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Noriyuki Sagata

Publications and source records attributed to Noriyuki Sagata.

14 recordsLinked to original sources
PubMed2006

Erp1/Emi2 is essential for the meiosis I to meiosis II transition in Xenopus oocytes.

Erp1 (also called Emi2), an inhibitor of the APC/C ubiquitin ligase, is a key component of cytostatic factor (CSF) responsible for Meta-II arrest in vertebrate eggs. Reportedly, however, Erp1 is expressed even during meiosis I in Xenopus oocytes. If so, it is a puzzle why normally maturing oocytes cannot arrest at Meta-I. Here, we show that actually Erp1 synthesis begins only around the end of meiosis I in Xenopus oocytes, and that specific inhibition of Erp1 synthesis by morpholino oligos prevents entry into meiosis II. Furthermore, we demonstrate that premature, ectopic expression of Erp1 at physiological Meta-II levels can arrest maturing oocytes at Meta-I. Thus, our results show the essential role for Erp1 in the meiosis I/meiosis II transition in Xenopus oocytes and can explain why normally maturing oocytes cannot arrest at Meta-I.

Animals↗
PubMed2005

Beta-TrCP recognizes a previously undescribed nonphosphorylated destruction motif in Cdc25A and Cdc25B phosphatases.

Beta-TrCP, the F-box protein of the SCF(beta-TrCP) ubiquitin ligase (SCF, Skp1/Cul1/F-box protein), recognizes the doubly phosphorylated DSG motif (DpSGPhiXpS) in various SCF(beta-TrCP) target proteins. The Cdc25A phosphatase, a key cell-cycle regulator in vertebrate cells, undergoes a rapid ubiquitin-dependent degradation in response to genotoxic stress. Beta-TrCP binds to the DSG motif of human Cdc25A in a manner dependent on Chk1 and other unknown kinases. However, Xenopus Cdc25A does not have a DSG motif at the corresponding site of human Cdc25A. Here, we report that both Xenopus Cdc25A and human Cdc25A have a previously undescribed nonphosphorylated DDG motif (DDGPhiXD) for recognition by beta-TrCP. When analyzed by using Xenopus eggs, the binding of beta-TrCP to the DDG motif is essential for the Chk1-induced ubiquitination and degradation of Xenopus Cdc25A and also plays a role in the degradation of human Cdc25A. The DDG motif also exists in human Cdc25B phosphatase (another key cell-cycle regulator), binds beta-TrCP strongly, and is essential for the ubiquitination and degradation of the (labile) phosphatase in normal conditions. We provide strong evidence that, in both Cdc25A and Cdc25B, the binding (efficiency) of beta-TrCP to the DDG motif is regulated by nearby residues, while ubiquitination is regulated by other events in addition to the beta-TrCP binding. Finally, our additional data suggest that beta-TrCP may recognize nonphosphorylated DDG-like motifs in many other proteins, including X11L (a putative suppressor of beta-amyloid production) and hnRNP-U (a pseudosubstrate of SCF(beta-TrCP)).

Amino Acid Motifs↗
PubMed2005

The Polo-like kinase Plx1 interacts with and inhibits Myt1 after fertilization of Xenopus eggs.

During the meiotic cell cycle in Xenopus oocytes, p90(rsk), the downstream kinase of the Mos-MAPK pathway, interacts with and inhibits the Cdc2 inhibitory kinase Myt1. However, p90(rsk) is inactivated after fertilization due to the degradation of Mos. Here we show that the Polo-like kinase Plx1, instead of p90(rsk), interacts with and inhibits Myt1 after fertilization of Xenopus eggs. At the M phase of the embryonic cell cycle, Cdc2 phosphorylates Myt1 on Thr478 and thereby creates a docking site for Plx1. Plx1 can phosphorylate Myt1 and inhibit its kinase activity both in vitro and in vivo. The interaction between Myt1 and Plx1 is required, at least in part, for normal embryonic cell divisions. Finally, and interestingly, Myt1 is phosphorylated on Thr478 even during the meiotic cell cycle, but its interaction with Plx1 is largely inhibited by p90(rsk)-mediated phosphorylation. These results indicate a switchover in the Myt1 inhibition mechanism at fertilization of Xenopus eggs, and strongly suggest that Plx1 acts as a direct inhibitory kinase of Myt1 in the mitotic cell cycles in Xenopus.

Animals↗
PubMed2004

Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism.

Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell cycle progression. In vertebrates, Chk1 and Chk2 phosphorylate Cdc25A at multiple N-terminal sites and target it for rapid degradation in response to genotoxic stress. Here we show that Chk1, but not Chk2, phosphorylates Xenopus Cdc25A at a novel C-terminal site (Thr504) and inhibits it from C-terminally interacting with various Cdk-cyclin complexes, including Cdk1-cyclin A, Cdk1-cyclin B, and Cdk2-cyclin E. Strikingly, this inhibition, rather than degradation itself, of Cdc25A is essential for the Chk1-induced cell cycle arrest and the DNA replication checkpoint in early embryos. 14-3-3 proteins bind to Chk1-phosphorylated Thr504, but this binding is not required for the inhibitory effect of Thr504 phosphorylation. A C-terminal site presumably equivalent to Thr504 exists in all known Cdc25 family members from yeast to humans, and its phosphorylation by Chk1 (but not Chk2) can also inhibit all examined Cdc25 family members from C-terminally interacting with their Cdk-cyclin substrates. Thus, Chk1 but not Chk2 seems to inhibit virtually all Cdc25 phosphatases by a novel common mechanism.

14-3-3 Proteins↗
PubMed2004

Regulation of Chk1 kinase by autoinhibition and ATR-mediated phosphorylation.

The checkpoint kinase Chk1 undergoes ATR-mediated phosphorylation and activation in response to unreplicated DNA, but the precise mechanism of Chk1 activation is not known. In this study, we have analyzed the domain structure of Xenopus Chk1 and explored the mechanism of its activation by ATR-mediated phosphorylation. We show that the C-terminal region of Xenopus Chk1 contains an autoinhibitory region (AIR), which largely overlaps with a bipartite, unusually long ( approximately 85-amino acid) nuclear localization signal. When coexpressed in oocytes or embryos, the AIR can interact with and inhibit the kinase domain of Chk1, but not full-length Chk1, suggesting an autoinhibitory intramolecular interaction in the Chk1 molecule. If linked with the preceding ATR phosphorylation domain that has either phospho-mimic mutation or genuine phosphorylation, however, the AIR can no longer interact with or inhibit the kinase domain, suggesting a conformational change of the AIR by ATR-mediated phosphorylation. Even in full-length Chk1, such phospho-mimic mutation can interfere with the autoinhibitory intramolecular interaction, but only if this interaction is somewhat weakened by an additional mutation in the AIR. These results provide significant insights into the mechanism of Chk1 activation at the DNA replication checkpoint.

Amino Acid Motifs↗
PubMed2003

The RRASK motif in Xenopus cyclin B2 is required for the substrate recognition of Cdc25C by the cyclin B-Cdc2 complex.

The FLRRXSK sequence is conserved in the second cyclin box fold of B-type cyclins. We show that this conserved sequence in Xenopus cyclin B2, termed the RRASK motif, is required for the substrate recognition by the cyclin B-Cdc2 complex of Cdc25C. Mutations to charged residues of the RRASK motif of cyclin B2 abolished its ability to activate Cdc2 kinase without affecting its capacity to bind to Cdc2. Cdc2 bound to the cyclin B2 RRASK mutant was not dephosphorylated by Cdc25C, and as a result, the complex was inactive. The cyclin B2 RRASK mutants can form a complex with the constitutively active Cdc2, but a resulting active complex did not phosphorylate a preferred substrate Cdc25C in vitro, although it can phosphorylate the non-specific substrate histone H1. The RRASK mutations prevented the interaction of Cdc25C with the cyclin B2-Cdc2 complex. Consistently, the RRASK mutants neither induced germinal vesicle breakdown in Xenopus oocyte maturation nor activated in vivo Cdc2 kinase during the cell cycle in mitotic extracts. These results suggest that the RRASK motif in Xenopus cyclin B2 plays an important role in defining the substrate specificity of the cyclin B-Cdc2 complex.

Amino Acid Sequence↗
PubMed2003

Expression of cell-cycle regulators during Xenopus oogenesis.

In full-grown Xenopus oocytes, cell-cycle regulators and an inactive form of maturation/M phase promoting factor (pre-MPF) are stored ready to bring about a specific cell cycle for oocyte maturation. We examined the expression pattern of these cell-cycle regulators as well as pre-MPF formation during oogenesis. Cdc2 and Cyclin B2 were already present in stage I oocytes and pre-MPF formation was also detected in stage I oocytes. Some negative regulators of MPF, Myt1 and Chk1, were synthesized early in oogenesis. In contrast, positive regulators of MPF, MEK, MAPK and Cdc25C, were mainly synthesized late in oogenesis. Northern blotting analysis suggested that the synthesis of these cell-cycle regulators was controlled by translation.

Animals↗
PubMed2003

Mr 25 000 protein, a substrate for protein serine/threonine kinases, is identified as a part of Xenopus laevis vitellogenin B1.

A phosphorylated protein with a molecular mass of 25 000 (pp25) previously purified from the cytosolic fraction of Xenopus laevis oocytes is an effective phosphate acceptor for casein kinases and protein kinase C. In this study, based on the partial amino acid sequence of pp25, a cDNA was isolated that encodes a new yolk precursor protein, Xenopus vitellogenin B1, which contained the sequence encoding pp25. Both mRNA and protein of vitellogenin B1 were expressed in all of the female organs examined. In agreement with a previous report, the amount of vitellogenin B1 protein in the liver increased after stimulation with estrogen. These results suggest that pp25 is a cytosolic non-crystallized yolk protein nutrient source, but it might also play a role in rapid development.

Amino Acid Sequence↗
PubMed2003

The C-terminal seven amino acids in the cytoplasmic retention signal region of cyclin B2 are required for normal bipolar spindle formation in Xenopus oocytes and embryos.

In many vertebrates, cyclin B has several subtypes, but the functional differences among them are largely unclear. Previously, we have shown that Xenopus cyclin B2, not cyclin B1, is involved in bipolar spindle formation through its cytoplasmic retention signal (CRS) region. However, identification of a nuclear export signal (NES) in the CRS region of cyclin B1 raised the possibility that an NES-like sequence (NELS) present in the CRS region of cyclin B2 might be involved in bipolar spindle formation. We show here that cyclin B2 is actually exported from the nucleus via its NELS, but that overexpression of the cyclin B2 CRS region, having a mutated NELS, still inhibits bipolar spindle formation in oocytes. In contrast, overexpression of the cyclin B2 CRS region lacking its C-terminal seven amino acids no longer inhibits bipolar spindle formation in oocytes or embryos. These results suggest strongly that the CRS region, especially its C-terminal seven acidic residues, of cyclin B2 is required for bipolar spindle formation in both the meiotic and mitotic cell divisions.

Active Transport, Cell Nucleus↗
PubMed2002

Requirement for both EDEN and AUUUA motifs in translational arrest of Mos mRNA upon fertilization of Xenopus eggs.

Translational arrest of maternal Mos mRNA upon fertilization of Xenopus eggs is a prerequisite for the initiation of embryonic divisions. Recent studies suggest that an embryo deadenylation element (EDEN) present in the 3' untranslated region (3'UTR) is sufficient for deadenylation (and, hence, probably for translational arrest) of Mos mRNA after fertilization. By directly monitoring translation of numerous Mos mRNA constructs in Xenopus eggs, however, we show here that the EDEN is necessary but not sufficient for translational arrest of Mos mRNA. We demonstrate that two AUUUA motifs, each located solitarily and distantly from the EDEN, are also required for the translational arrest of Mos mRNA after fertilization. Significantly, translational arrest of Eg2 mRNA, another EDEN-containing maternal mRNA, also requires a single AUUUA motif located far from the EDEN. Analysis of the poly(A) tails of various Mos mRNA constructs indicates that the EDEN alone confers only partial deadenylation on Mos mRNA, and that the AUUUA motifs act to enhance EDEN-directed deadenylation in a position-dependent manner. Finally, introduction of an excess of the EDEN, but not the AUUUA motifs, into eggs can restore translation of endogenous Mos mRNA. These results suggest that the EDEN, only together with appropriately positioned AUUUA motifs and a trans-acting factor(s), can efficiently deadenylate and hence translationally arrest Mos (as well as Eg2) mRNA after fertilization.

3' Untranslated Regions↗
PubMed2002

Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition.

In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication checkpoint is thought to be activated by the exponentially increased nucleo-cytoplasmic ratio. Here we show that the checkpoint kinase Chk1, but not Cds1 (Chk2), is activated transiently at the MBT in a maternal/zygotic gene product-regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre-MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1-independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chk1, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development.

Animals↗
PubMed2002

The existence of two distinct Wee1 isoforms in Xenopus: implications for the developmental regulation of the cell cycle.

In eukaryotic cells, the Wee1 protein kinase phosphorylates and inhibits Cdc2, thereby creating an interphase of the cell cycle. In Xenopus, the conventional Wee1 homolog (termed Xe-Wee1A, or Wee1A for short) is maternally expressed and functions in pregastrula embryos with rapid cell cycles. Here, we have isolated a second, zygotic isoform of Xenopus Wee1, termed Xe-Wee1B (or Wee1B for short), that is expressed in postgastrula embryos and various adult tissues. When ectopically expressed in immature oocytes, Wee1B inhibits Cdc2 activity and oocyte maturation (or entry into M phase) much more strongly than Wee1A, due to its short C-terminal regulatory domain. Moreover, ectopic Wee1B, unlike Wee1A, is very labile during meiosis II and cannot accumulate in mature oocytes due to the presence of PEST-like sequences in its N-terminal regulatory domain. Finally, when expressed in fertilized eggs, ectopic Wee1B but not Wee1A does affect cell division and impair cell viability in early embryos, due primarily to its very strong kinase activity. These results suggest strongly that the differential expression of Wee1A and Wee1B is crucial for the developmental regulation of the cell cycle in Xenopus.

Amino Acid Sequence↗