Disrupting the transforming activity of shrimp ras(Q(61)K) by deleting the CAAX box at the C-terminus.
BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus. Ras transcription and protein levels had increased significantly in the cells transfected with the S-ras plasmid, compared to cells transfected with a control plasmid pcDNA3.1. The bacterially expressed GTP-locked S-Ras(Q(61)K) is successfully prenylated by rat protein geranylgeranyltransferase I (PGGTase I) and then polymerized with tubulin, in agreement with findings for GTP-locked mammalian K(B)-Ras(Q(61)K) in vitro. Shrimp protein farnesyltransferase (PFTase) of shrimp did not prenylate the GTP-locked shrimp S-Ras(Q(61)K) (Lin and Chuang. 1998. J Exp Zool 281:565-573), whereas rat PFTase efficiently catalyzed the farnesylation of GTP-locked S-Ras(Q(61)K). To investigate the effect of geranylgeranylation on cellular transformation, we generated S-ras(Q(61)K) mutants with deletion of the CAAX box [S-ras(Q(61)K)(-caax)] or replacement of the CAAX box [S-ras(Q(61)K)(Kcaax)] or replacement of the arginine-rich domain [S-ras(Q(61)K)(K-Lys)] with corresponding sequences from rat K(B)-ras(Q(61)K). BALB/3T3 cells transfected with DNA encoding S-ras(Q(61)K), S-ras(Q(61)K)(KCAAX), S-ras(Q(61)K)(K-Lys) were transformed successfully, but S-ras(Q(61)K)(-CAAX) was defective in its ability to transform. Thus, prenylation at CAAX is required for transformation. Either the geranylgeranylated or the farnesylated S-Ras(Q(61)K) was endowed with abilities to transform. The arginine-rich region in S-Ras or the lysine-rich clusters from the rat K(B)-Ras appear not essential for activity to transform.
Huang
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CF
Division of Biochemistry and Molecular Sciences, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan 11529.
Chen
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CH
Chuang
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NN
D016475:3T3 Cells; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D033364:Decapoda / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D051231:Farnesyltranstransferase; D011905:Genes, ras / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D016296:Mutagenesis; D017368:Protein Prenylation; D051381:Rats; D014162:Transfection; D014404:Tubulin / Q000737:chemistry; D018631:ras Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D004273:DNA, Neoplasm; D014404:Tubulin; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D051231:Farnesyltranstransferase; D018631:ras Proteins