Genetic Analysis of the Kirsten-ras-Revertant 1 Gene: Potentiation of its Tumor Suppressor Activity by Specific Point Mutations

• Published in: Proceedings of the National Academy of Sciences - PNAS 1990-06, Vol.87 (11), p.4284-4288
• Main Authors: Kitayama, Hitoshi, Matsuzaki, Tomoko, Ikawa, Yoji, Noda, Makoto
• Format: Article
• Language: English
• Subjects: 3T3 cells; 550201 - Biochemistry- Tracer Techniques; ACID ANHYDRASES; AMIDES; Amino Acid Sequence; AMINO ACIDS; ANIMAL CELLS; Base Sequence; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; CARBOXYLIC ACIDS; Cell growth; Cell lines; Cell Transformation, Neoplastic - genetics; Cells; Complementary DNA; CONNECTIVE TISSUE CELLS; DNA; DNA Mutational Analysis; ENZYMES; FIBROBLASTS; Fundamental and applied biological sciences. Psychology; GENE MUTATIONS; GENE REGULATION; GENE REPRESSORS; GENES; Genes. Genome; Genetic mutation; GLUTAMINE; GLYCINE; GTP-ASES; GTP-Binding Proteins - genetics; HYDROLASES; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; MUTATIONS; Neoplasms, Experimental - pathology; NIH 3T3 cells; NUCLEIC ACIDS; NUCLEOPROTEINS; Oligonucleotides; Oncogene Protein p21(ras) - genetics; ONCOGENES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PROTEINS; rap GTP-Binding Proteins; RECOMBINANT DNA; SOMATIC CELLS; Structure-Activity Relationship; Suppression, Genetic; Transfection; TUMOR CELLS; Tumors; VALINE
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.11.4284

Kirsten-ras-revertant 1 (Krev-1) cDNA encodes a ras-related protein and exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when introduced into a v-K-ras-transformed mouse NIH 3T3 cell line, DT. Toward understanding the mechanism of action of Krev-1 protein, we constructed a series of point mutants of Krev-1 cDNA and tested their biological activities in DT cells and HT1080 human fibrosarcoma cells harboring the activated N-ras gene. Substitutions of the amino acid residues in the putative guanine nucleotide-binding regions (Asp17and Asn116), in the putative effector-binding domain (residue 38), at the putative acylation site (Cys181), and at the unique Thr61all decreased the transformation suppressor activity. On the other hand, substitutions such as Gly12to Val12and Gln63to Glu63were found to significantly increase the transformation suppressor/tumor suppressor activity of Krev-1. These findings are consistent with the idea that Krev-1 protein is regulated like many other G proteins by the guanine triphosphate/guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.