Mouse Emi2 as a distinctive regulatory hub in second meiotic metaphase

Development, Oct 2010

Toru Suzuki, Emi Suzuki, Naoko Yoshida, Atsuko Kubo, Hongmei Li, Erina Okuda, Manami Amanai, Anthony C. F. Perry

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Mouse Emi2 as a distinctive regulatory hub in second meiotic metaphase

Toru Suzuki 0 1 Emi Suzuki 0 Naoko Yoshida 0 Atsuko Kubo 0 Hongmei Li 0 Erina Okuda 0 Manami Amanai 0 Anthony C. F. Perry () 0 1 0 RIKEN Center for Developmental Biology , 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047 Japan 1 Laboratory of Mammalian Molecular Embryology, Bath Centre for Regenerative Medicine, and Development of Biology and Biochemistry, University of Bath , Bath BA2 7AY , UK SUMMARY The oocytes of vertebrates are typically arrested at metaphase II (mII) by the cytostatic factor Emi2 until fertilization. Regulatory mechanisms in Xenopus Emi2 (xEmi2) are understood in detail but contrastingly little is known about the corresponding mechanisms in mammals. Here, we analyze Emi2 and its regulatory neighbours at the molecular level in intact mouse oocytes. Emi2, but not xEmi2, exhibited nuclear targeting. Unlike xEmi2, separable N- and C-terminal domains of mouse Emi2 modulated metaphase establishment and maintenance, respectively, through indirect and direct mechanisms. The C-terminal activity was mapped to the potential phosphorylation target Tx5SxS, a destruction box (D-box), a lattice of Zn2+-coordinating residues and an RL domain. The minimal region of Emi2 required for its cytostatic activity was mapped to a region containing these motifs, from residue 491 to the C terminus. The cytostatic factor Mos-MAPK promoted Emi2-dependent metaphase establishment, but Mos autonomously disappeared from meiotically competent mII oocytes. The N-terminal Plx1-interacting phosphodegron of xEmi2 was apparently shifted to within a minimal fragment (residues 51-300) of mouse Emi2 that also contained a calmodulin kinase II (CaMKII) phosphorylation motif and which was efficiently degraded during mII exit. Two equimolar CaMKII isoform variants were present in mII oocytes, neither of which phosphorylated Emi2 in vitro, consistent with the involvement of additional factors. No evidence was found that calcineurin is required for mouse mII exit. These data support a model in which mammalian meiotic establishment, maintenance and exit converge upon a modular Emi2 hub via evolutionarily conserved and divergent mechanisms. INTRODUCTION The meiotic cell cycle of fertilizable oocytes in vertebrates is typically restrained at the second metaphase (mII) by a cytostatic factor (CSF) to prevent development without a paternal genome (parthenogenesis). The underlying mechanisms of mII arrest and exit are best understood in Xenopus and relatively poorly in mammals. In both, mII arrest correlates with the kinase activity of maturation promoting factor (MPF), a heterodimer of Cyclin B (CycB) and the cyclin-dependent kinase Cdc2 (Masui and Markert, 1971; Gautier et al., 1989; Gautier et al., 1990; Perry and Verlhac, 2008). MPF is active in both mitotic and meiotic cell cycles in vertebrates, but its prolonged stabilization by CSF is unique to mII and results in mII arrest. Exit from mII occurs when CycB undergoes destruction box(D-box-) dependent ubiquitylation by the anaphase-promoting complex, APC, an E3 ubiquitin ligase; this targets CycB for 26S proteasomal hydrolysis and eliminates MPF, thereby inducing metaphase exit (Glotzer et al., 1991; Peters, 2006). Arrest at mII is achieved by suspending APC activity, which is the function of CSF. One CSF responsible for this inhibition is the endogenous meiotic inhibitor 2, Emi2, the activity of which is essential for mII arrest as independently revealed in Xenopus (Schmidt et al., 2005) and the mouse (Shoji et al., 2006). Depletion of Emi2 from intact mouse oocytes causes mII release in a manner that requires the APC activator, Cdc20; one explanation of this is that Emi2 prevents Cdc20 from activating the APC (Shoji et al., 2006; Amanai et al., 2006). Xenopus Emi2 (xEmi2) is stabilized during mII by phosphorylation from xMos to xMek to xMAPK to xRsk to xEmi2 (Sagata et al., 1989; Bhatt and Ferrell, 1999; Gross et al., 2000; Inoue et al., 2007; Nishiyama et al., 2007a) (Fig. 1). xRsk phosphorylates xEmi2 at S335, T336, S342 and S344. Phosphorylation at S335 and T336 facilitates the binding of protein phosphatase 2A (xPP2A), which in turn dephosphorylates phosphoresidues at T545 and T551, and S213, T239, T252 and T267 (Wu et al., 2007b). Dephosphorylation of T545/T551 enhances binding of the xEmi2 C-terminal domain to the APC core component, xCdc27 (xAPC3) to inhibit the APC (Wu et al., 2007b) whereas dephosphorylation of the S213-T267 cluster stabilizes xEmi2 (Wu et al., 2007a). In Xenopus, xPP2A activity towards xEmi2 is thus stimulated by xMos via xRsk to promote mII arrest (Fig. 1). In the mouse, Mos null oocytes fail to activate the MAPK pathway but nevertheless often arrest or pause at mII with MPF activity initially unaffected, or progress through mII and then collapse back to mIII (Verlhac et al., 1996; Choi et al., 1996). Oocytes from Mos-null mice contain anomalously long, interphaselike microtubules during mI to mII and mII to mIII transitions (Verlhac et al., (...truncated)


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Toru Suzuki, Emi Suzuki, Naoko Yoshida, Atsuko Kubo, Hongmei Li, Erina Okuda, Manami Amanai, Anthony C. F. Perry. Mouse Emi2 as a distinctive regulatory hub in second meiotic metaphase, Development, 2010, pp. 3281-3291, 137/19, DOI: 10.1242/dev.052480