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Normal and transforming Ras are differently regulated for posttranslational modifications
T Yamada-Okabe1, R Doi, H Yamada-Okabe
1Department of Hygiene, Yokohama City University, School of Medicine Japan.
Abstract:
Point mutation of the c-H-ras gene significantly increases cellular transforming activities of Ras. Since posttranslational modification and subsequent membrane localization are essential for the biological activities of Ras, we examined whether or not the mutation also affects these two factors. The normal (Gly(12)) or the transforming (Val(12)) c-H-ras gene was expressed in NIH3T3 cells using a metallothionein promoter. Expression of either type of Ras was efficiently induced by the cadmium treatment of these cells, and immunoprecipitation of metabolically labeled cell extracts revealed that both normal and transforming Ras were expressed as four differently migrating forms on SDS-polyacrylamide gels, two of which were slower migrating cytosolic precursors and the other two were faster migrating membrane-bound forms. There was no significant difference in half lives between normal and transforming Ras; however, posttranslational modification was quite different between the two types of Ras. Transforming Ras was processed and became membrane-bound forms much more efficiently than normal Ras. Interestingly, posttranslational modification and membrane localization of Ras was significantly inhibited when the c-myc oncogene was co-expressed with Ras. In contrast to the c-myc oncogene, expression of either wild type or mutant p53 did not affect the posttranslational modification of Ras, suggesting that the c-myc oncogene specifically impairs the posttranslational modification of Ras.
Insights
Point mutations in the c-H-ras gene enhance Ras transforming activity. While both normal and mutated Ras localize to membranes, the mutated form does so more efficiently, but c-myc oncogene co-expression inhibits this process.
Area of Science:
- Molecular Biology
- Oncology
- Cellular Biology
Background:
- Ras proteins are crucial for cell signaling and transformation.
- Posttranslational modification and membrane localization are essential for Ras biological activity.
- Point mutations in the c-H-ras gene can lead to increased cellular transforming activities.
Purpose of the Study:
- To investigate how c-H-ras gene mutations affect Ras posttranslational modification and membrane localization.
- To compare the processing of normal (Gly12) versus transforming (Val12) Ras.
- To determine the impact of co-expressed oncogenes (c-myc, p53) on Ras modification and localization.
Main Methods:
- Expression of normal and mutant c-H-ras genes in NIH3T3 cells using a metallothionein promoter.
- Induction of gene expression with cadmium treatment.
- Immunoprecipitation of metabolically labeled cell extracts.
- Analysis of Ras forms by SDS-polyacrylamide gel electrophoresis.
Main Results:
- Both normal and transforming Ras were expressed and localized to the membrane.
- Transforming Ras exhibited more efficient processing and membrane-bound forms compared to normal Ras.
- Co-expression of the c-myc oncogene significantly inhibited Ras posttranslational modification and membrane localization.
- Expression of p53 (wild type or mutant) did not affect Ras modification or localization.
Conclusions:
- Ras point mutations enhance membrane localization, contributing to increased transforming activity.
- The c-myc oncogene specifically interferes with Ras posttranslational modification and membrane targeting.
- These findings highlight a potential mechanism by which c-myc cooperates with Ras in oncogenesis.