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Emi2 inhibition of the anaphase-promoting complex/cyclosome absolutely requires Emi2 binding via the C-terminal RL tail.

Ohe M, Kawamura Y, Ueno H, Inoue D, Kanemori Y, Senoo C, Isoda M, Nakajo N, Sagata N - Mol. Biol. Cell (2010)

Bottom Line: Furthermore, and importantly, the RL tail-mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C.Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail.We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan.

ABSTRACT
Emi2 (also called Erp1) inhibits the anaphase-promoting complex/cyclosome (APC/C) and thereby causes metaphase II arrest in unfertilized vertebrate eggs. Both the D-box and the zinc-binding region (ZBR) of Emi2 have been implicated in APC/C inhibition. However, it is not well known how Emi2 interacts with and hence inhibits the APC/C. Here we show that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. When expressed in Xenopus oocytes and egg extracts, Emi2 lacking the RL tail fails to interact with and inhibit the APC/C. The RL tail itself can directly bind to the APC/C, and, when added to egg extracts, either an excess of RL tail peptides or anti-RL tail peptide antibody can dissociate endogenous Emi2 from the APC/C, thus allowing APC/C activation. Furthermore, and importantly, the RL tail-mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C. Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail. We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR.

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Binding of the Emi2 RL motif to the APC/C at a site(s) distinct from that occupied by the Cdc20 IR domain. (A) CSF extracts were incubated with an excess (2 mM) of the indicated C-terminal RL motif peptides for 5 min and subjected to Cdc27 immunoprecipitation followed by Emi2 or Cdc20 immunoblotting. (B) CSF extracts were treated with calcium and then subjected to either control or Cdc27 immunoprecipitation followed by immunoblotting for the indicated proteins. On calcium treatment, Emi2 underwent hyperphosphorylation and degradation, as previously shown (Rauh et al., 2005). (C) CSF extracts were subjected to either control or Emi2 immunoprecipitation followed by immunoblotting for Cdc27 and Cdc20. The anti-Emi2 antibody used was anti-Emi2 (N-terminus) antibody, not anti-RL motif peptide antibody (which could not precipitate Cdc27 or Cdc20; data not shown, but see Figure 2F). Four, three, and four independent experiments were performed for A–C, respectively, and, for each a typical result is shown.
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Figure 3: Binding of the Emi2 RL motif to the APC/C at a site(s) distinct from that occupied by the Cdc20 IR domain. (A) CSF extracts were incubated with an excess (2 mM) of the indicated C-terminal RL motif peptides for 5 min and subjected to Cdc27 immunoprecipitation followed by Emi2 or Cdc20 immunoblotting. (B) CSF extracts were treated with calcium and then subjected to either control or Cdc27 immunoprecipitation followed by immunoblotting for the indicated proteins. On calcium treatment, Emi2 underwent hyperphosphorylation and degradation, as previously shown (Rauh et al., 2005). (C) CSF extracts were subjected to either control or Emi2 immunoprecipitation followed by immunoblotting for Cdc27 and Cdc20. The anti-Emi2 antibody used was anti-Emi2 (N-terminus) antibody, not anti-RL motif peptide antibody (which could not precipitate Cdc27 or Cdc20; data not shown, but see Figure 2F). Four, three, and four independent experiments were performed for A–C, respectively, and, for each a typical result is shown.

Mentions: It has been shown that Cdc20, an activator of the APC/C (Yu, 2007), also binds to the APC/C via the C-terminal tail called an IR domain (Vodermaier et al., 2003). We asked whether the RL tail of Emi2 would bind to the same site(s) within the APC/C as the IR domain of Cdc20, thereby competitively inhibiting the activation of the APC/C by Cdc20. When added to CSF extracts, an excess of Emi2 WT-RL motif peptides could not appreciably affect the association of Cdc20 with Cdc27 (or the APC/C), whereas it could largely inhibit Emi2-Cdc27 association (Figure 3A). Furthermore, upon degradation of Emi2 by calcium treatment of CSF extracts, the association of Cdc20 with Cdc27 was not affected (or increased) at all (Figure 3B). Moreover, in CSF extracts, Emi2 was bound to the APC/C complexed with Cdc20 (Figure 3C), consistent with previous results (Wu et al., 2007a). Thus, it appears that the RL tail of Emi2 binds to the APC/C at a site(s) distinct from that occupied by the IR domain of Cdc20. This notion, however, is consistent with the fact that most of the essential residues in the RL motif of Emi2 (Figures 1B and 2B) are not conserved in the IR domain of Cdc20 (Vodermaier et al., 2003).


Emi2 inhibition of the anaphase-promoting complex/cyclosome absolutely requires Emi2 binding via the C-terminal RL tail.

Ohe M, Kawamura Y, Ueno H, Inoue D, Kanemori Y, Senoo C, Isoda M, Nakajo N, Sagata N - Mol. Biol. Cell (2010)

Binding of the Emi2 RL motif to the APC/C at a site(s) distinct from that occupied by the Cdc20 IR domain. (A) CSF extracts were incubated with an excess (2 mM) of the indicated C-terminal RL motif peptides for 5 min and subjected to Cdc27 immunoprecipitation followed by Emi2 or Cdc20 immunoblotting. (B) CSF extracts were treated with calcium and then subjected to either control or Cdc27 immunoprecipitation followed by immunoblotting for the indicated proteins. On calcium treatment, Emi2 underwent hyperphosphorylation and degradation, as previously shown (Rauh et al., 2005). (C) CSF extracts were subjected to either control or Emi2 immunoprecipitation followed by immunoblotting for Cdc27 and Cdc20. The anti-Emi2 antibody used was anti-Emi2 (N-terminus) antibody, not anti-RL motif peptide antibody (which could not precipitate Cdc27 or Cdc20; data not shown, but see Figure 2F). Four, three, and four independent experiments were performed for A–C, respectively, and, for each a typical result is shown.
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Figure 3: Binding of the Emi2 RL motif to the APC/C at a site(s) distinct from that occupied by the Cdc20 IR domain. (A) CSF extracts were incubated with an excess (2 mM) of the indicated C-terminal RL motif peptides for 5 min and subjected to Cdc27 immunoprecipitation followed by Emi2 or Cdc20 immunoblotting. (B) CSF extracts were treated with calcium and then subjected to either control or Cdc27 immunoprecipitation followed by immunoblotting for the indicated proteins. On calcium treatment, Emi2 underwent hyperphosphorylation and degradation, as previously shown (Rauh et al., 2005). (C) CSF extracts were subjected to either control or Emi2 immunoprecipitation followed by immunoblotting for Cdc27 and Cdc20. The anti-Emi2 antibody used was anti-Emi2 (N-terminus) antibody, not anti-RL motif peptide antibody (which could not precipitate Cdc27 or Cdc20; data not shown, but see Figure 2F). Four, three, and four independent experiments were performed for A–C, respectively, and, for each a typical result is shown.
Mentions: It has been shown that Cdc20, an activator of the APC/C (Yu, 2007), also binds to the APC/C via the C-terminal tail called an IR domain (Vodermaier et al., 2003). We asked whether the RL tail of Emi2 would bind to the same site(s) within the APC/C as the IR domain of Cdc20, thereby competitively inhibiting the activation of the APC/C by Cdc20. When added to CSF extracts, an excess of Emi2 WT-RL motif peptides could not appreciably affect the association of Cdc20 with Cdc27 (or the APC/C), whereas it could largely inhibit Emi2-Cdc27 association (Figure 3A). Furthermore, upon degradation of Emi2 by calcium treatment of CSF extracts, the association of Cdc20 with Cdc27 was not affected (or increased) at all (Figure 3B). Moreover, in CSF extracts, Emi2 was bound to the APC/C complexed with Cdc20 (Figure 3C), consistent with previous results (Wu et al., 2007a). Thus, it appears that the RL tail of Emi2 binds to the APC/C at a site(s) distinct from that occupied by the IR domain of Cdc20. This notion, however, is consistent with the fact that most of the essential residues in the RL motif of Emi2 (Figures 1B and 2B) are not conserved in the IR domain of Cdc20 (Vodermaier et al., 2003).

Bottom Line: Furthermore, and importantly, the RL tail-mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C.Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail.We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan.

ABSTRACT
Emi2 (also called Erp1) inhibits the anaphase-promoting complex/cyclosome (APC/C) and thereby causes metaphase II arrest in unfertilized vertebrate eggs. Both the D-box and the zinc-binding region (ZBR) of Emi2 have been implicated in APC/C inhibition. However, it is not well known how Emi2 interacts with and hence inhibits the APC/C. Here we show that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. When expressed in Xenopus oocytes and egg extracts, Emi2 lacking the RL tail fails to interact with and inhibit the APC/C. The RL tail itself can directly bind to the APC/C, and, when added to egg extracts, either an excess of RL tail peptides or anti-RL tail peptide antibody can dissociate endogenous Emi2 from the APC/C, thus allowing APC/C activation. Furthermore, and importantly, the RL tail-mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C. Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail. We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR.

Show MeSH
Related in: MedlinePlus