Genetic analysis of the K-rev-1 transformation-suppressor gene.
Flat revertants with reduced malignancy in vivo can be isolated from Kirsten sarcoma virus-transformed NIH 3T3 cells (DT line) following transfection with a normal human fibroblast cDNA expression library. We have recovered from one such revertant a 1.8-kb cDNA clone, K-rev-1, that exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when transfected into DT cells. The K-rev-1 cDNA has the capacity to encode a protein with a calculated molecular weight of 21,000, having strong structural similarity to ras proteins (approximately 50% homology), especially in their guanosine triphosphate/guanosine diphosphate-binding, effector-binding, and membrane-attachment domains. Toward understanding the mechanism of action of K-rev-1 protein, we constructed a series of point mutants of K-rev-1 cDNA and tested their biological activities. Substitutions of the amino acid residues in the putative guanine nucleotide-binding regions (Asp17 and Asn116), in the putative effector-binding domain (residue 38), at the putative acylation site (Cys181), and at the unique Thr61 all decreased the transformation-suppressor activity. On the other hand, substitutions including Gly12 to Val12, Ala59 to Thr59, and Gln63 to Glu63 were found to significantly increase the transformation-suppressor activity of K-rev-1. These findings are consistent with the idea that K-rev-1 protein is regulated like many other G-proteins by guanine triphosphate/guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.
Kitayama
H
H
Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Ibaraki, Japan.
Matsuzaki
T
T
Sugimoto
Y
Y
Ikawa
Y
Y
Noda
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D005347:Fibroblasts; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology; D020690:GTPase-Activating Proteins; D016147:Genes, Tumor Suppressor; D011905:Genes, ras; D005816:Genetic Complementation Test; D006801:Humans; D007708:Kirsten murine sarcoma virus; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009374:Neoplasms, Experimental / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D014162:Transfection; D020668:rap GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D004247:DNA; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins