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Activation of intracellular kinases in Xenopus oocytes by p21ras and phospholipases: a comparative study
1Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Abstract:
Signal transduction induced by generations of second messengers from membrane phospholipids is a major regulatory mechanism in the control of cell proliferation. Indeed, oncogenic p21ras alters the intracellular levels of phospholipid metabolites in both mammalian cells and Xenopus oocytes. However, it is still controversial whether this alteration it is biologically significant. We have analyzed the ras-induced signal transduction pathway in Xenopus oocytes and have correlated its mechanism of activation with that of the three most relevant phospholipases (PLs). After microinjection, ras-p21 induces a rapid PLD activation followed by a late PLA2 activation. By contrast, phosphatidylcholine-specific PLC was not activated under similar conditions. When each of these PLs was studied for its ability to activate intracellular signalling kinases, all of them were found to activate maturation-promoting factor efficiently. However, only PLD was able to activate MAP kinase and S6 kinase II, a similar pattern to that induced by p21ras proteins. Thus, the comparison of activated enzymes after microinjection of p21ras or PLs indicated that only PLD microinjection mimetized p21ras signalling. Finally, inhibition of the endogenous PLD activity by neomycin substantially reduced the biological activity of p21ras. All these results suggest that PLD activation may constitute a relevant step in ras-induced germinal vesicle breakdown in Xenopus oocytes.
Insights
Phospholipase D (PLD) activation is a key step in ras-induced cell signaling in Xenopus oocytes. Inhibiting PLD reduces ras protein
Area of Science:
- Cell biology
- Molecular signaling
- Biochemistry
Background:
- Signal transduction via second messengers from membrane phospholipids regulates cell proliferation.
- Oncogenic p21ras affects phospholipid metabolite levels in mammalian cells and Xenopus oocytes, but its biological significance is debated.
Purpose of the Study:
- To investigate the ras-induced signal transduction pathway in Xenopus oocytes.
- To correlate ras activation with the mechanisms of three key phospholipases (PLs).
- To determine the role of PLD in ras-mediated signaling.
Main Methods:
- Microinjection of ras-p21 and various phospholipases (PLD, PLA2, PLC) into Xenopus oocytes.
- Assaying the activation of intracellular signaling kinases (MAP kinase, S6 kinase II, maturation-promoting factor).
- Inhibiting endogenous PLD activity using neomycin.
Main Results:
- Ras-p21 microinjection rapidly activated PLD, followed by PLA2, but not PLC.
- Only PLD activation mimicked the pattern of MAP kinase and S6 kinase II activation induced by p21ras.
- Inhibition of endogenous PLD by neomycin significantly reduced the biological activity of p21ras.
Conclusions:
- PLD activation is a critical component of the ras-induced signal transduction pathway in Xenopus oocytes.
- PLD activation plays a relevant role in ras-induced germinal vesicle breakdown.