequences of p42 mitogen-activated protein kinase (p42 MAPK) activation depend on the particular context in which the activation occurs. In oocytes, the activation of Mos, MEK, and p42 MAPK is required for progesterone-induced Cdc2 activation, and activated forms of any of these proteins can bring about Cdc2 activation in the absence of progesterone. However, in fertilized eggs, activation of the Mos/MEK/p42 MAPK pathway has the opposite effect, inhibiting Cdc2 activation and causing a G2 phase delay or arrest. In the present study, we have investigated the mechanism and physiological significance of the p42 MAPK-induced G2 phase arrest, using Xenopus egg extracts as a model system. We found that Wee1-depleted extracts were unable to arrest in G2 phase in response to Mos, and adding back Wee1 to the extracts restored their ability to arrest. This finding formally places Wee1 downstream of Mos/MEK/p42 MAPK. Purified recombinant p42 MAPK was found to phosphorylate recombinant Wee1 in vitro at sites that are phosphorylated in extracts. Phosphorylation by p42 MAPK resulted in a modest ( approximately 2-fold) increase in the kinase activity of Wee1 toward Cdc2. Titration experiments in extracts demonstrated that a twofold increase in Wee1 activity is sufficient to cause the delay in mitotic entry seen in Mos-treated extracts. Finally, we present evidence that the negative regulation of Cdc2 by Mos/MEK/p42 MAPK contributes to the presence of an unusually long G2 phase in the first mitotic cell cycle. Prematurely inactivating p42 MAPK in egg extracts resulted in a corresponding hastening of the first mitosis. The negative effect of p42 MAPK on Cdc2 activation may help ensure that the first mitotic cell cycle is long enough to allow karyogamy to be accomplished successfully.

Cyclin B Dissociation from CDK1 Precedes its Degradation Upon MPF Inactivation in Mitotic Extracts of Xenopus laevis Embryos

“…7 in ref. 44). Experimental activation of ERK2 to high levels during mitosis, however, stabilizes cyclin B and MPF activity arresting embryos and cell-free extracts in M-phase.…”

MAPK Pathway Activation Delays G2/M Progression by Destabilizing Cdc25B

“…The discovery that MEK1 but not MEK2 depletion blocked the EGFand TPA-induced destabilization of cdc25B and G 2 phase delay clearly demonstrates the involvement of MEK1 in this mechanism. In Xenopus extracts, MAPK activation causes a G 2 phase delay, attributed to ERK phosphorylation of Wee1, and phosphorylation of cdc25C by p90RSK, a downstream effector of ERK activity (42,43). There is contradictory evidence of a role for MAPK signaling in G 2 and mitosis in mammalian cells (5,6,8), but recent evidence suggests that some of this maybe attributed to cell lineage-dependent differences (44).…”

Starfish andXenopusoocyte maturation

“…At the same time the cap at its 5' end becomes methylated (Kuge et al 1998). The Pp39-mos protein produced at this point activates a MAPKKK which activates a MAPKK, which in turn activates a MAPK (Muslin et al 1993;Matten et al 1996;Radziwill et al 1996;Barkoff et al 2000;Ranginwale et al 2001;Walter et al 2000;Frank-Vaillant et al 2001) that with the aid of Hsp70 and Hsp90 (Fisher et al 2000) activates the Cdc-25 phosphatase. This finally activates the MPF (De Smedt et al 2002), thus inducing histone phosphorylation and germinal vesicle breakdown (GVBD) (Sagata et al 1988).…”