Induction of a G₂-Phase Arrest inXenopusEgg Extracts by Activation of p42 Mitogen-activated Protein Kinase

Abstract

Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G2-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G2-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G2 arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G2-phase during the first mitotic cell cycle.

Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

“…In mammalian somatic cells, the fate of wee1 is less clear: some studies found that wee1 protein was reduced during mitosis (Baldin and Ducommun, 1995;Watanabe et al, 1995), whereas others found that it was stable (McGowan and Russell, 1995;Parker et al, 1995). In Xenopus egg extracts, immunoblots of endogenous wee1 show that overall wee1 protein levels are essentially constant across the early embryonic cell cycles (Walter et al, 1997), a point that we confirm here. However, two highly cited reports concluded that wee1 is degraded in the early embryonic cell cycles (Michael and Newport, 1998;Ayad et al, 2003).…”

Aurora A, mitotic entry, and spindle bipolarity

“…First, addition of Aur-A protein during interphase does not lead to a detectable increase in Mos (H. Wu and J.V.R., unpublished data). Second, addition of Mos or MAP kinase blocks mitotic entry rather than accelerating it (36,37). CPEB-dependent translation of cyclin B1 is required for mitotic entry in egg extracts (38) and overexpression of Aur-A enhances polyadenylation of cyclin mRNA in somatic cells (17).…”

Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2Phase and Promotes the G2/M Cell Cycle Transition

“…In mitotic Xenopus egg extracts, MEK is active and required for normal mitotic progression (Abrieu et al, 1997;Walter et al, 1997;Bitangcol et al, 1998;Murakami and Vande Woude, 1998). MEK inhibition causes defects in both spindle formation (Horne and Guadagno, 2003) and spindle checkpoint control (Minshull et al, 1994;Takenaka et al, 1997;Chung and Chen, 2003).…”