Comparison of src-family cDNAs reveals distinct mechanisms underlying focus formation in transfected fibroblasts.
Despite the intensive study of both cellular transformation and src-family protein-tyrosine kinases, there have been no direct comparisons of transforming potency for normal members of this gene-family. In this study, the focus-forming activity of normal c-src, fyn, and lck cDNAs were compared in NIH 3T3 cell transfection assays. Focus formation was studied quantitatively, and individual foci were analyzed for phosphotyrosine content and expression of appropriate translational products. Each foci arising from c-src transfectants had a marked increase in phosphotyrosine content, and the majority of these foci expressed a c-src protein with an aberrant carboxyl terminus. Foci derived from lck transfectants also had a marked increase in phosphotyrosine content, and some foci expressed a lck protein with an aberrant carboxyl terminus. In contrast, foci from fyn-transfected cells were not distinguished from G418-selected mass cultures in terms of total phosphotyrosine content or expression of p59fyn. These studies support the previously published concept that overexpression of the normal fyn protein contributes to focus formation in transfected NIH 3T3 cells but suggest that the focus-forming activity observed after c-src or lck transfections is frequently attributable to mutational events. Because lck mutations have not been previously described in transformed foci, we characterized the lck transcript expressed in two foci and identified a novel point mutation that encodes a lck protein with increased in vivo kinase and focus-forming activity.
Sartor
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Clinical Pharmacology Branch, National Cancer Institute, Bethesda, Maryland 20892.
McLellan
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CA
Chiueh
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D002645:Chickens; D015870:Gene Expression; D016391:Genes, src; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015688:Oncogene Protein pp60(v-src) / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011519:Proto-Oncogenes; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; C494960:Fyn protein, mouse; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D015688:Oncogene Protein pp60(v-src); D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src)