Analysis of functional domains of the v-fms-encoded protein of Susan McDonough strain feline sarcoma virus by linker insertion mutagenesis.
The Susan McDonough strain of feline sarcoma virus contains an oncogene, v-fms, which is capable of transforming fibroblasts in vitro. The mature protein product of the v-fms gene (gp140fms) is found on the surface of transformed cells; this glycoprotein has external, transmembrane, and cytoplasmic domains. To assess the functional role of these domains in transformation, we constructed a series of nine linker insertion mutations throughout the v-fms gene by using a dodecameric BamHI linker. The biological effects of these mutations on the function and intracellular localization of v-fms-encoded proteins were determined by transfecting the mutated DNA into Rat-2 cells. Most of the mutations within the external domain of the v-fms-encoded protein eliminated focus formation on Rat-2 cells; three of these mutations interfered with the glycosylation of the v-fms protein and interfered with formation of the mature gp140fms. One mutation in the external domain led to cell surface expression of v-fms protein even in the absence of complete glycosylational processing. Cell surface expression of mutated v-fms protein is probably necessary, but is not sufficient, for cell transformation since mutant v-fms protein was found on the surface of several nontransformed cell lines. Mutations that were introduced within the external domain had little effect on in vitro kinase activity, whereas mutations within the cytoplasmic domain all had strong inhibitory effects on this activity.
Lyman
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SD
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Rohrschneider
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LR
D002451:Cell Compartmentation; D002472:Cell Transformation, Viral; D005455:Fluorescent Antibody Technique; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D009857:Oncogenes; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D005253:Sarcoma Viruses, Feline / Q000235:genetics*; D013329:Structure-Activity Relationship; D013499:Surface Properties
D009856:Oncogene Proteins, Viral; D011505:Protein-Tyrosine Kinases