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Mutations of the ras gene product p21 that abolish guanine nucleotide binding
Abstract:
We have constructed several point mutations affecting the GTP-binding site of p21, the ras-encoded protein. Both lysine (116K) and tyrosine (116Y) mutations of asparagine-116, which, by analogy with the crystal structure of elongation factor Tu (EF-Tu), has critical interactions with the guanine base, abolish GTP binding and transforming activities of p21. These activities are retained by proteins with a mutation at position 117 or 118. Both 116K and 116Y mutant p21s, when overproduced in Escherichia coli, are apparently devoid of GTP-binding and autokinase activities. Similarly, the mutant DNAs do not transform NIH 3T3 cells in a focus-forming assay. By cotransfection with pSV-neo, cell clones resistant to the neomycin analog G418 have been isolated. Cells transfected with 116K or 116Y mutant DNA are contact inhibited. In contrast to competent clones, the defective mutants have no detectable phosphorylated p21. The present results suggest that the basic structure of the GTP-binding site is conserved between p21 and EF-Tu and that this binding site is crucial for ras gene function.
Insights
Mutations in the GTP-binding site of ras-encoded p21 protein, specifically at asparagine-116, abolish GTP binding and transforming activities. This highlights the critical role of the GTP-binding site in ras gene function.
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- The ras oncogene encodes the p21 protein, a key regulator of cellular signaling.
- GTP-binding proteins, including p21, play crucial roles in cell growth and differentiation.
- Understanding the structure-function relationship of the GTP-binding site is vital for comprehending ras gene function.
Purpose of the Study:
- To investigate the role of specific amino acid residues within the GTP-binding site of p21 protein.
- To determine the impact of point mutations on GTP binding and transforming activities of p21.
- To explore the conservation of the GTP-binding site structure between p21 and elongation factor Tu (EF-Tu).
Main Methods:
- Site-directed mutagenesis was used to create point mutations in the GTP-binding site of p21.
- GTP binding assays were performed to assess the effect of mutations on GTP binding.
- Focus-forming assays with NIH 3T3 cells were employed to evaluate transforming activities.
- Autokinase activity and protein phosphorylation were analyzed in mutant p21 proteins.
Main Results:
- Mutations at asparagine-116 (116K and 116Y) abolished both GTP binding and transforming activities of p21.
- Mutations at positions 117 or 118 retained GTP binding and transforming activities.
- Overproduced 116K and 116Y mutant p21s showed no detectable GTP-binding or autokinase activities.
- Cells transfected with 116K or 116Y mutant DNA exhibited contact inhibition and lacked detectable phosphorylated p21.
Conclusions:
- The GTP-binding site of p21 is structurally conserved with that of EF-Tu.
- The identified residues within the GTP-binding site are crucial for ras gene function.
- Disruption of GTP binding by specific mutations leads to loss of transforming activity and altered cellular behavior.