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  • Emi protein consists of an N terminal regulatory region and

    2021-07-21

    Emi2 protein consists of an N-terminal regulatory region and a C-terminal functional region. The C-terminal region contains a destruction box (D-box), which likely competes with APC/C substrates (including cyclin B) for APC/C binding, and a zinc-binding region (ZBR), which somehow inhibits APC/C ubiquitin ligase activity (Miller et al., 2006, Schmidt et al., 2005). In addition, the well-conserved C-terminal YO-01027 tail, termed the RL tail, serves as a docking site for the APC/C, promoting the inhibitory interactions of the D-box and the ZBR with the APC/C (Ohe et al., 2010). In YO-01027 to these, the N-terminal region possesses signals required for rapid Emi2 degradation upon fertilization, including a calmodulin-dependent protein kinase II (CaMKII) phosphorylation site and a DSG destruction motif recognized by SCFβ-TrCP ubiquitin ligase (Hansen et al., 2006, Liu and Maller, 2005, Rauh et al., 2005). In Xenopus oocytes, the Mos-MAPK pathway upregulates both the stability and activity of Emi2, thus promoting Meta-II arrest (Inoue et al., 2007, Nishiyama et al., 2007). Rsk, the kinase immediately downstream from MAPK (or ERK2), directly phosphorylates Emi2 at residues in the central region (Inoue et al., 2007), and thereby recruits the protein phosphatase PP2A to Emi2 (J.Q. Wu et al., 2007, Q. Wu et al., 2007). Recruited PP2A likely antagonizes Emi2 N-terminal and C-terminal phosphorylations by Cdk1, which have been suggested to destabilize and inactivate Emi2, respectively (Hansen et al., 2007; J.Q. Wu et al., 2007). However, how Cdk1-mediated phosphorylation both destabilizes and inactivates Emi2 is largely unknown, although this issue is crucial for understanding the mechanism of Emi2 upregulation by the Mos-MAPK pathway. Here, we have explored the mechanisms of Emi2 inhibition by Cdk1 (as well as Emi2 upregulation by Mos) using Xenopus egg extracts. Our results show that multiple distinct kinases, including cyclin B1-Cdk1 itself, bind to the N-terminal Cdk1 sites of Emi2 and phosphorylate other critical sites to destabilize and inactivate Emi2. Mos/Rsk-recruited PP2A, identified here as PP2A-B56β/ε, keeps preferentially dephosphorylating the critical inhibitory sites of Emi2. Thus, Emi2 stability and activity are dynamically regulated by Emi2-bound multiple kinases and PP2A phosphatase, providing new insight into the mechanisms of Emi2 regulation in Meta-II arrest of vertebrate eggs. In addition, our data seem to provide general implications for Cdk1 substrate phosphorylation motifs in M phase regulation.
    Results
    Discussion Our results clarify precise, dynamic control mechanisms of Emi2 stability and activity in Meta-II arrest of Xenopus eggs, and, together with recently published data, show how Meta-II arrest is robustly maintained (Figure 7). In addition, our data seem to suggest a general role for Cdk1 substrate phosphorylation motifs in M phase regulation.
    Experimental Procedures Biochemical reagents, cDNAs, in vitro transcription/translation, antibodies, immunoblotting, in vitro kinase assays (including GST-Emi2 peptide fusion proteins), and pulldown assays are described in the Supplemental Information.
    Acknowledgments
    Introduction Members of the serine/threonine specific protein kinase CK1 were among the first protein kinases having been described in the literature [1], [2]. They are ubiquitously expressed, evolutionarily highly conserved and found in all eukaryotes ranging from yeast to humans [3], [4]. At present, seven mammalian CK1 isoforms have been characterised (α, β, γ1, γ2, γ3, δ, ε) [5], [6], [7], [8], [9] and more than 140 in vitro and in vivo substrates of CK1 are listed (reviewed in Ref. 4). CK1 plays important regulative roles in various cellular processes, including proliferation, apoptosis, cell differentiation, circadian rhythm, chromosome segregation and membrane trafficking [4], [10]. Deregulation of CK1 expression and activity contributes to the pathogenesis of neurodegenerative diseases and cancer. Therefore, CK1 isoforms are interesting new targets for the development of CK1 specific inhibitors and biological tools to inhibit CK1 activity which influences the regulation of microtubule dynamics and the spindle apparatus.