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Farnesylation of p21 Ras proteins in Xenopus oocytes
1Laboratory of Biochemical Physiology, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702-1201.
Abstract:
Unprocessed p21 Ras proteins microinjected into Xenopus oocytes were radiolabeled by coinjected [3H]farnesyl pyrophosphate, a direct farnesyl donor substrate for all known mammalian farnesyltransferases. Mevinolin, an inhibitor of HMG CoA reductase which reduces the levels of mevalonate and thus farnesyl pyrophosphate, blocked oncogenic H-Rasva112 induced germinal vesicle breakdown in oocytes. This mevinolin caused block was completely reversed by co-injected farnesyl pyrophosphate. The putative farnesyltransferase in Xenopus oocytes was identified to be similar to those found in mammalian cells in that it requires an intact CAAX box motif in addition to the conserved cysteine residue at the fourth position from the C-terminus of Ras proteins for its farnesylating activity. Peptide inhibitors of farnesyltransferase such as CVIM and TKCVIM were shown to inhibit farnesylation of microinjected Ras proteins thereby blocking its function namely the induction of oocyte maturation. These results demonstrate that Xenopus oocytes process bacterially produced mammalian Ras proteins in a manner similar to, if not identical with that in mammalian cells, thus validating the continued use of the Xenopus oocyte system for unraveling the functions of Ras proteins. Furthermore, our results indicate that the oocyte system may be a useful in vivo model for studying the farnesylation of human Ras proteins, its regulation, and the effects of farnesyltransferase inhibitors.
Insights
Xenopus oocytes can process mammalian Ras proteins, a key finding for understanding Ras function. This validates the oocyte system for studying Ras protein farnesylation and inhibitors.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Ras proteins are critical signaling molecules involved in cell growth and differentiation.
- Post-translational modification, specifically farnesylation, is essential for Ras protein function.
- Xenopus oocytes are a valuable model system for studying fundamental biological processes.
Purpose of the Study:
- To investigate the farnesylation of mammalian Ras proteins in Xenopus oocytes.
- To determine if Xenopus oocytes possess farnesyltransferase activity similar to mammalian cells.
- To evaluate the Xenopus oocyte as a model for studying farnesyltransferase inhibitors.
Main Methods:
- Microinjection of radiolabeled Ras proteins and [3H]farnesyl pyrophosphate into Xenopus oocytes.
- Inhibition of farnesylation using mevinolin (HMG CoA reductase inhibitor) and peptide inhibitors (CVIM, TKCVIM).
- Assessment of Ras protein function by monitoring oocyte maturation (germinal vesicle breakdown).
Main Results:
- Xenopus oocytes successfully farnesylated microinjected Ras proteins.
- Mevinolin inhibited Ras-induced oocyte maturation by reducing farnesyl pyrophosphate levels, an effect reversed by exogenous farnesyl pyrophosphate.
- Peptide inhibitors of farnesyltransferase blocked Ras farnesylation and function.
- The farnesyltransferase activity in oocytes requires an intact CAAX box motif, similar to mammalian cells.
Conclusions:
- Xenopus oocytes process mammalian Ras proteins similarly to mammalian cells, validating their use in Ras research.
- The oocyte system is a suitable in vivo model for studying Ras protein farnesylation and the efficacy of farnesyltransferase inhibitors.
- This study provides insights into the conserved mechanisms of Ras protein modification and function across species.