The Mos/MAPK pathway is involved in metaphase II arrest as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish.

Mos plays a crucial role in meiotic cell division in vertebrates. In Xenopus, Mos is involved in the initiation of oocyte maturation as an initiator and in the arrest at the metaphase II stage (MII) as a component of the cytostatic factor (CSF). The function of Mos is mediated by MAP kinase (MAPK). We investigated the function of the Mos/MAPK pathway during goldfish oocyte maturation induced by 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), a natural maturation-inducing hormone in fishes. Mos was absent in immature goldfish oocytes. It appeared before the onset of germinal vesicle breakdown (GVBD), increased to a maximum in mature oocytes arrested at MII and disappeared after fertilization. MAPK was activated after Mos synthesis but before maturation-promoting factor (MPF) activation, and its activity reached maximum at MII. Injection of either Xenopus or goldfish c-mos mRNA into one blastomere of 2-cell-stage Xenopus and goldfish embryos induced metaphase arrest, suggesting that goldfish Mos has a CSF activity. Injection of constitutively active Xenopus c-mos mRNA into immature goldfish oocytes induced MAPK activation, but neither MPF activation nor GVBD occurred. Conversely, the injection of goldfish c-mos antisense RNA inhibited both Mos synthesis and MAPK activation in the 17alpha,20beta-DP-treated oocytes, but these oocytes underwent GVBD. These results indicate that the Mos/MAPK pathway is not essential for initiating goldfish oocyte maturation despite its general function as a CSF. We discuss the general role of Mos/MAPK during oocyte maturation, with reference to the difference in contents of inactive MPF (pre-MPF) stored in immature oocytes.

Mos proto-oncogene function during oocyte maturation in Xenopus.

The function of the Xenopus c-mos proto-oncogene product (Mos(xe)) has been investigated during oocyte maturation. Experiments with a new antibody able to immunoblot Mos(xe) demonstrated the time course of MAP kinase (MAP K) activation in oocytes paralleled Mos(xe) accumulation, and in activated eggs the deactivation of MAP K paralleled the degradation of Mos(xe). Ablation of Mos synthesis by microinjection of antisense oligodeoxynucleotides abolished activation of MAP K by progesterone, but microinjection of GST-Mos fully restored both MAP K activation and germinal vesicle breakdown (GVBD). The Mos(xe) level at metaphase of Meiosis I (MI) was 2 - 3-fold less than that at metaphase of Meiosis II (MII), but MAP K activation was maximal at metaphase in both MI and MII. In the transition between MI and MII, both cyclin B and Mos(xe) levels rapidly declined in the presence of cycloheximide and injection of exogenous GST-Mos(xe) did not prevent degradation of either protein, although MAP K was activated. Microinjection of GST-Mos(xe) into oocytes was able to activate MAP K before GVBD and H1 kinase activation, and microinjection of constitutively-activated thiophosphorylated MAP K induced de novo synthesis of Mos(xe) before H1 kinase activation, suggesting the existence of a positive feedback loop between MAP K and Mos(xe) accumulation.

Mutations in the v-mos gene abolish its ability to induce differentiation but not transformation.

The v-mos oncogene product has the ability to induce differentiation in human monocytic leukemia U937 cells, thereby arresting cell proliferation, and also exhibits transforming activity in mouse NIH3T3 cells. Mutation in the v-mos gene consisting of one or two amino acid substitutions in the putative ATP-binding domain impaired its differentiation-inducing activity although mutant proteins showed rather higher levels of autophosphorylation in vitro. Macrophage-specific characteristics such as their morphology, expression of C3b receptor and Fc receptor, and production of interleukin-1 beta and tumor necrosis factor alpha, were equally diminished in cells transfected with mutant mos genes when compared to those with intact v-mos. The ability of the gene to arrest the proliferation of U937 cells was likewise diminished, while the transforming efficiency of the intact and mutant mos genes were essentially the same. These results suggest that the mos product functions differently in cell differentiation and transformation.

Characterization of MEK1 phosphorylation by the v-Mos protein.

Activation of MAP kinase/Erk Kinase (MEK) via direct phosphorylation by Mos may be crucial for cellular transformation by the activated c-mos or v-mos gene. Recent studies on a number of different protein kinases showed that phosphorylation within a subdomain of the catalytic domain may represent a common mode of activation. In this regard, activation of MEK1 by Raf involves phosphorylation of serine residues 218 and 222. Here we show that recombinant kinase-inactive MEK1 is phosphorylated by v-Mos with equal efficiency at both Ser 218 and Ser 222 in vitro. Tryptic phosphopeptide analysis of glutathione-S-transferase (GST)-MEK1 K97R and its alanine-for-serine mutants indicated that Ser 222 is the preferred phosphorylation site. Wild-type GST-MEK1 was phosphorylated at the same sites but contained a significantly lower amount of doubly phosphorylated species then its K97R kinase-inactive mutant. The ratio of GST-MEK1 species phosphorylated at two serines to those phosphorylated at one serine was similar in auto-phosphorylated and v-Mos-phosphorylated GST-MEK1. Consistent with the in vitro data, phosphopeptide mapping of MEK1 immunoprecipitated from mos transformed cells showed an increased amount of singly phosphorylated phosphopeptide compared to nontransformed cels. MEK1 was found to be more highly activated in NIH3T3 cells transformed by an activated c-mos or v-mos gene than in cells growing normally in medium containing serum. Our data indicate that Mos activated MEK1 in vitro as well as in vivo by phosphorylating Ser 222.

Overexpression of mos oncogene product in Swiss 3T3 cells induces apoptosis preferentially during S-phase.

When Swiss 3T3 cells are acutely infected with Moloney murine sarcoma virus containing the v-mos oncogene, 90% of the cells round up and detach from the monolayer (floating cells) and express high levels of v-Mos. The majority of the floating cells are generated between 30 and 70 h post infection when the cellular level of Mos reaches approximately 0.1% of the total protein. Seventy percent of the floating cells exclude trypan blue but are growth arrested with 2C or 4C DNA content, whereas the remaining floating cells with < 2C DNA content, are dead or dying, and show characteristic apoptotic phenotypes. The apoptotic cells are most likely generated from cells in S-phase since these cells are absent from the viable floating cell population and the percentage of cells with < 2C DNA approximated the expected S-phase fraction of logarithmically growing cells. In addition, 5'-bromo-2'-deoxyuridine-labeling studies showed that approximately 50% of the floating cells with typical apoptotic phenotypes were metabolically-labelled with the drug. These analyses show that cell populations in different stages of the cell cycle are differently affected by high levels of v-Mos expression and cells in S-phase appear to be uniquely sensitive and undergo apoptosis.

Mos and the cell cycle: the molecular basis of the transformed phenotype.

The product of the mos proto-oncogene is a serine/threonine kinase that is expressed at high levels in germ cells. Mos is a regulator of meiotic maturation, and is required for the initiation and progression of oocyte meiotic maturation that leads to the production of unfertilized eggs. Mos is also a component of cytostatic factor, an activity that is believed to arrest oocyte maturation at meiotic metaphase II. There is evidence showing that the Mos protein is associated with tubulin in unfertilized eggs and transformed cells, raising the possibility that it is involved in the microtubular reorganization that occurs during M-phase. Inappropriate expression of its M-phase activity during interphase of the cell cycle may be responsible for its transforming activity.