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Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis
D J Clanton1, Y Y Lu, D G Blair
1Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Maryland 21701.
Abstract:
Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.
Insights
Point mutations in the glycine-rich sequence of rasH p21 oncogene affect its GTP-binding and autokinase activities. Certain mutations confer transforming ability without ligand binding, highlighting the sequence
Area of Science:
- Oncogene research
- Molecular biology
- Protein biochemistry
Background:
- The v-rasH oncogene encodes the p21 protein, crucial for cellular signaling.
- The glycine-rich region (GXGXXGK) is a conserved motif in nucleotide-binding proteins, including ras p21.
- Understanding mutations in this region is key to deciphering oncogenic transformation mechanisms.
Purpose of the Study:
- To investigate the functional significance of specific glycine residues within the ras p21 nucleotide-binding consensus sequence.
- To determine how point mutations in this region affect GTP-binding, autokinase activity, and cellular transformation.
- To elucidate the role of ligand binding in p21 oncogenic activity.
Main Methods:
- Oligonucleotide-directed mutagenesis was used to create point mutations in the v-rasH oncogene's glycine-rich sequence.
- Mutant proviral DNA was inserted into the pSV2neo plasmid for transfection into NIH 3T3 cells.
- GTP-binding and autokinase activities of mutant p21 proteins were assessed.
- Cellular transformation was evaluated by growth in soft agar and tumor formation in nude mice.
Main Results:
- Substitution of glycine at positions 10, 13, or 15 with valine abolished GTP-binding and autokinase activities.
- Mutations at positions 33, 51, or 59 did not affect GTP-binding.
- Mutants at positions 13 and 15 lost transforming ability, while other GTP-binding mutants retained it.
- A G10V mutant, lacking GTP-binding and autokinase activity, fully transformed NIH 3T3 cells, inducing tumors in mice.
Conclusions:
- The glycine-rich consensus sequence is critical for regulating p21 activities.
- Specific mutations can confer oncogenic transforming activity to p21 independent of GTP binding.
- These findings suggest that p21 can adopt an active conformation through mechanisms other than ligand binding.