Constant regulation of both the MPF amplification loop and the Greatwall-PP2A pathway is required for metaphase II arrest and correct entry into the first embryonic cell cycle

Thierry Lorca ( 1 Cyril Bernis 0 1 Suzanne Vigneron 1 Andrew Burgess 1 Estelle Brioudes 1 Jean-Claude Labb 1 Anna Castro ) 1 0 Present address: Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego , La Jolla, CA 92093-0347 , USA 1 Universites Montpellier 2 et 1, Centre de Recherche de Biochimie Macromoleculaire , CNRS UMR 5237, IFR 122, 1919 Route de Mende, 34293 Montpellier CEDEX 5 , France - Summary Recent results indicate that regulating the balance between cyclin-BCdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-BCdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the ce different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data n indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist e ic regarding the regulation of this balance during M phase and interphase. Here, we demonstrate that constant regulation of the cyclinS BCdc2 amplification loop is required for correct mitotic division and to promote correct timing of mitotic entry. Our results show lle that removal of Cdc25 from metaphase-II-arrested oocytes promotes mitotic exit, whereas depletion of either Myt1 or Wee1 in C interphase egg extracts induces premature mitotic entry. We also provide evidence that, besides the cyclin-BCdc2 amplification loop, fo the Greatwall-PP2A pathway must also be tightly regulated to promote correct first embryonic cell division. When PP2A is prematurely l inhibited in the absence of cyclin-BCdc2 activation, endogenous cyclin-ACdc2 activity induces irreversible aberrant mitosis in ruan cwyhcilcihn Athearnedisc,yfcilrisnt, Bp,artthiuals tpraronmsieontitnpghporsepmhoatruyrleatiaonnd orfapmiditoetxiict sfurobmstrmateitsosainsd., second, subsequent rapid and complete degradation of o J Introduction Entry and exit of mitosis are regulated by the cyclin-BCdc2 complex, also known as the M-phase-promoting factor (MPF). Mitotic entry is induced by the activation of this complex by phosphorylation (Nurse, 1990), whereas mitotic exit is promoted by inactivation through the ubiquitin-dependent degradation of its cyclin B regulatory subunit (Glotzer et al., 1991). During G2, cyclin-BCdc2 is maintained in an inactive state by phosphorylation of the catalytic Cdc2 subunit on two inhibitory residues: threonine 14 and tyrosine 15 (Draetta and Beach, 1988; Gould and Nurse, 1989; Krek and Nigg, 1991). The identity of the kinases responsible for these phosphorylation events was first shown in yeast. Different genetic studies indicated that the protein kinases encoded by the genes wee1 and myt1 acted as negative regulators of cyclin-BCdc2 (Lundgren et al., 1991; Russell and Nurse, 1987a). Subsequent studies performed with human and Xenopus Wee1 and Myt1 proteins demonstrated a role for these two kinases in the phosphorylation of Thr14 and Tyr15 of Cdc2 (Mueller et al., 1995a; Mueller et al., 1995b; Parker and PiwnicaWorms, 1992). The subsequent activation of the cyclin-BCdc2 complex at mitotic entry is promoted by the dephosphorylation of these two inhibitory residues of Cdc2 by the phosphatase Cdc25 (Gautier et al., 1991; Kumagai and Dunphy, 1991; Millar et al., 1991; Strausfeld et al., 1991). At the end of G2, abrupt dephosphorylation of Tyr15 and Thr14 residues by Cdc25 triggers initial activation of cyclin-BCdc2, which in turn further activates Cdc25 and inactivates Wee1 and Myt1 by phosphorylation, resulting in full activation of the cyclin-BCdc2 complex (Hoffmann et al., 1993; Izumi et al., 1992; Mueller et al., 1995b; Russell and Nurse, 1987b; Smythe and Newport, 1992). This positive-feedback mechanism is called the MPF amplification loop. Besides the direct regulation of cyclin-BCdc2 by this amplification loop, other feedback mechanisms also indirectly regulate cyclin-BCdc2 activation. In this regard, the Polo kinase Plx1 participates in the activation of cyclin-BCdc2 by directly phosphorylating and activating the Cdc25 phosphatase (Abrieu et al., 1998; Kumagai and Dunphy, 1996; Qian et al., 1998; Qian et al., 2001), and by promoting ubiquitin-dependent degradation of Wee1 (Watanabe et al., 2004) or inactivation of Myt1 (Nakajima et al., 2003). The current model proposes that mitotic entry is promoted by cyclin-BCdc2 activation through the MPF amplification loop (Nurse, 1990), whereas mitotic exit is triggered by the inactivation of this complex through ubiquitin-dependent degradation of cyclin B (Murray et al., 1989). However, the involvement of other phosphatases in mitosis entry and exit has also been reported. In this regard, it has been shown in interphase Xenopus egg extracts that the inhibition of PP2A by okadaic acid (OA) promotes cyclinBCdc2 activation and blocks Cdc25 dephosphorylation, suggesting that PP2A negatively regulates MPF by maintaining Cdc25 in its inactive dephosphorylated form (Clarke et al., 1993; Felix et al., 1990). Accordingly, in fission yeast, decreased activity of PP2A induces premature mitosis, probably through the activation of Cdc25 (Kinoshita et al., 1990; Kinoshita et al., 1993). The role of PP2A in mitotic exit has also been suggested. Thus, inhibition of PP2A in Xenopus extracts from metaphase-II-arrested oocytes induces cyclin B degradation and exit from mitosis (Lorca et al., 1991). In addition, a decrease in PP2A activity promotes chromosome nondisjunction in fission yeast (Kinoshita et al., 1990). Moreover, a mutant CDC55 in budding yeast, which encodes a PP2A regulatory subunit, displays premature separation of sister chromatids and mitotic exit in the presence of an active spindleassembly checkpoint (Minshull et al., 1996). Mitotic exit in this mutant takes place in the absence of cyclin B degradation through the inactivation of Cdc28 on its inhibitory residues. Finally, the activation of Cdc14 is also required in budding yeast to promote cyclin-BCdc2 substrate dephosphorylation and mitotic exit (DAmours and Amon, 2004; Stegmeier and Amon, 2004). However, the Cdc14 phosphatase does not play a major role in late mitosis in fission yeast or in higher eukaryotes (Trautmann and ce McCollum, 2002). en Recent results have shed light on the mechanisms by which ic PP2A can control mitotic entry and exit (Castilho et al., 2009; lleS iMndoicchaitdeatheatt aPlP.,2A20i0n9d;ucVeisgndeerpohnosepthoarl.y,la2t0io0n9)o.f TMhPesFe snuebwstradtaetsa. C To promote mitotic entry and to maintain the mitotic state, this fo phosphatase must be inhibited by the recently identified Greatwall l kinase. At mitotic exit, however, PP2A must be activated again to ruan aplr.o, m20o0te9;cyVcilginne-BronCedtc2al.s,u2b0s0tr9a)t.e Tdheupsh,oistpahpopryealartsiotnha(tCraesgtuillhao (...truncated)