Keita Ohsumi

Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs.

Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins—regulatory proteins of meiosis and mitosis—for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.

Residual Cdc2 activity remaining at meiosis I exit is essential for meiotic M–M transition in Xenopus oocyte extracts

To investigate the regulatory mechanisms of the cell cycle transition from M phase to M phase in meiotic cycles, a Xenopus oocyte extract that performs the M–M transition has been developed. Using the meiotic extract, we found that a low level of Cdc2 activity remained at the exit of meiosis I (MI), due to incomplete degradation of cyclin B. The inactivation of the residual Cdc2 activity induced both entry into S phase and tyrosine phosphorylation on Cdc2 after MI. Quantitative analysis demonstrated that a considerable amount of Wee1 was present at the MI exit and Cdc2 inhibitory phosphorylation during this period was suppressed by the dominance of Cdc2 over Wee1. Consistently, the addition of more than a critical amount of Wee1 to the extract induced Cdc2 inhibitory phosphorylation, changing the M–M transition into an M–S–M transition. Thus, the Cdc2 activity remaining at MI exit is required for suppressing entry into S phase during the meiotic M–M transition period.

Developmentally regulated, alternative splicing of the Rpn10 gene generates multiple forms of 26S proteasomes.

The 26S proteasome is a multisubunit protein‐ destroying machinery that degrades ubiquitin‐tagged proteins. To date only a single species of Rpn10, which possibly functions as a multiubiquitin chain‐binding subunit, has been identified in various organisms. Here we report that mouse Rpn10 mRNAs occur in at least five distinct forms, named Rpn10a to Rpn10e, and that they are generated from a single gene by developmentally regulated, alternative splicing. Rpn10a is ubiquitously expressed, whereas Rpn10e is expressed only in embryos, with the highest levels of expression in the brain. Both forms of Rpn10 are components of the 26S proteasome, with an apparently similar affinity for multiubiquitylated [125I]lysozyme in vitro. However, they exert markedly divergent effects on the destruction of B‐type cyclin in Xenopus egg extracts. Thus, the 26S proteasome occurs in at least two functionally distinct forms: one containing a ubiquitously expressed Rpn10a and the other a newly identified, embryo‐specific Rpn10e. While the former is thought to perform proteolysis constitutively in a wide variety of cells, the latter may play a specialized role in early embryonic development.

Identification and analyses of the Xenopus TERT gene that encodes the catalytic subunit of telomerase

The Xenopus telomerase catalytic component gene, xTERT (Xenopus telomerase reverse transcriptase), has been cloned. The production of xTERT recombinant protein together with the proposed Xenopus telomerase RNA (xTR) (Chen et al., 2000. Cell 100, 503-514) in a rabbit reticulocyte lysate system led to the reconstitution of active telomerase, indicating that both products are functional telomerase components. Both xTERT expression and telomerase activity are high from the early to the late blastula stage. However, they are decreased at the gastrula stage and thereafter, suggesting that the xTERT expression level is the primary mechanism for regulating telomerase activity in Xenopus development. This is the first report of a non-mammalian vertebrate TERT gene. Sequence comparison of xTERT with human and mouse TERTs has uncovered four regions conserved in the amino-terminal halves of vertebrate TERT proteins, the functions of which will be discussed herein.

Remodeling of Human Sperm Chromatin Mediated by Nucleoplasmin from Amphibian Eggs

The molecular events associated with decondensation of human sperm nuclei were analyzed by incubating sperm with egg extracts from an amphibian, Bufo japonicus. Acid‐urea‐Triton polyacrylamide gel electrophoresis (AUT‐PAGE) showed that the nuclear basic proteins of human sperm consist mainly of protamines (HPI, HPII) with minor amounts of nucleosomal histones. On incubation of lysolecithin (LC)‐ and dithiothreitol (DTT)‐treated human sperm with the egg extract, the nuclei lost HPI and HPII within 15 min in association with extensive nuclear decondensation, and the acquirement of a whole set of nucleosomal histones. Incubation of LC‐DTT‐sperm with nucleoplasmin purified from Bufo eggs also induced nuclear decondensation and loss of protamines within 30 min. Native‐PAGE and Western blot analyses of incubation medium indicated tight association of the released protamines to nucleoplasmin, strongly suggesting that protamines are removed from sperm nuclei not enzymatically but by their specific binding to nucleoplasmin. On incubation of LC‐DTT‐sperm with nucleoplasmin and exogenous nucleosomal core histones, micrococcal nuclease‐protected DNA fragments were released, although their unit repeat length was slightly less than that of somatic nucleosomes. Thus remodeling of human sperm during fertilization can be mimicked under defined conditions with nucleoplasmin and exogenous histones.

Cell‐cycle‐dependent Xenopus TRF1 recruitment to telomere chromatin regulated by Polo‐like kinase

Telomeres are regulated by a homeostatic mechanism that includes telomerase and telomeric repeat binding proteins, TRF1 and TRF2. Recently, it has been hypothesized that telomeres assume distinct configurations in a cell‐cycle‐dependent manner, although direct biochemical evidence is lacking. Here we demonstrated that Xenopus TRF1 (xTRF1) associates with telomere chromatin specifically in mitotic Xenopus egg extracts, and dissociates from it upon mitotic exit. Both the N‐terminal TRF‐homology (TRFH) domain and the linker region connecting the TRFH domain and the C‐terminal Myb domain are required for this cell‐cycle‐dependent association of xTRF1 with chromatin. In contrast, Xenopus TRF2 (xTRF2) associates with chromatin throughout the cell cycle. We showed that Polo‐like kinase (Plx1) phosphorylates xTRF1 in vitro. Moreover, the mitotic xTRF1–chromatin association was significantly impaired when Plx1 was immunodepleted from the extracts. Finally, high telomerase activities were detected in association with replicating interphase chromatin compared with mitotic chromatin. These results indicate that telomere chromatin is actively regulated by cell‐cycle‐dependent processes, and provide an insight for understanding how telomeres undergo DNA metabolisms during the cell cycle.

Oocyte extracts for the study of meiotic M-M transition.

In meiotic cell cycles, meiosis I (MI) is followed by meiosis II (MII) without an intervening S phase, whereas in mitotic cell cycles, an S phase necessarily alternates with an M phase. For the study of mitotic cell cycles, extracts prepared from unfertilized and parthenogenetically activated Xenopus eggs have been very useful as they can perform the progression of mitotic cycles in vitro. To establish a cell-free system to study the regulatory mechanisms of meiotic transition from MI to MII, extracts have been prepared from maturing Xenopus oocytes isolated from ovaries, stimulated with progesterone to induce the resumption of meiosis, and arrested at meiotic metaphase I by cold treatment. In oocyte extracts, the activity of cyclin B-Cdc2 complexes, the M phase inducer, fluctuates in the same manner as it does in maturing oocytes during the MI to MII transition period. By the use of oocyte extracts, it has been found that incomplete inactivation of Cdc2 at the end of MI is required for meiotic M-M transition. The meiotic extract should provide a useful tool to elucidate molecular mechanisms of meiotic M to M transition, including a role of Mos/mitogen-activated protein kinase cascade in the suppression of S phase entry after MI exit. In this chapter, we describe methods for the preparation and the uses of meiotic extracts. As a comparison, we also include a protocol for the preparation of mitotic extracts.

An indirect role for cyclin B-Cdc2 in inducing chromosome condensation in Xenopus egg extracts

We have studied the cytoplasmic mechanism that induces metaphase chromosome condensation in cell-free Xenopus egg extracts. To analyze the mechanism responsible for inducing chromosome condensation separately from those responsible for sperm chromatin remodeling and nuclear envelope disassembly, we used Xenopus sperm chromatin that had already been remodeled to nucleosomal chromatin by incubating demembranated sperm with egg extracts added with lysolecithin. We found that inhibition of cyclin B-Cdc2 with butyrolactone I abolished chromosome condensation of the remodeled sperm chromatin by M-phase egg extracts, but incubation of the chromatin with active cyclin B-Cdc2 alone did not induce chromosome condensation, indicating a requirement for cytoplasmic factor(s) in addition to cyclin B-Cdc2 for the induction of chromosome condensation. We further demonstrated that if the cyclin B-Cdc2-dependent phosphorylation state was protected against dephosphorylation by a preincubation of M-phase extracts with ATP-gamma-S, chromosome condensation and phosphorylation of chromosomal histone H1 occurred even when extracts were depleted of cyclin B-Cdc2 activity. The chromosome condensation seen in the absence of cyclin B-Cdc2 was completely inhibited with another protein kinase inhibitor, 6-dimethylaminopurine, implying that a protein kinase other than cyclin B-Cdc2 was involved in the induction of chromosome condensation. These results strongly suggest that a cyclin B-Cdc2-dependent protein kinase cascade is involved in inducing chromosome condensation and the phosphorylation of chromosomal histone H1.

Dependence of removal of sperm‐specific proteins from Xenopus sperm nuclei on the phosphorylation state of nucleoplasmin

In Xenopus laevis, nucleoplasmin from fully grown oocytes is not highly phosphorylated, but is more extensively phosphorylated during oocyte maturation to retain this state until mid‐blastula transition. Incubation of demembranated sperm with nucleoplasmin from oocytes or mature eggs revealed that egg nucleoplasmin is twice as potent as oocyte nucleoplasmin in removing sperm‐specific basic proteins from chromatin (protamine‐removing activity: PRA). Dephosphorylation of egg nucleoplasmin by alkaline phosphatase induced a remarkable decline of PRA in nucleoplasmin. Treatment of oocyte nucleoplasmin with cdc2 protein kinase induced an increase of the extent of phosphorylation, but to a level lower than that exhibited by egg nucleoplasmin, suggesting the involvement of other unspecified kinase(s) in phosphorylating nucleoplasmin during oocyte maturation. Incubation of sperm with cdc2 kinase induced selective phosphorylation of sperm‐specific basic proteins, accompanied by their enhanced removal from sperm chromatin upon exposure to high‐salt solutions. These results suggest that removal of sperm‐specific basic proteins from sperm chromatin in fertilized eggs is facilitated by phosphorylation of both nucleoplasmin and sperm‐specific basic proteins.