Palmitoylated cysteine 192 is required for RhoB tumor-suppressive and apoptotic activities.
RhoA and RhoB share 86% amino acid sequence identity, yet RhoA promotes whereas RhoB suppresses malignant transformation. Amino acids 29, 100, 116, 123, 129, 140-143, 141, 146, 152, 154, 155, 173, 181, 183-187, 189, 190, 191, 192, and 193 in RhoB were mutated to the corresponding RhoA residues to determine those critical for RhoB tumor-suppressive activity. Of all the mutants made, only the cysteine 192 (one of two palmitoylation sites) and cysteine 193 (the prenylation site) point mutations abolish RhoB functions. In contrast, mutation of the other palmitoylation site, cysteine 189, did not affect RhoB functions. Moving cysteine 192 to position 190 did not affect RhoB function either. Mutation of cysteine 192 to glycine, alanine, or serine blocks the ability of RhoB to suppress transforming growth factor beta type II receptor, p2lwaf, and AP-1 promoter transcriptional activities. Furthermore, mutations of cysteines 192 and 193, but not 189, mislocalize RhoB and prevent RhoB from inhibiting anchorage-dependent and anchorage-independent tumor growth and colony formation as well as prevent it from inducing apoptosis. The cysteine 192 RhoB mutant is farnesylated and geranylgeranylated as efficiently as wild type RhoB. A RhoA-(1-180)/RhoB-(181-196) chimera inhibited tumor cell proliferation and induced apoptosis as efficiently as RhoB. These results demonstrate that the presence of neither cysteine 193 nor cysteine 192 alone is sufficient and that both palmitoylated cysteine 192 and prenylated cysteine 193, but not palmitoylated cysteine 189, are required for RhoB tumor-suppressive and proapoptotic activities.
Wang
De-An
DA
Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Interdisciplinary Oncology, Tampa, Florida 33612, USA.
Sebti
Said M
SM
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D017209:Apoptosis; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004396:Coloring Agents / Q000494:pharmacology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000494:pharmacology; D016147:Genes, Tumor Suppressor; D005998:Glycine / Q000737:chemistry; D006801:Humans; D020287:In Situ Nick-End Labeling; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019308:Palmitic Acid / Q000737:chemistry; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D017368:Protein Prenylation; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000737:chemistry; D020743:rhoB GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D004396:Coloring Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017931:DNA Primers; D013778:Tetrazolium Salts; D013844:Thiazoles; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D019308:Palmitic Acid; D012694:Serine; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; C022616:thiazolyl blue; D003545:Cysteine; D000409:Alanine; D005998:Glycine