The APC/C Inhibitor XErp1/Emi2 Is Essential for Xenopus Early Embryonic Divisions

Abstract

Mitotic divisions result from the oscillating activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 activity is terminated by the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets cyclin B for destruction. In somatic divisions, the early mitotic inhibitor 1 (Emi1) and the spindle assembly checkpoint (SAC) regulate cell cycle progression by inhibiting the APC/C. Early embryonic divisions lack these APC/C-inhibitory components, which raises the question of how those cycles are controlled. We found that the APC/C-inhibitory activity of XErp1 (also known as Emi2) was essential for early divisions in Xenopus embryos. Loss of XErp1 resulted in untimely destruction of APC/C substrates and embryonic lethality. XErp1's APC/C-inhibitory function was negatively regulated by Cdk1 and positively by protein phosphatase 2A (PP2A). Thus, Cdk1 and PP2A operate at the core of early mitotic cell cycles by antagonistically controlling XErp1 activity, which results in oscillating APC/C activity.

Protein phosphatase 1 is essential for Greatwall inactivation at mitotic exit

“…To gain insights into the mechanism of Gwl inactivation at mitotic exit, we used a previously established Xenopus embryo extract system [17]. Interphasic extract was prepared from embryos that were in the process of exiting the second mitotic division as evidenced by cleavage furrow ingression ( Fig 1A).…”

“…The electrophoretic mobility of kinase-dead 35 S-FlagGwl G41S was markedly less affected by the addition of D90 (lanes 14, 15 and 21) consistent with the finding that Gwl phosphorylates itself in an autocatalytic manner. Addition of roscovitine resulted in rapid dephosphorylation of 35 S-Flag-Gwl G41S (lanes [15][16][17][18][19][20]. Notably, however, the presence of I-2 during mitotic exit had no detectable effect on the dephosphorylation of 35 S-Flag-Gwl G41S (lanes 21-26).…”

“…Anti-XErp1 and anti-Cdc27 antibodies were described before [17]. Anti-cyclin B2 antibody was purchased from Biozol (MBL-K0189-3).…”

“…Embryo extract was prepared as described before [17]. When indicated, 6xHis-cyclin BD90 (aa91-397) was added to a concentration of 75 nM, roscovitine to 1 mM and PP1 Inhibitor-2 to 4 lM.…”

Organization of early frog embryos by chemical waves emanating from centrosomes

“…Whether mitotic exit is a self-sustaining autocatalytic reaction that can generate waves, and if so what determines their velocity, remain to be determined. Feedback between Cdk1 and the anaphase-promoting complex/ cyclosome (APC/C) through XErp1 [38,49,50] presents a potential autocatalytic mechanism for a mitotic exit, but it is not clear why this different reaction would propagate with the same velocity as the entry wave. An alternative is that the exit wave is a consequence of local changes in the cytoplasm that follow the mitotic entry wave with a fixed time delay [33], and therefore does not need to be autocatalytic or capable of wave propagation on its own.…”