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Decreased GTPase activity of K-ras mutants deriving from human functional adrenocortical tumours
Abstract:
Our previous studies have shown that seven out of 15 patients with adrenocortical tumours contained K-ras gene mutation. In addition, the mutation type was a multiple-site mutation, and the hot spots were located at codons 15, 16, 18 and 31, which were different from those reported before (codons 12, 13 and 61). To understand whether the mutation hot spots in human adrenocortical tumours were associated with activation of K-Ras oncogene and the alterations of its biocharacteristics, mutant K-Ras genes were cloned from tumour tissues and then constructed with expression vector pBKCMV. Mutant K-Ras genes were expressed at high levels in Escherichia coli and the resultant K-Ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. The purified K-Ras protein from E. coli were then measured for their intrinsic GTPase activity and the GTPase activity in the presence of GTPase-activating protein for Ras. The results showed that the wild-type cellular K-Ras protein (p21BN) exhibits about ten times higher intrinsic GTPase activity than the activated protein (p21BM3) encoded by mutant K-Ras gene, which mutated at codon 60. With regards to the codon 15, 16, 18 and 31 mutant K-Ras proteins (p21BM2), the GTPase activity in the presence of GAP is much lower than that of the normal K-Ras protein, whereas the intrinsic GTPase activity is nearly the same as that of the normal K-Ras protein. These results indicated that mutations at these hot spots of K-Ras gene were indeed activated K-Ras oncogene in adrenocortical tumours; however, their association with tumors needs further experiments to prove.
Insights
New K-ras gene mutations in adrenocortical tumors were identified at codons 15, 16, 18, and 31. These mutations activate the K-Ras oncogene, impacting its GTPase activity and potentially contributing to tumor development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Previous studies identified K-ras gene mutations in 7/15 adrenocortical tumors.
- These mutations occurred at novel hot spots (codons 15, 16, 18, 31), differing from previously reported sites (codons 12, 13, 61).
Purpose of the Study:
- To investigate if K-ras gene mutation hot spots in adrenocortical tumors are associated with K-Ras oncogene activation.
- To analyze the alterations in K-Ras protein biocharacteristics resulting from these specific mutations.
Main Methods:
- Cloning of mutant K-ras genes from tumor tissues.
- Expression of mutant K-Ras proteins in Escherichia coli.
- Assessing intrinsic GTPase activity and GTPase-activating protein (GAP)-mediated activity of purified K-Ras proteins.
Main Results:
- Mutant K-Ras proteins (p21BM2) from codons 15, 16, 18, and 31 exhibited significantly lower GTPase activity in the presence of GAP compared to wild-type K-Ras (p21BN).
- Intrinsic GTPase activity of these mutant proteins was comparable to wild-type.
- A K-ras mutation at codon 60 resulted in an activated protein (p21BM3) with ten times lower intrinsic GTPase activity than wild-type.
Conclusions:
- Mutations at the identified hot spots (codons 15, 16, 18, 31) in the K-ras gene activate the K-Ras oncogene in adrenocortical tumors.
- Further research is required to definitively establish the association between these activated K-Ras mutations and adrenocortical tumor development.