Yoshinori Kanemori

Emi2 Inhibition of the Anaphase-promoting Complex/Cyclosome Absolutely Requires Emi2 Binding via the C-Terminal RL Tail

Both the D-box and the zinc-binding region (ZBR) of Emi2 are implicated in APC/C inhibition. This article shows that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. The RL tail apparently promotes the inhibitory interactions of the D-box and the ZBR with the APC/C. The RL tail thus serves as a docking site for the APC/C.

Dynamic Regulation of Emi2 by Emi2-Bound Cdk1/Plk1/CK1 and PP2A-B56 in Meiotic Arrest of Xenopus Eggs

In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Mos and Emi2, an inhibitor of the APC/C ubiquitin ligase. In Xenopus, Cdk1 phosphorylates Emi2 and both destabilizes and inactivates it, whereas Mos recruits PP2A phosphatase to antagonize the Cdk1 phosphorylation. However, how Cdk1 phosphorylation inhibits Emi2 is largely unknown. Here we show that multiple N-terminal Cdk1 phosphorylation motifs bind cyclin B1-Cdk1 itself, Plk1, and CK1δ/ε to inhibit Emi2. Plk1, after rebinding to other sites by self-priming phosphorylation, partially destabilizes Emi2. Cdk1 and CK1δ/ε sequentially phosphorylate the C-terminal APC/C-docking site, thereby cooperatively inhibiting Emi2 from binding the APC/C. In the presence of Mos, however, PP2A-B56β/ε bind to Emi2 and keep dephosphorylating it, particularly at the APC/C-docking site. Thus, Emi2 stability and activity are dynamically regulated by Emi2-bound multiple kinases and PP2A phosphatase. Our data also suggest a general role for Cdk1 substrate phosphorylation motifs in M phase regulation.

Emi2 Inhibition of the APC/C Absolutely Requires Emi2 Binding via the C-terminal RL Tail

Both the D-box and the zinc-binding region (ZBR) of Emi2 are implicated in APC/C inhibition. This article shows that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. The RL tail apparently promotes the inhibitory interactions of the D-box and the ZBR with the APC/C. The RL tail thus serves as a docking site for the APC/C.

Two Functional Forms of ACRBP/sp32 Are Produced by Pre-mRNA Alternative Splicing in the Mouse

ABSTRACT ACRBP/sp32 is a binding protein specific for the precursor (pro-ACR) and intermediate forms of sperm serine protease ACR. In this study, we examined the expression pattern, localization, and possible role of mouse ACRBP in spermatogenic cells and epididymal sperm. Unlike other mammalian ACRBPs, two forms of Acrbp mRNA—wild-type Acrbp-W and variant Acrbp-V5 mRNAs—were generated by alternative splicing of Acrbp in the mouse. ACRBP-W was synthesized in pachytene spermatocytes and haploid spermatids and immediately processed into a mature protein, ACRBP-C, by removal of the N-terminal half. The intron 5-retaining splice variant mRNA produced a predominant form of ACRBP, ACRBP-V5, that was present in pachytene spermatocytes and round spermatids, but was absent in elongating spermatids. ACRBP-W and ACRBP-V5 were both colocalized with pro-ACR in the acrosomal granules of early round spermatids, whereas the sperm acrosome contained only ACRBP-C. Glutathione S-transferase pull-down assays revealed that ACRBP-V5 and ACRBP-C possess a different domain capable of binding each of two segments in the C-terminal region of pro-ACR. Moreover, autoactivation of pro-ACR was remarkably accelerated by the presence of ACRBP-C. These results suggest that ACRBP-V5 and ACRBP-C may function in the transport/packaging of pro-ACR into acrosomal granules during spermiogenesis and in the promotion of ACR release from the acrosome during acrosomal exocytosis, respectively.

Possible involvement of mitogen- and stress-activated protein kinase 1, MSK1, in metaphase-II arrest through phosphorylation of EMI2 in mouse oocytes

Ovulated oocytes are arrested at the metaphase of second meiotic division. The metaphase-II arrest in Xenopus oocytes is regulated by RSKs located downstream of the Mos-MAPK pathway. In mice, other kinase(s) besides RSKs may be responsible for the metaphase-II arrest, because RSK1/RSK2/RSK3-triple knockout mice exhibit no obvious phenotype. Here, we show the subcellular localization and possible role of mitogen- and stress-activated kinase 1, MSK1 known as another downstream kinase of the Mos-MAPK pathway, in the mouse oocytes. Immunostaining analysis indicated that MSK1 is present in the germinal vesicle (GV) and cytoplasm of oocytes at the GV and metaphase-II stages, respectively. An active, phosphorylated form of MSK1 was predominantly localized to the metaphase-II spindle. The inhibition of the MSK1 activity failed to maintain the sister chromatid alignment within the metaphase-II plate. Importantly, MSK1 exhibited the ability to phosphorylate four Ser/Thr residues of meiotic cell-cycle regulator EMI2. The phosphorylation was required for up-regulation of the EMI2 activity in the oocytes. These results suggest that mouse MSK1 may play a key role in the metaphase-II arrest through phosphorylation of EMI2.

Biogenesis of sperm acrosome is regulated by pre-mRNA alternative splicing of Acrbp in the mouse

Significance Mammalian sperm possess a Golgi-derived exocytotic organelle, the acrosome, located on the apical region of the head. Proper biogenesis of the acrosome is essential for the fertilization process because the aberrant acrosome formation results in the sterility or subfertility of males. Here, we show that the acrosome formation is governed by two forms of proacrosin-binding protein ACRBP, wild-type ACRBP-W and variant ACRBP-V5, which are generated by pre-mRNA alternative splicing of Acrbp. ACRBP-V5 is involved in the formation and configuration of the acrosomal granule during early spermiogenesis, whereas the inactive status of proacrosin in the acrosome is maintained by ACRBP-W until acrosomal exocytosis. Proper biogenesis of a sperm-specific organelle, the acrosome, is essential for gamete interaction. An acrosomal matrix protein, ACRBP, is known as a proacrosin-binding protein. In mice, two forms of ACRBP, wild-type ACRBP-W and variant ACRBP-V5, are generated by pre-mRNA alternative splicing of Acrbp. Here, we demonstrate the functional roles of these two ACRBP proteins. ACRBP-null male mice lacking both proteins showed a severely reduced fertility, because of malformation of the acrosome. Notably, ACRBP-null spermatids failed to form a large acrosomal granule, leading to the fragmented structure of the acrosome. The acrosome malformation was rescued by transgenic expression of ACRBP-V5 in ACRBP-null spermatids. Moreover, exogenously expressed ACRBP-W blocked autoactivation of proacrosin in the acrosome. Thus, ACRBP-V5 functions in the formation and configuration of the acrosomal granule during early spermiogenesis. The major function of ACRBP-W is to retain the inactive status of proacrosin in the acrosome until acrosomal exocytosis.

Possible Role of p38 MAPK-MNK1-EMI2 Cascade in Metaphase-II Arrest of Mouse Oocytes

ABSTRACT The Mos-MAPK signaling pathway involving the Mos-MEK1/2-ERK1/2-RSK1/2/3 or MSK1-EMI2 cascade is directly linked to metaphase-II arrest of vertebrate oocytes. In this study, we examined whether p38, a member of the MAPK subfamily, is regulated under the control of Mos and contributes to metaphase-II arrest in the mouse oocyte. Morpholino oligonucleotide-mediated depletion of Mos revealed a remarkable decrease in phosphorylation of p38. Simultaneous treatment of oocytes with two chemical inhibitors of p38 and MEK1/2 induced both release from metaphase II and degradation of cyclin B1, whereas the treatment with each of these two inhibitors had little effect. Moreover, phosphorylation of EMI2 was dramatically abolished by addition of the two inhibitors. Indeed, MNK1, a kinase downstream of p38, exhibited the ability to phosphorylate EMI2. These results suggest that in addition to the Mos-MEK1/2 pathway, the Mos-mediated p38 pathway may be implicated in metaphase-II arrest.

Surfing and Swimming of Ejaculated Sperm in the Mouse Oviduct

ABSTRACT To accomplish fertilization in the oviductal ampulla, ejaculated sperm are required to migrate through the female reproductive tract. However, this fundamental process largely remains unknown. In this study, we focused on the role of oviductal smooth muscle (myosalpinx) contractions in the sperm migration. Administration of prifinium bromide, padrin, to mice effectively suppressed myosalpinx contractions, resulting in a decreased rate of fertilization in a dose-dependent manner, and an abrogation of high-speed back-and-forth/shuttling flows of oviductal fluids around the isthmus. Regardless of padrin administration, no shuttling flows were found near the ampulla. In the isthmus, sperm formed a tight assemblage that was synchronized with the shuttling flows. The sperm assemblage was gradually loosened and then completely abolished near the ampulla. No sperm assemblage was formed in the isthmus when padrin was administrated. These results suggest that myosalpinx contractions play important roles in the formation of sperm assemblage in the isthmus, and in the transport of the assemblage to the middle region of the oviduct. It is also suggested that the motility of sperm is essential for the migration of sperm from the middle oviductal region to the ampulla.

SCFFbl12 Increases p21Waf1/Cip1 Expression Level through Atypical Ubiquitin Chain Synthesis

ABSTRACT The cyclin-dependent kinase (CDK) inhibitor p21 is an unstructured protein regulated by multiple turnover pathways. p21 abundance is tightly regulated, and its defect causes tumor development. However, the mechanisms that underlie the control of p21 level are not fully understood. Here, we report a novel mechanism by which a component of the SCF ubiquitin ligase, Fbl12, augments p21 via the formation of atypical ubiquitin chains. We found that Fbl12 binds and ubiquitinates p21. Unexpectedly, Fbl12 increases the expression level of p21 by enhancing the mixed-type ubiquitination, including not only K48- but also K63-linked ubiquitin chains, followed by promotion of binding between p21 and CDK2. We also found that proteasome activator PA28γ attenuates p21 ubiquitination by interacting with Fbl12. In addition, UV irradiation induces a dissociation of p21 from Fbl12 and decreases K63-linked ubiquitination, leading to p21 degradation. These data suggest that Fbl12 is a key factor that maintains adequate intracellular concentration of p21 under normal conditions. Our finding may provide a novel possibility that p21s fate is governed by diverse ubiquitin chains.