Mutational activation of pp60(c-src) leads to a tumorigenic phenotype in a preneoplastic Syrian hamster embryo cell line.
Previous studies indicated that overexpression of wild-type avian c-src cannot induce neoplastic transformation of NIH 3T3 cells. In this study, we isolated and characterized novel spontaneously derived transforming mutants of avian pp60(c-src) from a Syrian hamster embryo-derived cell line, 10W, transfected with the avian c-src gene. Seventeen independently derived transfected 10W cell clones were injected into athymic nude mice. After a latency period, tumors eventually arose and were established in culture. The tumorigenic phenotype was always accompanied by the presence of the avian c-src DNA and functional expression of pp60(c-src). However, most of the tumor-derived cell lines expressed an electrophoretically altered form of pp60(c-src), suggesting mutations in src. Consistent with this hypothesis, DNAs isolated from the tumor-derived lines, but not the parental 10W cell lines, morphologically transformed NIH 3T3 cells in a focus-forming assay. We characterized pp60(c-src) in detail from three of the tumor-derived lines: 4AT, 4BT, and E2T. Two of these lines contained mutations within the exogenous c-src coding region. Line 4AT has an internal repeat of 29 amino acids immediately following Gln-513, which disrupts the spacing between the end of the kinase domain and Tyr-527, the negative regulatory site in pp60(c-src). Line 4BT has a 5-bp deletion following Phe-520, which results in loss of Tyr-527. However, the DNA sequence of the coding region of pp60(c-src) from a third line, E2T, was completely wild type. Cyanogen bromide cleavage analyses of the altered pp60(c-src) from lines 4AT and 4BT showed that Tyr-527, the site of negative regulation of c-src, is not phosphorylated, but Tyr-416, the site of in vitro autophosphorylation, is phosphorylated. However, in line E2T, Tyr-527 was phosphorylated, and Tyr-416 was phosphorylated to a lesser extent. Additionally, two proteins that indicate activation of src, p85 cortactin and p120(cas), are phosphorylated in at least six of the tumor-derived cell lines, although to a lesser extent in line E2T. These results suggest that dephosphorylation of Tyr-527 and phosphorylation of Tyr-416 correlate with activation of pp60(c-src) in the tumor-derived lines 4AT and 4BT, respectively. However, in line E2T, the high levels of pp60(c-src), in combination with a partial activation of the pp60(c-src) protein as indicated by phosphorylation of Tyr-416, appear to be involved in the neoplastic process, rather than mutation.
Lansing
T J
TJ
Department of Cancer Biology, Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina 27709, USA.
Turk
B F
BF
Kanner
S B
SB
Gilmer
T M
TM
D016475:3T3 Cells / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D008647:Mesocricetus; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011230:Precancerous Conditions / Q000235:genetics*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D019000:Phosphotyrosine; D014443:Tyrosine; D004247:DNA; D016392:Proto-Oncogene Proteins pp60(c-src)