SAC proteins were identified in budding yeast mutants that lost ability to metaphase arrest after addition of spindle poisons [29
]. SAC proteins function in arresting cells in metaphase by inhibiting the APC/C until all chromosomes are biorientated and so under tension from spindle microtubules (reviews see [31
]). Vertebrate homologues of the SAC proteins Bub1 (Budding uninhibited by benzimidazole 1) Mad1 and Mad2 (Metaphase arrest deficient 1 and 2) have been suggested to affect MetII arrest in Xenopus
eggs. Immunodepletion of these SAC proteins from egg extracts have all been demonstrated to block CSF arrest [34
SAC components have been implicated as the downstream effectors the c-Mos/MEK/MAPK/p90rsk pathway, long thought to be essential for frog CSF arrest. c-Mos (pp39mos), a proto-oncogene from a family of kinases functioning in signal transduction regulating cell growth and differentiation [36
], is highly expressed during germ cell maturation, and has proposed roles throughout frog oocyte maturation [37
]. Functioning as a MAPK kinase kinase (MEKK), c-Mos is important for activation of the MAPK kinase, MEK1 [44
]. MEK1 serves as the upstream activator of MAPK [47
], which switches on the 90-kD ribosomal protein S6 kinase (p90rsk [50
]). At fertilization c-Mos is degraded [43
], whilst MEK1, MAPK and p90rsk are inactivated shortly afterwards [51
The c-Mos ...p90rsk signaling cascade has been shown to aid directly MPF activation and stabilization [53
] making it an ideal CSF candidate. Microinjection of c-Mos RNA into two-cell embryos results in metaphase arrest, and immunodepletion of c-Mos causes a loss of cleavage-arresting activity [43
]. Similarly, an injection of an active form of MAPK [56
] or constitutively-active rsk [57
], into blastomeres of two-cell embryos arrests the injected blastomere in metaphase. Indeed in frog, p90rsk has been suggested to be the only MAPK substrate needed for cyclin B re-accumulation on entry to MetII, MetII spindle formation, and CSF arrest [57
]. This is supported by the fact that c-Mos protein is unable to establish CSF arrest in frog egg extracts immunodepleted of p90rsk [59
The SAC component Bub1 is phosphorlyated and activated by p90rsk [60
]. Bub1 [34
], Mad1 and Mad2 [35
] all appear to be required downstream of c-Mos given that the immunodepletion of these proteins blocked the establishment of CSF arrest by c-Mos in frog egg extracts. Such studies suggest a model in which CSF arrest by c-Mos is mediated by Bub1, Mad1 and Mad2 proteins.
From the above it appears that a well-defined CSF pathway has been identified in frog. However, studying SAC components maybe somewhat misleading with respect to identifying CSF. Although CSF activity and the SAC are similar in being able to induce metaphase arrest through APC/C inhibition, they may use different signalling pathways. Any arrest must be reversed by Ca2+
to prove physiological relevance with respect to CSF. A further issue is how CSF arrest can be achieved at MetII but not at MI metaphase (MetI) since many components of the c-Mos pathway are present and active at MetI. For example, c-Mos, MAPK, p90rsk and Bub1 are essential for suppression of S-phase between meiotic divisions [43
] yet do not block eggs at MetI. A possible explanation is the involvement of cyclin E/cdk2, both of which are synthesized during MII [63
] and inhibit the APC/C.
In frog eggs cyclin E/Cdk2 activity has been reported to play an essential role in CSF arrest. Cyclin E/Cdk2, like c-Mos, can establish metaphase arrest in egg extracts [34
]. Cdk2 antisense prevents CSF arrest [64
] and recombinant cyclin E/Cdk2 causes metaphase arrest in egg extracts even in the absence of c-Mos [34
]. The two pathways (c-Mos and cyclin E/cdk2) are therefore suggested to be independent of each other but both appear to inhibit the APC/C. CSF activity therefore may result from the coexpression of cyclin E/Cdk2 with the c-Mos/MEK/MAPK/p90rsk pathway. However, the role of cyclin E/cdk2 in CSF arrest remains to be fully elucidated since inhibiting cdk2 [65
], and ablation of cyclin E [66
] have both been reported not disrupt CSF arrest.
Once CSF arrest has been established then many of the above proteins seem no longer required for maintenance (p90rsk, Mad2, Bub1 and cyclin E/cdk2 are all dispensable for maintenance [34
]). This suggests that these proteins act upstream or independently of other effectors of CSF activity. SAC proteins may be essential to improve the efficiency of APC/C inhibition on entry into MetII arrest, yet appear redundant in the maintenance of arrest.
Whilst the c-Mos/MEK/MAPK/p90rsk/(SAC proteins) pathway is well established in the frog, its role in mammalian eggs is less clear. Although eggs from c-Mos knockout mice eventually undergo parthenogenetic activation [67
], they do MetII arrest, remaining there for 2–4 h, before going on to exit MII [69
]. This suggests that whilst c-Mos is critical for protracted MetII arrest, it is not required for its establishment. Loss of MEK or MAPK activity also results in parthenogenesis [21
] suggesting as in frog they are downstream components of the c-Mos pathway. However, p90rsk plays no essential role in mouse because eggs from Rsk knockouts arrest at MetII [70
]. Furthermore SAC proteins do not mediate CSF activity since mouse eggs expressing dominant negative mutants of Bub1 and Mad2 arrest at MetII [71
]. Therefore the c-Mos/MEK/MAPK pathway acting independently of p90rsk is likely only to be involved in helping maintaining MetII arrest in mammals, rather than having a direct role in its establishment.