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N Sagata

Publications and source records attributed to N Sagata.

At least 37 records · Page 2Linked to original sources
PubMed1995

MAP kinase activation is essential for oncogenic transformation of NIH3T3 cells by Mos.

The c-mos proto-oncogene product, Mos, is a serine/threonine protein kinase that controls the meiotic cell cycle in vertebrate oocytes. Both in vivo and in vitro, Mos can activate mitogen-activated protein kinase (MAPK) most probably by direct phosphorylation of MAPK kinase (MAPKK). In many cell types transformed by diverse oncogene products such as Raf, MAPK is constitutively activated, suggesting that the MAPK pathway may mediate oncogenic signalling by many oncogene products. Using mouse NIH3T3 cells, we examined whether oncogenic transformation by Mos is mediated by MAPK activation. Coexpression of a kinase-defective (dominant-negative) mutant of Mek1, one of the MAPKK isoforms, completely suppressed transformation by Mos. By contrast, coexpression of wild-type Mek1 markedly enhanced the transforming efficiency of Mos. Moreover, overexpression of the dominant-negative Mek1 reverted the transformation phenotype of Mos-transformed cells. These results indicate that in NIH3T3 cells the Mek1/MAPK pathway is necessary and sufficient for transformation (and its maintenance) by Mos. Transformation of NIH3T3 cells by Raf or Ras was also suppressed by the dominant-negative Mek1, but significantly less efficiently than that by Mos, suggesting the existence of multiple signalling pathways for Raf and Ras oncoproteins.

3T3 Cells↗
PubMed1995

Degradation of c-Fos by the 26S proteasome is accelerated by c-Jun and multiple protein kinases.

c-Fos is associated with c-Jun to increase the transcription of a number of target genes and is a nuclear proto-oncoprotein with a very short half-life. This instability of c-Fos may be important in regulation of the normal cell cycle. Here we report a mechanism for degradation of c-Fos. Coexpression of c-Fos and c-Jun in HeLa cells caused marked increase in the instability of c-Fos, whereas v-Fos, the retroviral counterpart of c-Fos, was stable irrespective of the coexpression of c-Jun. Interestingly, deletion of the C-terminal PEST region of c-Fos, which is altered in v-Fos by a frameshift mutation, greatly enhanced its stability, with loss of the effect of c-Jun on its stability. c-Fos synthesized in vitro was degraded by the 26S proteasome in a ubiquitin-dependent fashion. Simple association with c-Jun had no effect on the degradation of c-Fos, but the additions of three protein kinases, mitogen-activated protein kinase, casein kinase II, and CDC2 kinase, resulted in marked acceleration of its degradation by the proteasome-ubiquitin system, though only in the presence of c-Jun. In contrast, v-Fos and c-Fos with a truncated PEST motif were not degraded, suggesting that they escaped from down-regulation by breakdown. These findings indicate a new oncogenic pathway induced by acquisition of intracellular stability of a cell cycle modulatory factor.

Amino Acid Sequence↗
PubMed1994

Parthenogenetic activation of oocytes in c-mos-deficient mice.

In Xenopus the c-mos proto-oncogene product (Mos) is essential for the initiation of oocyte maturation, for the progression from meiosis I to meiosis II and for the second meiotic metaphase arrest, acting as an essential component of the cytostatic factor CSF. Its function in mouse oocytes is unclear, however, as is the biological significance of c-mos mRNA expression in testes and several somatic tissues. We have generated c-mos-deficient mice by gene targeting in embryonic stem cells. These mice grew at the same rate as their wild-type counterparts and reproduction was normal in the males, but the fertility of the females was very low. The c-mos-deficient female mice developed ovarian teratomas at a high frequency. Oocytes from these females matured to the second meiotic metaphase both in vivo and in vitro, but were activated without fertilization. The results indicate that in mice Mos plays a role in the second meiotic metaphase arrest, but does not seem to be essential for the initiation of oocyte maturation, spermatogenesis or somatic cell cycle.

Animals↗
PubMed1994

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.

Animals↗
PubMed1993

Mos is degraded by the 26S proteasome in a ubiquitin-dependent fashion.

Mos, the c-mos proto-oncogene product, is a key regulator of cell cycle progression. Recently, rapid turnover of Mos in an early stage of meiotic maturation of Xenopus oocytes was found to be mediated by the ubiquitin pathway, but the protease responsible for its breakdown was not identified. In the present study, we found that 35S-labeled Mos synthesized in an in vitro transcription/translation system was degraded ATP- and time-dependently by the 26S proteasome, but not by the 20S proteasome, in the presence of a ubiquitin-ligation system. The 26S proteasome did not degrade a mutant Mos in which Ser3 was replaced by Asp3 that is metabolically stable in oocytes, indicating a similarity in the proteolytic events in vivo to those observed in vitro in the present work. This is the first demonstration that the proteasome catalyzes the ATP-dependent degradation of a naturally occurring, short-lived oncoprotein by the ubiquitin pathway. This finding suggests that the proteasome may regulate the intracellular stability of various oncoproteins.

Adenosine Triphosphate↗
PubMed1993

Degradation of Mos by the N-terminal proline (Pro2)-dependent ubiquitin pathway on fertilization of Xenopus eggs: possible significance of natural selection for Pro2 in Mos.

The c-mos proto-oncogene product (Mos), an essential component of the cytostatic factor responsible for meiotic arrest in vertebrate eggs, undergoes specific proteolysis soon after fertilization or activation of Xenopus eggs. To determine the degradation pathway of Mos on egg activation, various Mos mutants were expressed in Xenopus eggs and their degradation on egg activation was examined. Mos degradation absolutely required its penultimate proline (Pro2) residue and dephosphorylation of the adjacent serine (Ser3) residue. These degradation signals were essentially the same as those of Mos in meiosis I of Xenopus oocyte maturation, where Mos has been shown to be degraded by the 'second-codon rule'-based ubiquitin pathway. To test whether Mos degradation on egg activation is also mediated by the ubiquitin pathway, we attempted to identify and abrogate a specific ubiquitination site(s) in Mos. We show that the major ubiquitination site in Mos is a Lys34 residue and that replacement of this residue with a non-ubiquitinatable Arg residue markedly enhances the stability of Mos on egg activation. These results indicate that the degradation of Mos on egg activation or fertilization is mediated primarily by the N-terminal Pro2-dependent ubiquitin pathway, as in meiosis I of oocyte maturation. The N-terminal Pro2 residue of Mos appears to be naturally selected primarily for its degradation on fertilization, rather than that in meiosis I.

Amino Acid Sequence↗
PubMed1993

[CSF and Mos].

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Cell Cycle↗
PubMed1992

The 'second-codon rule' and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes.

The c-mos proto-oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N-terminal residue, or by a rule referred to here as the 'second-codon rule'. We demonstrate that unstable Mos is degraded by the ubiquitin-dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N-terminus of Mos from being recognized by a ubiquitin-protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N-terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle.

Amino Acid Sequence↗
PubMed1992

Differential occurrence of CSF-like activity and transforming activity of Mos during the cell cycle in fibroblasts.

The Xenopus c-mos proto-oncogene product, Mosxe, possesses cytostatic factor (CSF) activity to arrest maturing oocytes in metaphase II and has weak transforming activity in mouse NIH3T3 cells. We show that Mosxe mutants bearing 'stabilizing' penultimate N-terminal amino acids are strongly transforming and can retard progression through the G2-M phases in Mosxe-transformed cells, probably via their CSF activity. On the other hand, a cyclin-Mosxe fusion protein, which undergoes abrupt degradation at the end of mitosis and is restored to its normal levels only after the G1 phase, transforms cells much less efficiently than a mutated cyclin-Mosxe fusion protein that is stable during M-G1 transition. Moreover, in low-serum medium, cells transformed by the unstable cyclin-Mosxe require a long period to enter the S phase, in contrast with the rapid entry into the S phase of cells transformed by the stable cyclin-Mosxe. These results provide strong evidence that unlike the physiological CSF activity, the transforming activity of Mos is exerted in the G1 phase of the cell cycle.

3T3 Cells↗
PubMed1991

Independent inactivation of MPF and cytostatic factor (Mos) upon fertilization of Xenopus eggs.

In vertebrates, mature eggs are arrested at the second meiotic metaphase by the cytostatic factor (CSF), now known to be the c-mos proto-oncogene product (Mos). Fertilization or egg activation triggers a transient increase in the cytoplasmic free calcium and releases the meiotic arrest by inactivating maturation/mitosis-promoting factor (MPF). CSF or Mos, which is also inactivated by the calcium transient, seems to stabilize MPF in mature eggs and CSF-injected embryos. Thus, it was assumed that CSF inactivation is the primary cause of MPF inactivation on meiotic release. We have directly compared the degradation kinetics of CSF (Mos) and MPF during meiotic release, using the same batch of Xenopus eggs. We report here that, at the molecular level, cyclin subunits of MPF are degraded before Mos is degraded and, at the physiological level, that MPF activity is inactivated before CSF activity during activation of Xenopus eggs. These results, in conjunction with circumstantial evidence, support the novel view that a calcium transient on fertilization induces a CSF-independent pathway for MPF inactivation, whereas CSF inactivation during meiotic release serves only to allow the fertilized egg to enter mitosis.

Animals↗
PubMed1991

Correlation between physiological and transforming activities of the c-mos proto-oncogene product and identification of an essential Mos domain for these activities.

Using Xenopus eggs and NIH3T3 cells as assay systems, we have compared the physiological (i.e., maturation-inducing and cleavage-arresting) and in vitro transforming activities of the c-mos genes from various species as well as their mutant genes. These analyses show that the three biological activities all depend upon the intrinsic protein kinase activity of Mos and correlate well with each other. Furthermore, our results demonstrate that a well conserved N-terminal 14-amino acid sequence of Mos, termed the Mos-box, is essential for all three activities. These results indicate that the in vitro transforming activity of Mos can be ascribed to the same kinase activity of Mos that exerts the physiological activities.

Amino Acid Sequence↗
PubMed1990

mos proto-oncogene function.

Maturation promoting factor (MPF) is a cytoplasmic activity that causes oocytes arrested in prophase to resume meiosis. An inactive form of MPF termed pre-MPF exists in fully grown oocytes. In Xenopus oocytes, progesterone induces maturation and pre-MPF activation. These early maturation events require protein synthesis. We have shown that p39mos synthesis is rapidly induced in progesterone-treated Xenopus oocytes during the protein synthesis sensitive period and prior to activation of pre-MPF or germinal vesicle breakdown (GVBD). p39mos may qualify, therefore, as an 'initiator' of maturation. Mouse oocytes undergoing meiotic maturation also express p39mos. Microinjection of antisense mos oligodeoxynucleotides into fully grown mouse and Xenopus oocytes results in the block of meiotic maturation. In Xenopus, antisense-injected oocytes not only lack p39mos, but also lack MPF and fail to undergo GVBD. In the mouse, the microinjected oocytes progress through GVBD, but fail to produce the first polar body; cytogenetic analysis shows they are arrested at the bivalent chromosome stage of metaphase I. This and additional studies with Xenopus oocytes indicate that p39mos is also required throughout maturation. We have shown that p39mos is indistinguishable from the protein product constitutively expressed in NIH/3T3 cells transformed with activated c-mos. It is likely that its activity as a transforming gene may be due to activation of pre-MPF activities in interphase in the somatic cell cycle.

Animals↗
PubMed1989

Specific proteolysis of the c-mos proto-oncogene product by calpain on fertilization of Xenopus eggs.

The Xenopus c-mos proto-oncogene product, pp39mos, accumulates in the unfertilized egg during maturation, is hyperphosphorylated and exhibits protein kinase activity. On fertilization, or soon after the completion of meiosis, the accumulated pp39mos undergoes selective proteolysis. Using an in vitro protease assay system, we show here that this specific proteolysis is caused by the calcium-dependent cysteine protease, calpain.

Animals↗
PubMed1989

The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs.

The c-mos proto-oncogene product, pp39mos, is present in unfertilized Xenopus eggs, and disappears on fertilization. Microinjection of synthetic mos RNA into two-cell embryos induces cleavage arrest at metaphase. By contrast, egg cytosol extracts, when immunodepleted of endogenous pp39mos, lose their cleavage-arresting activity in injected embryos. These results demonstrate that Mos protein is the cytostatic factor CSF, long known as an endogenous meiotic inhibitor in vertebrate eggs.

Animals↗
PubMed1989

The product of the mos proto-oncogene as a candidate "initiator" for oocyte maturation.

The endogenous c-mos product, pp39mos, is required for progesterone-induced meiotic maturation in Xenopus oocytes. Treatment of oocytes with progesterone induced a rapid increase in pp39mos that preceded both the activation of maturation promoting factor (MPF) and germinal vesicle breakdown (GVBD). Microinjection of synthetic mos RNA into oocytes activated MPF and induced GVBD in the absence of progesterone. Thus, the mos proto-oncogene product may qualify as a candidate "initiator" protein of MPF and is at least one of the "triggers" for G2 to M transition.

Animals↗
PubMed1988

Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes.

The c-mos proto-oncogene is expressed as a maternal mRNA in oocytes and early embryos of Xenopus laevis, but its translation product pp39mos is detectable only during progesterone-induced oocyte maturation. Microinjection of mos-specific antisense oligonucleotides into oocytes not only prevents expression of pp39mos, but also blocks germinal vesicle breakdown, indicating that it functions during reinitiation of meiotic division.

Animals↗
PubMed1988

Immunogenicity of a recombinant vaccinia virus expressing envelope a glycoprotein of bovine leukaemia virus.

We constructed a recombinant vaccinia virus (RVV) expressing envelope (env) glycoprotein (gp51) of bovine leukaemia virus (BLV): the expression of gp51 was detected by Western blot of the lysates of rabbit kidney cells infected with the RVV. The rabbits inoculated intradermally with the RVV alone failed to induce detectable anti-gp51 antibodies even 10 weeks after immunization. However, when these animals were boosted with inactivated BLV virion in saline, significant levels of anti-gp51 antibodies were induced as shown both in Western blot and immunodiffusion analyses. In these animals, antibodies against gag product (p24) were not detected. On the other hand, the rabbits inoculated with wild-type vaccinia virus and boosted similarly three times with the BLV virion in saline did not induce detectable anti-gp51 antibodies at all. The present experiment revealed that the RVV possessed the capability to endow immunological memory without inducing apparent anti-gp51 antibody responses, meaning that the RVV activated helper T cells far more strongly than B cells. The applicability of the RVV to vaccine development is discussed.

Animals↗