Genetic modifiers of EGFR dependence in non-small cell lung cancer.

Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival.

The effects of demecolcine, alone or in combination with sucrose on bovine oocyte protrusion rate, MAPK1 protein level and c-mos gene expression level.

AIMS: Our study aims to clarify the effects of demecolcine, alone or in combination with sucrose on bovine oocyte protrusion rate, MAPK1 protein level and c-mos gene expression level. METHODS: The effects of the demecolcine concentration, treatment duration, and synergistic effects with sucrose solution on the rate of membrane protrusions of bovine oocytes were investigated. Using real-time fluorescent quantitative PCR, western blot analysis, and immunofluorescence assays, the expression of the maternal c-mos gene, the protein level of mitogen-activated protein kinase 1 (MAPK1), and the change in the localization of spindles and nuclei during the demecolcine treatment were analyzed in bovine oocytes. RESULTS: Treatment of bovine oocytes with both demecolcine (0.6 µg/mL) and sucrose (0.05 M) for 1 h led to the highest rate of membrane protrusions, and synergistic effects were also observed. Real-time fluorescent quantitative PCR analysis revealed that the demecolcine treatment up-regulated the expression of the maternal c-mos gene. Western blot analysis indicated that the demecolcine treatment enhanced the protein level of MAPK1 in bovine oocytes. Immunofluorescence analysis indicated that the spindles and nuclei were localized at the place of the membrane protrusions. CONCLUSIONS: The present results suggest that demecolcine might contribute to the activation of the Mos/MAPK pathway and affect spindle structure. These results provide a reference for more efficient generation of enucleated bovine oocytes.

Ringo/cyclin-dependent kinase and mitogen-activated protein kinase signaling pathways regulate the activity of the cell fate determinant Musashi to promote cell cycle re-entry in Xenopus oocytes.

Cell cycle re-entry during vertebrate oocyte maturation is mediated through translational activation of select target mRNAs, culminating in the activation of mitogen-activated protein kinase and cyclin B/cyclin-dependent kinase (CDK) signaling. The temporal order of targeted mRNA translation is crucial for cell cycle progression and is determined by the timing of activation of distinct mRNA-binding proteins. We have previously shown in oocytes from Xenopus laevis that the mRNA-binding protein Musashi targets translational activation of early class mRNAs including the mRNA encoding the Mos proto-oncogene. However, the molecular mechanism by which Musashi function is activated is unknown. We report here that activation of Musashi1 is mediated by Ringo/CDK signaling, revealing a novel role for early Ringo/CDK function. Interestingly, Musashi1 activation is subsequently sustained through mitogen-activated protein kinase signaling, the downstream effector of Mos mRNA translation, thus establishing a positive feedback loop to amplify Musashi function. The identified regulatory sites are present in mammalian Musashi proteins, and our data suggest that phosphorylation may represent an evolutionarily conserved mechanism to control Musashi-dependent target mRNA translation.

Porcine Aurora A accelerates Cyclin B and Mos synthesis and promotes meiotic resumption of porcine oocytes.

Full-grown oocytes arrested at germinal vesicle stage contain many dormant maternal mRNAs, and Aurora A has been reported to play a key role for the translation of these maternal mRNAs in Xenopus oocytes. Although the presence of Aurora A has been reported in mammals, the functions of Aurora A on the protein synthesis and the meiotic resumption have never been elucidated in mammalian oocytes. In the present study, the effects of porcine Aurora A on meiotic resumption of porcine oocytes were examined. At first, we cloned porcine Aurora A from total RNA of immature porcine oocytes by RT-PCR and obtained full-length cDNA that was 77%, 86% and 54% homologous with mouse, human and Xenopus Aurora A, respectively. The Aurora A mRNA and large amounts of protein were present throughout maturation period in porcine oocytes. The overexpression of porcine Aurora A by the mRNA injection into immature porcine oocytes had no effects on Cyclin B synthesis and meiotic resumption. Therefore we constructed a mutated Aurora A (AA-Aurora A), which was replaced the expecting inhibitory phosphorylation sites, serines 283 and 284, to non-phosphorylatable alanines. The oocytes expressed AA-Aurora A were accelerated their Cyclin B synthesis and Rsk phosphorylation, an indicator of Mos synthesis, then their meiotic resumption was promoted significantly. These results suggest for the first time in mammalian oocytes that mammalian Aurora A stimulates the protein synthesis and promotes the meiotic resumption. In addition, we identified the inhibitory phosphorylation sites of porcine Aurora A, and indicate the presence of phosphorylation-dependent regulation mechanisms in mammalian Aurora A.

Upregulation of meiosis-specific genes in lymphoma cell lines following genotoxic insult and induction of mitotic catastrophe.

BACKGROUND: We have previously reported that p53 mutated radioresistant lymphoma cell lines undergo mitotic catastrophe after irradiation, resulting in metaphase arrest and the generation of endopolyploid cells. A proportion of these endopolyploid cells then undergo a process of de-polyploidisation, stages of which are partially reminiscent of meiotic prophase. Furthermore, expression of meiosis-specific proteins of the cancer/testis antigens group of genes has previously been reported in tumours. We therefore investigated whether expression of meiosis-specific genes was associated with the polyploidy response in our tumour model. METHODS: Three lymphoma cell lines, Namalwa, WI-L2-NS and TK6, of varying p53 status were exposed to a single 10 Gy dose of gamma radiation and their responses assessed over an extended time course. DNA flow cytometry and mitotic counts were used to assess the kinetics and extent of polyploidisation and mitotic progression. Expression of meiotic genes was analysed using RT-PCR and western blotting. In addition, localisation of the meiotic cohesin REC8 and its relation to centromeres was analysed by immunofluorescence. RESULTS: The principal meiotic regulator MOS was found to be significantly post-transcriptionally up-regulated after irradiation in p53 mutated but not p53 wild-type lymphoma cells. The maximum expression of MOS coincided with the maximal fraction of metaphase arrested cells and was directly proportional to both the extent of the arrest and the number of endopolyploid cells that subsequently emerged. The meiotic cohesin REC8 was also found to be up-regulated after irradiation, linking sister chromatid centromeres in the metaphase-arrested and subsequent giant cells. Finally, RT-PCR revealed expression of the meiosis-prophase genes, DMC1, STAG3, SYCP3 and SYCP1. CONCLUSION: We conclude that multiple meiotic genes are aberrantly activated during mitotic catastrophe in p53 mutated lymphoma cells after irradiation. Furthermore, we suggest that the coordinated expression of MOS and REC8 regulate the extent of arrested mitoses and polyploidy.

c-mos immunoreactivity aids in the diagnosis of gestational trophoblastic lesions.

C-mos is an important proto-oncogene involved in the mitogen-activating protein kinase pathway. This study was designed to explore c-mos immunoreactivity in gestational trophoblastic lesions and compare it with immunoreactivity in normal placentas as well as other gynecological lesions and germ cell tumors using antibody P-19. The immunohistochemical distribution of c-mos in 159 cases of gynecological lesions and 26 germ cell tumors using formalin-fixed, paraffin-embedded tissues was evaluated. The lesions included 45 (32 complete and 13 partial) hydatidiform moles, 17 choriocarcinomas, 5 placental site trophoblastic tumors, 18 squamous cell carcinomas and 5 adenocarcinomas of the cervix, 11 endometrial carcinomas, 9 ovarian carcinomas, 4 primary peritoneal papillary serous carcinomas, 9 low-grade endometrial stromal sarcomas, 4 epithelioid leiomyomas, 6 leiomyosarcomas, and 26 gem cell tumors (3 embryonal carcinomas, 5 yolk sac tumors, 6 immature teratomas, and 3 mature teratomas from the ovary; 9 testicular seminomas). Twenty-six normal placentas also were included for comparison. Among cases of gestational trophoblastic diseases, c-mos immunoreactivity was found in all hydatidiform moles and choriocarcinomas, but in none of the placental site trophoblastic tumors. The c-mos staining pattern was similar in trophoblastic diseases and normal placentas with strong expression in syncytiotrophoblast, moderate expression in villous intermediate trophoblast, and predominantly negative expression in implantation site intermediate trophoblast, chorionic-type intermediate trophoblast, and villous cytotrophoblast. All the nontrophoblastic tumors, including carcinomas, sarcomas, and germ cell tumors, were negative for c-mos expression. Immunohistochemical detection of c-mos is useful in differentiating choriocarcinoma from placental site trophoblastic tumor and nontrophoblastic tumors of the female genital tract that may sometimes cause problems in differential diagnosis.

Definitive expression of c-mos in late meiotic prophase leads to phosphorylation of a 34 kda protein in cultured rat spermatocytes.

To investigate the role of c-mos in rat spermatogenesis, expression of c-mos, MAP kinase kinase (MAPKK), MAP kinase (MAPK), cdc2 and protein kinase A (PKA) by spermatogenic cell culture of 14 day-old rats was examined. MAPKK and PKA expressions were constitutive, whereas the expression of MAPK and cdc2 in spermatogonia initially decreased, but later increased on meiotic maturation of spermatocytes. c-mos expression was definitive of late meiotic prophase. c-mos immunoprecipitates prepared from the c-mos-enriched fraction (pI9.0-9.6) could form complex(es) in the cultured spermatogenic cell lysates. In vitro phosphorylation of the c-mos immune complexes revealed a 34 kDa protein that was phosphorylated at serine and threonine residues as a target of the c-mos signal. Its pI value was 4.4-4.5, and cdc2 was not detected, making it different from cdc2 (p34). These results suggest that the phosphorylation of the 34 kDa protein by the c-mos signal may play a crucial role in the meiotic division of rat spermatocytes.

cAMP-Dependent PKA negatively regulates polyadenylation of c-mos mRNA in rat oocytes.

Activation of members of the MAPK family, Erk 1 and 2, in oocytes resuming meiosis is regulated by Mos. The cAMP-dependent PKA-mediated cAMP action that inhibits the resumption of meiosis also prevents MAPK activation. We hypothesized that PKA interferes with the MAPK signaling pathways at the level of Mos. We also assumed that this regulatory cascade may involve p34cdc2. To test our hypothesis we explored the role of PKA and p34cdc2 in regulating Mos expression. Rat oocytes that resume meiosis spontaneously served as our experimental model. We found that meiotically arrested rat oocytes express the c-mos mRNA with no detectable Mos protein. The presence of Mos was initially demonstrated at 6 h after meiosis reinitiation and was associated with its mRNA polyadenylation. (Bu)(2)cAMP inhibited Mos expression as well as c-mos mRNA polyadenylation. Both these cAMP actions were reversed by the highly selective inhibitor of the catalytic subunit of PKA, 4-cyano-3-methylisoquinoline. Polyadenylation of c-mos mRNA was also prevented by roscovitine, which is a potent inhibitor of p34cdc2. Ablation of MAPK activity by two specific MAPK signaling pathway inhibitors, either PD 98059 or U0126, did not interfere with Mos accumulation. Our results suggest that translation of Mos in rat oocytes is negatively regulated by a PKA-mediated cAMP action that inhibits c-mos mRNA polyadenylation and involves suppressed activity of p34cdc2. We also demonstrate that stimulation of Mos synthesis in the rat does not require an active MAPK.

Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha-ras-transformed human breast cell lines.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted approximately 16-24 h (IC(50) approximately 0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supplementing the culture medium with anti-TGFbeta or soluble TGF beta(II) receptor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by approximately 50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA approximately 50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for approximately 72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGF beta supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGF beta(II) receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

The kinase Eg2 is a component of the Xenopus oocyte progesterone-activated signaling pathway.

Quiescent Xenopus oocytes are activated by progesterone, which binds to an unidentified surface-associated receptor. Progesterone activates a poorly understood signaling pathway that results in the translational activation of mRNA encoding Mos, a MAP kinase kinase kinase necessary for the activation of MAP kinase and MPF, the resumption of meiosis, and maturation of the oocyte into the sperm-responsive egg. We have designed a screen to identify early signaling proteins based on the premise that some of these proteins would be phosphorylated or otherwise modified within minutes of progesterone addition. This screen has revealed Eg2, a Ser/Thr kinase. We find that Eg2 is phosphorylated soon after progesterone stimulation and provide evidence that it functions in the signaling pathway. Overexpression of Eg2 via mRNA microinjection shortens the time between progesterone stimulation and the appearance of new Mos protein, accelerates activation of MAP kinase and advances entry into the meiotic cell cycle. Finally, overexpression of Eg2 dramatically reduces the concentration of progesterone needed to trigger oocyte activation. These results argue that the kinase Eg2 is a component of the progesterone-activated signaling pathway that releases frog oocytes from cell cycle arrest.

Meiotic maturation in Xenopus requires polyadenylation of multiple mRNAs.

Cytoplasmic polyadenylation of specific mRNAs commonly is correlated with their translational activation during development. Here, we focus on links between cytoplasmic polyadenylation, translational activation and the control of meiotic maturation in Xenopus oocytes. We manipulate endogenous c-mos mRNA, which encodes a protein kinase that regulates meiotic maturation. We determined that translational activation of endogenous c-mos mRNA requires a long poly(A) tail per se, rather than the process of polyadenylation. For this, we injected 'prosthetic' poly(A)_synthetic poly(A) tails designed to attach by base pairing to endogenous c-mos mRNA that has had its own polyadenylation signals removed. This prosthetic poly(A) tail activates c-mos translation and restores meiotic maturation in response to progesterone. Thus the role of polyadenylation in activating c-mos mRNA differs from its role in activating certain other mRNAs, for which the act of polyadenylation is required. In the absence of progesterone, prosthetic poly(A) does not stimulate c-mos expression, implying that progesterone acts at additional steps to elevate c-Mos protein. By using a general inhibitor of polyadenylation together with prosthetic poly(A), we demonstrate that these additional steps include polyadenylation of at least one other mRNA, in addition to that of c-mos mRNA. These other mRNAs, encoding regulators of meiotic maturation, act upstream of c-Mos in the meiotic maturation pathway.

Elevated level of cyclin D1 in mos-transformed cells.

Mos is a germ cell-specific serine/threonine protein kinase that plays an important role during meiotic divisions of oocytes. Upon expression in somatic cells, Mos causes cell cycle perturbations leading to neoplastic transformation. Mos activates the MAP kinase pathway in both oocytes and transformed somatic cells. To determine the mechanism of cell cycle perturbation in mos-transformed cells, we examined the status of some key regulators of G1 phase. We provide evidence that Mos causes an elevation in the level of cyclin D1 in NIH/3T3 cells. As expected from the increased cyclin D1 level, mos transformation of NIH/3T3 cells caused an increase in the protein kinase activities of cyclin D1-Cdk4 and cyclin E-Cdk2 and induced hyperphosphorylation of the retinoblastoma protein. Of importance, the level of cyclin D1 was also elevated in eye lens of the c-mos-transgenic mice compared to normal mice. Our results indicate that the mechanism of cellular transformation by Mos involves an elevation in the level of cyclin D1 in somatic cells.

Recurring themes in oocyte maturation.

I am pleased to contribute to this special issue of Biology of the Cell in honor of Yoshio Masui. Oocyte maturation remains a small enough and young enough field that the authors assembled for this issue can trace the entire development of the field over the last 30 years, beginning with the early demonstration by Masui (1967) and others, that steroids can induce complete maturation of denuded oocytes in vitro. Not very long after that Masui and Markert (1971) published the seminal paper that identified MPF and CSF activity. In this article I intend to highlight recurring themes in oocyte maturation that continue to be actively investigated, almost all of which derive from studies pursued at some point in time by Masui and his colleagues.

Germinal vesicle material is dispensable for oscillations in cdc2 and MAP kinase activities, cyclin B degradation and synthesis during meiosis in Xenopus oocytes.

We have investigated at a molecular level the requirements for germinal vesicle (nuclear) material during the course of meiosis in Xenopus oocytes. We present the localization of some cell cycle proteins in stage VI oocytes; most of those analyzed are cytoplasmic, although some (MAD, 26S proteasome) are distributed between the cytoplasm and the germinal vesicle. By analyzing changes in individual oocytes, we find that the unphosphorylated form of cyclin B2 disappears and the phosphorylated form is then degraded in both nucleated and enucleated oocytes. Enucleated oocytes are also capable of resynthesizing both cyclin B1 and cyclin B2 after the initial degradation and of reactivating cdc2 kinase. Synthesis of mos protein and activation of MAP kinase concomitant with cdc2-cyclin B reactivation are also unaffected by prior removal of the germinal vesicle.

Analysis of the mechanism(s) of metaphase I arrest in strain LT mouse oocytes: participation of MOS.

Oocytes of almost all vertebrates become arrested at metaphase II to await fertilization. Arrest is achieved with the participation of a protein complex known as cytostatic factor (CSF) that stabilizes histone H1 kinase activity. MOS and mitogen-activated protein kinase (MAPK) are important components of CSF. Strain LT/Sv mice, and strains related to LT/Sv, produce a high percentage of atypical oocytes that are arrested at metaphase I when normal oocytes have progressed to metaphase II. The potential role of MOS in metaphase I arrest was investigated using strain LT/Sv and LT-related recombinant inbred strains, LTXBO and CX8-4. MOS and MAPK are produced and functional in maturing LT oocytes. Two experimental paradigms were used to reduce or delete MOS in LT oocytes and assess effects on metaphase I arrest. First, sense and antisense Mos oligonucleotides were microinjected into metaphase I-arrested oocytes. Antisense, but not sense, Mos oligonucleotides promoted the activation of metaphase I-arrested oocytes. Second, mice carrying a Mos null mutation were crossed with LT mice, the null mutation was backcrossed three times to LT mice, and Mos(+/-) N3 mice were intercrossed to produce Mos(-/-), Mos(+/-) and Mos(+/+) N3F1 mice. Oocytes of all three Mos genotypes of N3F1 mice sustained meiotic arrest for 17 hours indicating that metaphase I arrest is not initiated by a MOS-dependent mechanism. However, unlike Mos(+/+) and Mos(+/-) CX8-4 N3F1 oocytes, metaphase I arrest of Mos(-/-) CX8-4 N3F1 oocytes was not sustained after 17 hours and became reversed gradually. These results, like the antisense Mos oligonucleotide microinjection experiments, suggest that MOS participates in sustaining metaphase I arrest in LT oocytes.

Comparative effects of insulin on the activation of the Raf/Mos-dependent MAP kinase cascade in vitellogenic versus postvitellogenic Xenopus oocytes.

Xenopus postvitellogenic oocytes resume meiosis in vitro upon exposure to insulin or insulin-like growth factor 1 (IGF-1) via a ras-dependent pathway, whereas stage IV (600 micron < diameter < 1000 micron) oocytes cannot. The aim of the present study was to determine which event(s) of the transduction pathway from IGF-1 receptor to maturation-promoting factor (MPF) activation is deficient in the small, vitellogenic, oocytes to explain their inability to undergo germinal vesicle breakdown (GVB) after insulin treatment. We thus analyzed the effect of insulin on the Ras/Raf-dependent mitogen-activated protein kinase cascade because of its crucial role prior to MPF activation. The effect of insulin on pp39mos synthesis in stage IV oocytes was also studied since this protein kinase participates in the mitogen-activated protein kinase (MAPK) pathway as a MAPKK kinase like Raf. Contrary to what is observed in postvitellogenic oocytes, MAPK was not activated in insulin-treated stage IV oocytes even 20 hr after the stimulation. This was not caused by the absence of MAPK activators like MEK (MAPKK), Raf, or Ras, but rather by the inability of insulin to activate Ras. Interestingly, injection of constitutively active raf mRNA as well as oncogenic Ras protein, Ha-Ras lys12, in stage IV oocytes resulted in MAPK activation, whereas neither Mos accumulation nor GVB occurred, suggesting that the Ras --> Raf --> MAPKK --> MAPK cascade was functional but that MAPK activation alone was not sufficient for the mitogenic signal to proceed further down in the pathway leading to MPF activation. Treatment of stage IV oocytes with insulin did not stimulate Mos synthesis either, indicating a dysfunction in the "Mos synthesis machinery." The present results show that incompetence of Xenopus stage IV oocytes to activate MPF in response to insulin is primarily due to the inability of the peptide to activate Ras and to stimulate pp39mos synthesis and secondarily to a deficiency in the mitogenic pathway that connects MAPK to MPF activation.

Expression of Mos proto-oncoprotein in bovine oocytes during maturation in vitro.

The c-mos proto-oncogene product Mos is believed to be an active component of the cytostatic factor that stabilizes and sustains the activity of maturation-promoting factor. Mos has been found to be responsible for the metaphase arrest of oocytes at the second meiotic division in both Xenopus and the mouse. In this study, we have demonstrated, by Western blot and immunoprecipitation analysis, that an approximately 39-kDa protein, identified as Mos, was present in in vitro-matured (metaphase II stage) bovine oocytes but disappeared in parthenogenetically activated oocytes. The oocytes actively synthesized p39mos at the metaphase II stage (between 22 and 26 h of in vitro maturation [IVM]), whereas little p39mos synthesis was detected during the first 4 h of IVM and it was nondetectable during aging at 44-48 h of IVM, when oocytes lose the capability of normal development after fertilization. Ethanol activation of mature oocytes led to the disappearance of p39mos. beta-Tubulin, but not p34cdc2, was co-precipitated with Mos when extracts of metaphase II-stage bovine oocytes were incubated with Mos antiserum. These results demonstrated that Mos is present and actively synthesized in mature bovine oocytes and that oocytes aged beyond the optimal time for fertilization seem to lose the ability to synthesize the Mos protein. beta-Tubulin was found to be associated with Mos, which suggests a possible role for the cytoskeletal protein in maintaining the meiotic arrest in mature bovine oocytes.

The Ras-GTPase-activating protein SH3 domain is required for Cdc2 activation and mos induction by oncogenic Ras in Xenopus oocytes independently of mitogen-activated protein kinase activation.

The Ras-GTPase-activating protein (RasGAP) is an important modulator of p21ras - dependent signal transduction in Xenopus oocytes and in mammalian cells. We investigated the role of the RasGAP SH3 domain in signal transduction with a monoclonal antibody against the SH3 domain of RasGaP. This antibody prevented the activation of the maturation-promoting factor complex (cyclin B-p34cdc2) by oncogenic Ras. The antibody appears to be specific because as little as 5 ng injected per oocyte reduced the level of Cdc2 activation by 50% whereas 100 ng of nonspecific immunoglobulin G did not affect Cdc2 activation. The antibody blocked the Cdc2 activation induced by oncogenic Ras but not that induced by progesterone, which acts independently of Ras. A peptide corresponding to positions 317 to 326 of a sequence in the SH3 domain of human RasGAP blocked Cdc2 activation, whereas a peptide corresponding to positions 273 to 305 of a sequence in the N-terminal moiety of the SH3 domain of RasGAP had no effect. The antibody did not block the mitogen-activated protein (MAP) kinase cascade (activation of MAPK/ERK kinase [MEK], MAP kinase, and S6 kinase p90rsk). Surprisingly, injection of the negative MAP kinase mutant protein ERK2 K52R (containing a K-to-R mutation at position 52) blocked the Cdc2 activation induced by oncogenic Ras as well as blocking the activation of MAP kinase. Thus, MAP kinase is also implicated in the regulation of Cdc2 activity. In this study, we further investigated the regulation of the synthesis of the c-mos oncogene product, which is necessary for the activation of Cdc2. We report that the synthesis of the c-mos oncogene product, which is necessary for the activation antibody to the SH3 domain of RasGAP and by injecting the negative MAP kinase mutant protein ERK2 K52R. These results suggest that oncogenic Ras activates two signaling mechanisms: the MAP kinase cascade and a signaling pathway implicating the SH3 domain of RasGAP. These mechanisms might control Mos protein expression implicated in Cdc2 activation.

Primate reproductive organs reveal a novel pattern of proto-oncogene c-mos and transcription factor Oct-3 mRNA expression.

In mice, expression of the transcription factor Oct-3 and the proto-oncogene c-mos is limited to germ cells, suggesting a specific role for these factors in gamete physiology and early embryonic development. We have studied the expression pattern of Oct-3 and c-mos in various reproductive as well as control tissues in the cynomolgus monkey, using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern analysis. Analogously with the data from the mouse model, strong expression of Oct-3 and c-mos could be detected in monkey ovary and oocytes. Unexpectedly, strong expression of c-mos was demonstrable in the pituitary gland and the amount of mRNA expression in the pituitary was roughly equal to that found in the ovary. Of the tissues examined, the testicular expression of c-mos was the most intense. Weak signal for c-mos mRNA was also seen in hypothalamus and brain; however, all other tissue types examined were negative for c-mos expression. In addition to the oocytes, expression of Oct-3 mRNA was detected in the ovarian granulosa cells, fallopian tube, myometrium, cervix, breast, liver, adrenal gland, pituitary, hypothalamus, brain cortex, prostate, and in testis. Thus, in the cynomolgus monkey, Oct-3 is predominantly, but not specifically, expressed in reproductive tissues. In the female monkey reproductive organs, the expression of c-mos seems to be germ cell specific. Therefore, further characterization of c-mos and Oct-3 functions in primate reproductive physiology, especially in gametogenesis and early embryonic development, is highly warranted.

Expression of c-mos protein in cultured rat spermatogenic cells and evidence that rat testicular 43 kDa c-mos protein has an elongated amino acid sequence at the N-terminus.

Expression of c-mos protein in rat spermatogenesis was examined by means of a cell culture system. A commercially available antibody (Cambridge Research Biochemicals, OA-11-861) was used to detect the protein. The 43 kDa c-mos (43K c-mos) protein was detectable in spermatogenic cells close to the occurrence of the first meiotic divisions (a few days before appearance of spermatids) during postnatal development, which was nearly consistent with expression in vivo. The results suggested that the c-mos protein has a role in meiotic maturation of spermatogenic cells. The 43K c-mos protein was partially purified from rat testes and the protein band was identified. The 43K c-mos protein had a pI value of around 9.0-9.6 having a hydrophobic nature, and was phosphorylated in vitro on serine. Neither ubiquitination nor glycosylation were detected. N-Terminal amino acid sequencing showed that the 43K c-mos protein has a Asp-Glu-Gly-Gly-Asn-Leu-Gln-sequence located 5' upstream (99 bases upstream) of the rat c-mos coding sequence reported. The amino acid analysis revealed a nearly consistent composition with that deduced from the DNA sequence. These results suggested that rat testicular 43K c-mos protein is translated from a 5' upstream sequence of the predicted consensus AUG start codon, probably by an unusual translational rule.