scholarly journals H-ras(val12) induces cytoplasmic but not nuclear events of the cell cycle in small Xenopus oocytes.

1990 ◽  
Vol 1 (7) ◽  
pp. 543-554 ◽  
Author(s):  
A D Johnson ◽  
R J Cork ◽  
M A Williams ◽  
K R Robinson ◽  
L D Smith
Keyword(s):  
Xenopus Oocytes ◽  
Meiotic Maturation ◽  
Cortical Granule ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Endogenous Protein ◽  
Stage 4 ◽  
Nuclear Events ◽  
Activation Response ◽  
Vitelline Envelope

Microinjection of H-ras(val12) protein into fully grown Xenopus oocytes has been shown to induce meiotic maturation. In the present study, mRNA encoding the mutant ras protein was injected into both fully grown (stage 6) and growing (stage 4) oocytes. The mRNA induced nuclear breakdown in stage 6 oocytes, as expected. However, the mRNA induced neither nuclear breakdown nor maturation promoting factor when injected into stage 4 oocytes. Instead, the response in stage 4 oocytes included an activation pulse of calcium, cortical granule breakdown, elevation of the vitelline envelope, and abortive cleavage furrows, all of which are characteristics of the activation response in mature eggs. In addition, the injected mRNA led to increased rates of endogenous protein synthesis and the migration of subcortical organelles into the oocyte interior. These observations are discussed relative to the suggestion that oncogenic ras protein leads to an increase in both diacylglycerol and inositol trisphosphate, which then regulate the various cytoplasmic events described.

Role of phosphatidylinositide metabolism in ras-induced Xenopus oocyte maturation

1990 ◽  
Vol 10 (3) ◽  
pp. 923-929
Author(s):  
B T Pan ◽  
G M Cooper
Keyword(s):  
Xenopus Oocytes ◽  
Second Messengers ◽  
Phospholipid Metabolism ◽  
Meiotic Maturation ◽  
Ras Oncogene ◽  
Ras Protein ◽  
32P Incorporation ◽  
Kinetics Of ◽  
Oncogene Protein

Microinjection of Xenopus oocytes with ras protein (p21) was used to investigate the role of phospholipid metabolism in ras-induced meiotic maturation. Induction of meiosis by ras was compared with induction by progesterone, insulin, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Neomycin, which specifically binds to phosphatidylinositides and inhibits their metabolism, blocked meiotic maturation induced by ras or insulin but not by progesterone or TPA. In addition, p21 and TPA, but not insulin or progesterone, stimulated the incorporation of 32Pi into oocyte lipids. ras protein specifically stimulated 32P incorporation into phosphatidylinositides, whereas both ras and TPA stimulated 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. The stimulatory effect of p21 on phosphatidylinositide metabolism correlated with the dose response and kinetics of ras-induced meiotic maturation. In addition, the ras oncogene protein was more potent than the proto-oncogene protein both in inducing meiotic maturation and in stimulating phosphatidylinositide metabolism. These results indicate that phosphatidylinositide turnover is required for ras-induced meiosis and suggest that phosphatidylinositide-derived second messengers mediate the biological activity of ras in Xenopus oocytes.

Modulation of maturation and ribosomal protein S6 phosphorylation in Xenopus oocytes by microinjection of oncogenic ras protein and protein kinase C

1990 ◽  
Vol 10 (3) ◽  
pp. 880-886
Author(s):  
T Kamata ◽  
H F Kung
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ribosomal Protein ◽  
Xenopus Oocytes ◽  
Protein A ◽  
Ras Proteins ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Ribosomal Protein S6 ◽  
Kinase C

Using Xenopus oocytes as a model system, we investigated the possible involvement of ras proteins in the pathway leading to phosphorylation of ribosomal protein S6. Our results indicate that microinjection of oncogenic T24 H-ras protein (which contains valine at position 12) markedly stimulated S6 phosphorylation on serine residues in oocytes, whereas normal ras protein (which contains glycine at position 12) was without effect. The S6 phosphorylation activity in the cell extract from T24 ras protein-injected oocytes was increased significantly. In addition, injection of protein kinase C potentiated the induction of maturation and S6 phosphorylation by the oncogenic ras protein. A similar potentiation was detected when T24 ras protein-injected oocytes were incubated with active phorbol ester. These findings suggest that ras proteins activate the pathway linked to S6 phosphorylation and that protein kinase C has a synergistic effect on the ras-mediated pathway.

scholarly journals Role of phosphatidylinositide metabolism in ras-induced Xenopus oocyte maturation.

1990 ◽  
Vol 10 (3) ◽  
pp. 923-929 ◽  
Author(s):  
B T Pan ◽  
G M Cooper
Keyword(s):  
Xenopus Oocytes ◽  
Second Messengers ◽  
Phospholipid Metabolism ◽  
Meiotic Maturation ◽  
Ras Oncogene ◽  
Ras Protein ◽  
32P Incorporation ◽  
Kinetics Of ◽  
Oncogene Protein

Microinjection of Xenopus oocytes with ras protein (p21) was used to investigate the role of phospholipid metabolism in ras-induced meiotic maturation. Induction of meiosis by ras was compared with induction by progesterone, insulin, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Neomycin, which specifically binds to phosphatidylinositides and inhibits their metabolism, blocked meiotic maturation induced by ras or insulin but not by progesterone or TPA. In addition, p21 and TPA, but not insulin or progesterone, stimulated the incorporation of 32Pi into oocyte lipids. ras protein specifically stimulated 32P incorporation into phosphatidylinositides, whereas both ras and TPA stimulated 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. The stimulatory effect of p21 on phosphatidylinositide metabolism correlated with the dose response and kinetics of ras-induced meiotic maturation. In addition, the ras oncogene protein was more potent than the proto-oncogene protein both in inducing meiotic maturation and in stimulating phosphatidylinositide metabolism. These results indicate that phosphatidylinositide turnover is required for ras-induced meiosis and suggest that phosphatidylinositide-derived second messengers mediate the biological activity of ras in Xenopus oocytes.

scholarly journals Oncogenic ras protein induces meiotic maturation of amphibian oocytes in the presence of protein synthesis inhibitors

FEBS Letters ◽  
1988 ◽  
Vol 234 (2) ◽  
pp. 426-430 ◽  
Author(s):  
Catherine C. Allende ◽  
M.Victoria Hinrichs ◽  
Eugenio Santos ◽  
Jorge E. Allende
Keyword(s):  
Protein Synthesis ◽  
Meiotic Maturation ◽  
Protein Synthesis Inhibitors ◽  
Ras Protein ◽  
Oncogenic Ras

scholarly journals Modulation of maturation and ribosomal protein S6 phosphorylation in Xenopus oocytes by microinjection of oncogenic ras protein and protein kinase C.

1990 ◽  
Vol 10 (3) ◽  
pp. 880-886 ◽  
Author(s):  
T Kamata ◽  
H F Kung
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ribosomal Protein ◽  
Xenopus Oocytes ◽  
Protein A ◽  
Ras Proteins ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Ribosomal Protein S6 ◽  
Kinase C

Using Xenopus oocytes as a model system, we investigated the possible involvement of ras proteins in the pathway leading to phosphorylation of ribosomal protein S6. Our results indicate that microinjection of oncogenic T24 H-ras protein (which contains valine at position 12) markedly stimulated S6 phosphorylation on serine residues in oocytes, whereas normal ras protein (which contains glycine at position 12) was without effect. The S6 phosphorylation activity in the cell extract from T24 ras protein-injected oocytes was increased significantly. In addition, injection of protein kinase C potentiated the induction of maturation and S6 phosphorylation by the oncogenic ras protein. A similar potentiation was detected when T24 ras protein-injected oocytes were incubated with active phorbol ester. These findings suggest that ras proteins activate the pathway linked to S6 phosphorylation and that protein kinase C has a synergistic effect on the ras-mediated pathway.

Induction of neurite formation in PC12 cells by microinjection of proto-oncogenic Ha-ras protein preincubated with guanosine-5'-O-(3-thiotriphosphate)

1987 ◽  
Vol 7 (12) ◽  
pp. 4553-4556
Author(s):  
T Satoh ◽  
S Nakamura ◽  
Y Kaziro
Keyword(s):  
Nerve Growth Factor ◽  
Pc12 Cells ◽  
Morphological Differentiation ◽  
The Other ◽  
P21 Protein ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Neurite Formation ◽  
Pheochromocytoma Pc12 ◽  
Oncogenic Protein

Rat pheochromocytoma (PC12) cells differentiate to neuronal cells in response to nerve growth factor. It has been shown that microinjection of oncogenic but not proto-oncogenic p21 protein induces morphological differentiation in PC12 cells (D. Bar-Sagi and J. R. Feramisco, Cell 42:841-848, 1985). In this paper we describe a recombinant human proto-oncogenic Ha-ras protein which can effectively induce neurite extension of PC12 cells when microinjected as a complex with guanosine-5'-O-(3-thiotriphosphate). The protein was found to be less effective when complexed with GTP. On the other hand, an oncogenic ras protein coinjected with guanosine-5'-O-(2-thiodiphosphate) was entirely inactive. These results indicate that the binary p21-GTP complex, but not the p21-GDP complex, is effective in inducing differentiation in PC12 cells, irrespective of the oncogenic or the proto-oncogenic protein.

scholarly journals Induction of neurite formation in PC12 cells by microinjection of proto-oncogenic Ha-ras protein preincubated with guanosine-5'-O-(3-thiotriphosphate).

1987 ◽  
Vol 7 (12) ◽  
pp. 4553-4556 ◽  
Author(s):  
T Satoh ◽  
S Nakamura ◽  
Y Kaziro
Keyword(s):  
Nerve Growth Factor ◽  
Pc12 Cells ◽  
Morphological Differentiation ◽  
The Other ◽  
P21 Protein ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Neurite Formation ◽  
Pheochromocytoma Pc12 ◽  
Oncogenic Protein

Rat pheochromocytoma (PC12) cells differentiate to neuronal cells in response to nerve growth factor. It has been shown that microinjection of oncogenic but not proto-oncogenic p21 protein induces morphological differentiation in PC12 cells (D. Bar-Sagi and J. R. Feramisco, Cell 42:841-848, 1985). In this paper we describe a recombinant human proto-oncogenic Ha-ras protein which can effectively induce neurite extension of PC12 cells when microinjected as a complex with guanosine-5'-O-(3-thiotriphosphate). The protein was found to be less effective when complexed with GTP. On the other hand, an oncogenic ras protein coinjected with guanosine-5'-O-(2-thiodiphosphate) was entirely inactive. These results indicate that the binary p21-GTP complex, but not the p21-GDP complex, is effective in inducing differentiation in PC12 cells, irrespective of the oncogenic or the proto-oncogenic protein.

scholarly journals Characterization of p96h2bk: immunoreaction with an anti-Erk(extracellular-signal-regulated kinase) peptide antibody and activity in Xenopus oocytes and eggs

1998 ◽  
Vol 335 (1) ◽  
pp. 43-50
Author(s):  
Dong-Hua CHEN ◽  
Chin-Tin CHEN ◽  
Yong ZHANG ◽  
Mei-Ann LIU ◽  
Roberto CAMPOS-GONZALEZ ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Xenopus Oocytes ◽  
Protein Kinase Activity ◽  
Cell Stage ◽  
Extracellular Signal Regulated Kinase ◽  
Oncogenic Ras ◽  
Cycle Arrest ◽  
Extracellular Signal ◽  
M Phase

We have shown previously that oncogenic Ras induces cell cycle arrest in activated Xenopus egg extracts [Pan, Chen and Lin (1994) J. Biol. Chem. 269, 5968–5975]. The cell cycle arrest correlates with the stimulation of a protein kinase activity that phosphorylates histone H2b in vitro (designated p96h2bk) [Chen and Pan (1994) J. Biol. Chem. 269, 28034–28043]. We report here that p96h2bk is likely to be p96ram, a protein of approx. 96 kDa that immunoreacts with a monoclonal antibody (Mk-1) raised against a synthetic peptide derived from a sequence highly conserved in Erk1/Erk2 (where Erk is extracellular-signal-regulated kinase). This is supported by two lines of evidence. First, activation/inactivation of p96h2bk correlates with upward/downward bandshifts of p96ram in polyacrylamide gels. Secondly, both p96h2bk and p96ram can be immunoprecipitated by antibody Mk-1. We also studied the activity of p96h2bk/p96ram in Xenopus oocytes and eggs. p96h2bk/p96ram was inactive in stage 6 oocytes, was active in unfertilized eggs, and became inactive again in eggs after fertilization. Since stage 6 oocytes are at G2-phase of the cell cycle, unfertilized eggs arrest at M-phase and eggs exit M-phase arrest after fertilization, the results thus indicate that p96h2bk/p96ram activity is cell cycle dependent. Moreover, microinjection of oncogenic Ras into fertilized eggs at the one-cell stage arrests the embryos at the two-cell stage, and this induced arrest is correlated with an inappropriate activation of p96h2bk/p96ram. The data are consistent with the concept that inappropriate activation of p96h2bk/p96ram plays a role in the cell cycle arrest induced by oncogenic Ras.

scholarly journals Whole-Mount Immunofluorescence for Visualizing Endogenous Protein and Injected RNA in Xenopus Oocytes

2018 ◽  
Vol 2018 (10) ◽  
pp. pdb.prot097022
Author(s):  
Samantha P. Jeschonek ◽  
Kimberly L. Mowry
Keyword(s):  
Xenopus Oocytes ◽  
Endogenous Protein ◽  
Whole Mount
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