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Structure of the activated c-raf-1 gene from human stomach cancer
K Shimizu1, Y Nakatsu, S Nomoto
1Molecular Genetics Section, Faculty of Science, Kyushu University, Fukuoka, Japan.
Abstract:
We previously isolated a novel human transforming gene from a primary stomach cancer and identified it as an activated version of the c-raf-1 gene which is the human homologue of v-raf, a viral oncogene encoding a serine/threonine-specific protein kinase. Analyses of cDNA and genomic clones of this gene revealed that it was generated by substitution of 5'-sequence (exons 1-5) of the normal c-raf-1 gene with an unrelated human sequence. We identified the region in the genomic clone where the rearrangement had occurred. The rearranged EcoRI fragment was detected in all the primary transformants obtained from two independent transfections, suggesting that the recombination had occurred in the primary cancer. By sequence analysis of cDNA, the putative product of the transforming gene was inferred to have a hydrophobic stretch ahead of the ser/thr-protein kinase domain of the c-raf-1 gene product. We introduced one of the cDNA which contains the 1.6-kb open reading frame into the pUC9 vector. An autophosphorylating, 58 kd protein was induced in Escherichia coli cells bearing the plasmid upon induction. Since ser/thr-protein kinase activity of the normal c-raf protein has not been evidenced, these results suggest that the truncation/replacement of the amino-terminal domain of the c-raf-1 protein leads to constitutive activation of the protein kinase probably residing on the downstream domain.
Insights
A novel transforming gene, an activated c-raf-1 (RAF proto-oncogene serine/threonine kinase), was found in stomach cancer. Its altered structure activates the protein kinase, suggesting a role in cancer development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The c-raf-1 gene encodes a serine/threonine-specific protein kinase and is homologous to the viral oncogene v-raf.
- Activated oncogenes play a crucial role in cancer development.
- Identifying novel transforming genes can elucidate cancer mechanisms.
Purpose of the Study:
- To characterize a novel human transforming gene isolated from stomach cancer.
- To determine the molecular basis of its transforming activity.
- To investigate the functional consequences of its altered structure.
Main Methods:
- Isolation and analysis of cDNA and genomic clones of the transforming gene.
- DNA sequencing to identify rearrangement sites and protein domains.
- Expression of the gene in Escherichia coli to study protein activity.
Main Results:
- The transforming gene is an activated version of c-raf-1, resulting from the substitution of its 5'-sequence with an unrelated human sequence.
- The rearrangement occurred in the primary stomach cancer.
- The altered c-raf-1 protein exhibits autophosphorylating activity and possesses a serine/threonine-specific protein kinase domain.
- Expression in E. coli produced an autophosphorylating 58 kDa protein.
Conclusions:
- Truncation or replacement of the amino-terminal domain of c-raf-1 leads to constitutive activation of its protein kinase activity.
- This constitutive activation is likely mediated by the downstream kinase domain.
- The activated c-raf-1 gene may contribute to stomach cancer development.