Transformation-resistant mos revertant is unable to activate MAP kinase kinase in response to v-mos or v-raf.
To study the mechanism by which v-mos induces cell transformation, we generated a transformed rat cell line (DTM) containing two functional copies of mos, one encoding the p37v-mos of the m1 wild-type strain of Moloney murine sarcoma virus (Mo-MuSV) and the other the p85gag-mos fusion protein of the ts110 mutant of Moloney murine sarcoma virus. Subsequently, we isolated a revertant cell line (F-1) following transfection of DTM with a mutant retroviral construct (pIC4Neo) carrying a selectable marker. Like DTM, the F-1 revertant contained two integrated copies of v-mos, expressed mos containing viral RNA, and contained rescuable transforming viruses. The revertant did not grow in soft agar, showed a greatly reduced ability to form tumors in nude mice, and exhibited organized tubulin and actin structures similar to those found in normal cells. Revertant cells were resistant to retransformation by v-mos and v-raf but could be retransformed by v-ras. MAP kinase (ERK-2) and MAP kinase kinase (MKK-1) activity, which are constitutively elevated in v-mos- and v-raf-transformed cells, exhibits levels in the F-1 revertant similar to those seen in nontransformed cells. F-1 and normal REF-1 cells express elevated levels of protein phosphatases in comparison to DTM cells. In vivo treatment with okadaic acid, a potent protein phosphatase inhibitor, leads to an increase in MKK-1 and MAP kinase activity in F-1 cells but not in REF-1. The results support the hypothesis that mos acts through the MAP kinase cascade (MKK-1 and ERK-2) to induce cell transformation and that blocking v-mos activation of that cascade (possibly because of increased levels of phosphatase) prevents transformation.
Topol
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LZ
Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Maryland 21702.
Marx
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Calothy
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Blair
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DG
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed / Q000166:cytology / Q000201:enzymology; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002472:Cell Transformation, Viral / Q000235:genetics*; D003598:Cytoskeletal Proteins / Q000737:chemistry; D015342:DNA Probes / Q000235:genetics; D004789:Enzyme Activation; D004988:Ethers, Cyclic / Q000494:pharmacology; D011905:Genes, ras; D020929:Mitogen-Activated Protein Kinase Kinases; D019319:Okadaic Acid; D017232:Oncogene Proteins v-mos / Q000235:genetics*; D027384:Oncogene Proteins v-raf; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection / Q000235:genetics; D018631:ras Proteins / Q000502:physiology
D003598:Cytoskeletal Proteins; D015342:DNA Probes; D004988:Ethers, Cyclic; D017232:Oncogene Proteins v-mos; D015682:Retroviridae Proteins, Oncogenic; D019319:Okadaic Acid; D011494:Protein Kinases; D027384:Oncogene Proteins v-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D018631:ras Proteins