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Effects of two major activating lesions on the structure and conformation of human ras oncogene products
Abstract:
ras oncogenes are frequently activated in human tumors by mutations at codon 12 or 61 in their coding sequences. To investigate how these subtle alterations exert such profound effects on the biologic activities of these genes, we studied structural and conformational properties of human ras-oncogene-encoded 21-kDa proteins (p21s). We observed striking differences in the electrophoretic mobilities of the proteins under reducing and nonreducing conditions. These findings imply that intramolecular disulfide bonds affect native p21 conformation. The two activating lesions were shown to induce distinctly different alterations in p21 electrophoretic mobility that were unmasked only under reducing conditions. These results suggest that regions of the molecule containing such alterations are either not exposed or under conformational constraints in the native p21 molecule. We confirmed the opposing effects on protein mobility induced by the two activating lesions by using a recombinant gene containing both lesions. The recombinant gene's high-titer transforming activity further established that the two lesions do not negatively complement one another with respect to transforming-gene function. Our findings of distinct alterations in electrophoretic mobilities of position-12- and position-61-altered p21 molecules should be applicable to the rapid immunologic diagnosis of ras oncogenes in human malignancies.
Insights
Mutations in ras oncogenes at codon 12 or 61 alter the conformation of ras p21 proteins. These distinct structural changes, revealed by electrophoretic mobility shifts, are key for diagnosing ras oncogenes in tumors.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- Ras oncogenes are frequently mutated in human cancers, particularly at codons 12 and 61.
- These mutations lead to altered ras p21 protein function, driving tumor development.
Purpose of the Study:
- To investigate the structural and conformational changes in human ras p21 proteins induced by activating mutations at codons 12 and 61.
- To explore the implications of these conformational changes for ras oncogene diagnosis.
Main Methods:
- Studied structural and conformational properties of human ras-oncogene-encoded 21-kDa proteins (p21s).
- Analyzed electrophoretic mobilities of p21 proteins under reducing and nonreducing conditions.
- Utilized a recombinant gene containing both codon 12 and 61 lesions to confirm effects.
Main Results:
- Observed significant differences in p21 electrophoretic mobilities under reducing versus nonreducing conditions, indicating intramolecular disulfide bonds influence native p21 conformation.
- Activating mutations at codons 12 and 61 induced distinct alterations in p21 electrophoretic mobility, unmasked under reducing conditions.
- These alterations suggest specific molecular regions are either inaccessible or conformationally constrained in native p21.
Conclusions:
- The distinct electrophoretic mobility alterations of codon 12 and 61 mutated p21 molecules provide a basis for rapid immunologic diagnosis of ras oncogenes in human malignancies.
- Ras oncogene mutations at codons 12 and 61 induce conformationally distinct p21 proteins.