Michitaka Isoda

FoxM1-driven cell division is required for neuronal differentiation in early Xenopus embryos.

In vertebrate embryogenesis, neural induction is the earliest step through which the fate of embryonic ectoderm to neuroectoderm becomes determined. Cells in the neuroectoderm or neural precursors actively proliferate before they exit from the cell cycle and differentiate into neural cells. However, little is known about the relationship between cell division and neural differentiation, although, in Xenopus, cell division after the onset of gastrulation has been suggested to be nonessential for neural differentiation. Here, we show that the Forkhead transcription factor FoxM1 is required for both proliferation and differentiation of neuronal precursors in early Xenopus embryos. FoxM1 is expressed in the neuroectoderm and is required for cell proliferation in this region. Specifically, inhibition of BMP signaling, an important step for neural induction, induces the expression of FoxM1 and its target G2-M cell-cycle regulators, such as Cdc25B and cyclin B3, thereby promoting cell division in the neuroectoderm. Furthermore, G2-M cell-cycle progression or cell division mediated by FoxM1 or its target G2-M regulators is essential for neuronal differentiation but not for specification of the neuroectoderm. These results suggest that FoxM1 functions to link cell division and neuronal differentiation in early Xenopus embryos.

The extracellular signal-regulated kinase-mitogen-activated protein kinase pathway phosphorylates and targets Cdc25A for SCFβ-TrCP- dependent degradation for cell cycle arrest

The extracellular signal-regulated kinase (ERK) pathway is generally mitogenic, but, upon strong activation, it causes cell cycle arrest by a not-yet fully understood mechanism. In response to genotoxic stress, Chk1 hyperphosphorylates Cdc25A, a positive cell cycle regulator, and targets it for Skp1/Cullin1/F-box protein (SCF)(beta-TrCP) ubiquitin ligase-dependent degradation, thereby leading to cell cycle arrest. Here, we show that strong ERK activation can also phosphorylate and target Cdc25A for SCF(beta-TrCP)-dependent degradation. When strongly activated in Xenopus eggs, the ERK pathway induces prominent phosphorylation and SCF(beta-TrCP)-dependent degradation of Cdc25A. p90rsk, the kinase downstream of ERK, directly phosphorylates Cdc25A on multiple sites, which, interestingly, overlap with Chk1 phosphorylation sites. Furthermore, ERK itself phosphorylates Cdc25A on multiple sites, a major site of which apparently is phosphorylated by cyclin-dependent kinase (Cdk) in Chk1-induced degradation. p90rsk phosphorylation and ERK phosphorylation contribute, roughly equally and additively, to the degradation of Cdc25A, and such Cdc25A degradation occurs during oocyte maturation in which the endogenous ERK pathway is fully activated. Finally, and importantly, ERK-induced Cdc25A degradation can elicit cell cycle arrest in early embryos. These results suggest that strong ERK activation can target Cdc25A for degradation in a manner similar to, but independent of, Chk1 for cell cycle arrest.

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.

Identification of non-Ser/Thr-Pro consensus motifs for Cdk1 and their roles in mitotic regulation of C2H2 zinc finger proteins and Ect2

The cyclin B-dependent protein kinase Cdk1 is a master regulator of mitosis and phosphorylates numerous proteins on the minimal consensus motif Ser/Thr-Pro (S/T-P). At least in several proteins, however, not well-defined motifs lacking a Pro in the +1 position, referred herein to as non-S/T-P motifs, have been shown to be phosphorylated by Cdk1. Here we show that non-S/T-P motifs in fact form consensus sequences for Cdk1 and probably play roles in mitotic regulation of physiologically important proteins. First, we show, by in vitro kinase assays, that previously identified non-S/T-P motifs all harbour one or more C-terminal Arg/Lys residues essential for their phosphorylation by Cdk1. Second, using Arg/Lys-scanning oriented peptide libraries, we demonstrate that Cdk1 phosphorylates a minimal sequence S/T-X-X-R/K and more favorable sequences (P)-X-S/T-X-[R/K]2–5 as its non-S/T-P consensus motifs. Third, on the basis of these results, we find that highly conserved linkers (typically, T-G-E-K-P) of C2H2 zinc finger proteins and a nuclear localization signal-containing sequence (matching P-X-S-X-[R/K]5) of the cytokinesis regulator Ect2 are inhibitorily phosphorylated by Cdk1, well accounting for the known mitotic regulation and function of the respective proteins. We suggest that non-S/T-P Cdk1 consensus motifs identified here may function to regulate many other proteins during mitosis.

Emi2 mediates meiotic MII arrest by competitively inhibiting the binding of Ube2S to the APC/C

In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Emi2, a direct inhibitor of the APC/C ubiquitin ligase. Two different ubiquitin-conjugating enzymes, UbcH10 and Ube2S, work with the APC/C to target APC/C substrates for degradation. However, their possible roles and regulations in unfertilized/fertilized eggs are not known. Here we use Xenopus egg extracts to show that both UbcH10 and Ube2S are required for rapid cyclin B degradation at fertilization, when APC/C binding of Ube2S, but not of UbcH10, increases several fold, coincidently with (SCF(β-TrCP)-dependent) Emi2 degradation. Interestingly, before fertilization, Emi2 directly inhibits APC/C-Ube2S binding via the C-terminal tail, but on fertilization, its degradation allows the binding mediated by the Ube2S C-terminal tail. Significantly, Emi2 and Ube2S bind commonly to the APC/C catalytic subunit APC10 via their similar C-terminal tails. Thus, Emi2 competitively inhibits APC/C-Ube2S binding before fertilization, while its degradation on fertilization relieves the inhibition for APC/C activation.

Essential role of the Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope

Cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout (KO) mice sterile. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are sterile and display meiotic defects virtually identical to those observed in Cdk2 KO mice including non-homologous chromosome pairing, unrepaired double-strand breaks, undetectable sex-body and pachytene arrest. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA KO spermatocytes display severely affected telomere tethering as well as impaired distribution of Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an important activator of Cdk2 at meiotic telomeres, and provide genetic evidence for a physiological function of mammalian Cdk2 that is not dependent on cyclins.

Targeting p38α Increases DNA Damage, Chromosome Instability, and the Anti-tumoral Response to Taxanes in Breast Cancer Cells

Breast cancer is the second leading cause of cancer-related death among women. Here we report a role for the protein kinase p38α in coordinating the DNA damage response and limiting chromosome instability during breast tumor progression, and identify the DNA repair regulator CtIP as a p38α substrate. Accordingly, decreased p38α signaling results in impaired ATR activation and homologous recombination repair, with concomitant increases in replication stress, DNA damage, and chromosome instability, leading to cancer cell death and tumor regression. Moreover, we show that pharmacological inhibition of p38α potentiates the effects of taxanes by boosting chromosome instability in murine models and patient-derived xenografts, suggesting the potential interest of combining p38α inhibitors with chemotherapeutic drugs that induce chromosome instability.

New insights into Cdk2 regulation during meiosis

Cyclin dependent kinases (CDKs) are proline-directed serine/ threonine kinases that play a central role in regulating cell cycle progression. Since the activity of CDKs depends on the binding of regulatory subunit named cyclins, the analysis of the CDK functions that have been studied so far has usually gone in parallel to the study of cyclins. However, the proteins referred to as RINGO or Speedy, which have no amino acid sequence homology to cyclins, have been found to function as atypical CDK activators. The first RINGO protein was identified as a new activator of Cdk1 and Cdk2, which induced the meiotic maturation of frog (Xenopus) oocytes. Interestingly, CDKs activated by Xenopus RINGO were found to phosphorylate different sites on the kinase Myt1 than those phosphorylated by cyclinCDK complexes. In fact, Myt1 is much more efficiently inhibited by RINGO-CDK than by cyclin-CDK, supporting the idea that RINGO-activated CDKs may have different functions. RINGO proteins are conserved in metazoans and several mammalian RINGO family members have been identified. The best-studied mammalian family member is RingoA, whose mRNA is highly expressed in mouse gonads, suggesting that RingoA might regulate meiotic progression, as reported for Xenopus RINGO in oocytes. Genetic studies published during the past decade have provided good evidence that Cdk1 is the only CDK family member that is essential for mouse cell proliferation, and genetic inactivation of Cdk1 has been shown to cause mouse embryonic lethality. In contrast, Cdk2 is not essential for somatic cell proliferation in mouse but it is indispensable for male and female meiosis. Interestingly, the meiotic phenotypes that are observed in Cdk2 knockout spermatocytes and oocytes have not been reported in any of the mice deficient for particular cyclins. A recent report has shown that the combined deletion of cyclins E1 and E2 produces similar meiotic phenotypes in spermatocytes as the deletion of Cdk2. However, cyclins E1 and E2 do not co-localize with Cdk2 during prophase I of meiosis, neither at telomeres nor at crossing-over sites, suggesting that they are unlikely to be involved in Cdk2 activation at these specific locations. These findings support the existence of a cyclin-independent mechanism of Cdk2 activation in germ cells. We have recently reported that RingoA knockout mice are born with the expected Mendelian frequency and do not show any overt differences compared to their wild-type littermates. However, they are sterile and have hypoplastic testes and ovaries, supporting an important role for RingoA in mammalian meiotic progression. Importantly, spermatocytes and oocytes that are deficient in RingoA show identical phenotypes as those deficient in Cdk2, namely they are mostly arrested in a pachytene-like stage of prophase I with non-homologous chromosome pairing, telomere detachment from the inner nuclear membrane (INM) and telomere fusion (Fig. 1). In addition, RingoA-deficient spermatocytes and oocytes show strong accumulation of gH2AX, which is a hallmark of unrepaired double strand breaks. The arrested germ cells then undergo apoptosis probably due to the pachytene checkpoint, which is activated by the accumulation of unrepaired DNA. RingoA localizes to the telomeric regions in pachytene spermatocytes, as it has been reported for Cdk2, co-localizes with Cdk2 from the leptotene stage and then disappears from telomeres as cells enter the diplotene stage. RingoA and Cdk2 also co-localize along the asynapsed sex chromosome arms in pachytene spermatocytes. Interestingly, RingoA-deficient spermatocytes show impaired localization to telomeres of Sun1, a protein required for telomere tethering to the INM, whereas the telomeric localization of TERB1, another protein required for telomere-INM tethering, is maintained. RingoA-Cdk2 can phosphorylate in vitro the Nterminus of Sun1, which is the domain involved in binding to TERB1, so it is possible that RingoA-Cdk2 regulates the tethering of telomeres to the INM through phosphorylation of Sun1, but the detailed physiological mechanism remains unclear. In contrast to the co-localization of RingoA and Cdk2 in telomeres and sex chromosomes, RingoA does not co-localize with Cdk2 at crossing-over sites. Since Cdk2 has been reported to bind the laterecombination marker protein Mlh1, Cdk2 might participate in late-recombination events through the binding to a regulatory subunit other than RingoA. In addition to the regulation of Sun1 localization, RingoA seems to control chromatin methylation. RingoA-deficient spermatocytes show decreased levels of the histone H3