Deletions in the C-terminal coding region of the v-sis gene: dimerization is required for transformation.

Hannink, Mark; Sauer, M K; Donoghue, Daniel J.

doi:10.1128/mcb.6.4.1304

Cited by 36 publications

(39 citation statements)

References 30 publications

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“…This vector provides high levels of expression from the SV40 late promoter. pJC119-derived plasmids containing deletions in the region encoding the C terminus of the v-sis protein have been described previously (22). Plasmids pMH74 and pMH101, which contain point mutations in the sequence encoding the signal peptide of the v-sis protein, have also been described (20).…”

Section: Methodsmentioning

confidence: 99%

“…Additional C-terminal deletions were constructed by using BAL 31 exonuclease as previously described (22). Briefly, pMS001 (22) was digested at its unique BamHI site and blunted with the Klenow fragment of Escherichia coli DNA polymerase I in the presence of deoxynucleoside triphosphates, and BAL 31 exonuclease was used to create deletions in the 3' portion of the v-sis coding region.…”

Section: Methodsmentioning

confidence: 99%

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Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.

Lee¹,

Maher²,

Hannink³

et al. 1987

Mol. Cell. Biol.

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The v-vis gene encodes p28sis, the transforming protein of simian sarcoma virus. This gene resulted from a fusion of the env gene of simian sarcoma-associated virus and the woolly monkey gene for the B chain of platelet-derived growth factor (PDGF). Previous work has shown that the v-sis gene product undergoes signal sequence cleavage, glycosylation, dimerization, and proteolytic processing to yield a secreted form of the protein. It transport across the endoplasmic reticulum is blocked by the introduction of a charged amino acid residue within the signal sequence, the protein does not dimerize, is not secreted, and is no longer transforming as assayed by focus-forming ability in NIH 3T3 cells. Instead, this mutant protein localizes to the nucleus as demonstrated by both indirect immunofluorescence and cell fractionation. Using a series of deletion mutations, we delimited an amino acid sequence within this protein which is responsible for nuclear localization. This region is completely conserved in the predicted human c-sis protein, although it lies outside of regions required for transformation by the v-sis gene product. This nuclear transport signal is contained within amino acid residues 237 to 255, RVTIRTVRVRRPPKGKHRK. An amino acid sequence containing these residues is capable of directing cytoplasmic v-sis mutant proteins to the nucleus. This sequence is also capable of directing less efficient nuclear transport of a normally cytoplasmic protein, pyruvate kinase. Pulse-chase experiments indicate that the half-lives of nuclear and cytoplasmic v-sis mutant proteins are approximately 35 min. Using the heat-inducible hsp70 promoter from Drosophila melanogaster, we showed that the nuclear v-sis protein accumulates in the nucleus within 30 min of induction. The identification of a nuclear transport signal in the v-sis gene product raises interesting questions regarding the possibility of some function for PDGF or PDGF-related molecules in the nucleus.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.

Lee¹,

Maher²,

Hannink³

et al. 1987

Mol. Cell. Biol.

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“…Transfections modified DEAE-dextran protocol (17). For CAT assays, Rat-1 cells were cotransfected with 10 p,g of pPLCAT and 10 ,ug of the respective plasmids containing the v-and vic-rel genes.…”

Section: Methodsmentioning

confidence: 99%

Transactivation of gene expression by nuclear and cytoplasmic rel proteins.

Hannink

Temin

1989

Mol. Cell. Biol.

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Transcriptional activation of gene expression by oncogenic proteins can lead to cellular transformation. It has recently been demonstrated that the protein encoded by the v-rel oncogene from reticuloendotheliosis virus strain T can transactivate gene expression from certain promoters in a cell-specific manner. We have examined the cytological location, transforming properties, and transactivation properties of proteins encoded by chimeric turkey v-rel/chicken c-rel genes. We found that whereas the v-rel protein was nuclear in both chicken embryo and rat fibroblasts, the presence of the C terminus of the c-rel protein inhibited nuclear localization of the rel protein in these fibroblasts. Cytoplasmic rel proteins containing C-terminal c-rel sequences transactivated gene expression from the polyomavirus late promoter as efficiently as did similar rel proteins located in the nucleus. These results indicate that the cellular location of rel proteins is not important for transactivation of gene expression and suggest that transactivation by rel proteins is indirect, perhaps by affecting an intracellular signal transduction pathway that eventually results in the alteration of gene expression. The transforming properties of the rel protein were unaltered by the presence of the c-rel C terminus, but, as previously reported for turkey c-rel sequences, substitution of chicken c-rel sequences for internal v-rel sequences reduced the transforming activity of the rel protein and eliminated the immortalization ability. However, all of the chimeric v/c-rel proteins were able to transactivate gene expression, indicating that transactivation does not correlate with transformation. These results suggest that transactivation may be necessary but is not sufficient for transformation by rel proteins.Recent evidence demonstrates that alteration of gene expression by oncogenic proteins can lead to cellular transformation. There are several mechanisms by which oncogenic proteins can alter gene expression. Certain oncogenes are thought to encode proteins that function as direct transactivators of transcription (23). A prototype of this group of oncogenic proteins is the v-jun oncogene. The v-and c-jun proteins are located in the nucleus, bind to DNA in a sequence-specific manner, and interact with other cellular proteins involved in transcription (3, 4). Both the oncogenic and the cellular proto-oncogenic jun proteins are able to activate transcription (3, 4), and cellular transformation by these oncogenic proteins probably results from subtle differences between the activation of genes by the oncogenic proteins and by the proto-oncogenic proteins. Alterations in gene expression have also been shown to result from expression of oncogenic proteins that are not directly involved in transcription. Autocrine expression of the v-sis oncogene results in the activation of transcription of many genes, including c-fos and c-myc (1,15,16,25,27). The v-mos oncogene has been shown to alter expression from the alpha-2 collagen promoter (32), and bot...

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“…PDGF BB has been identified as the human homolog of the v-sis oncogene product (4,5,19). The minimal v-sis transforming domain spans 89 amino acids (8,11), contains eight cysteine residues, four of which are involved in intrachain disulfide linkages essential for PDGF function (7,18), and is identical in sequence to PDGF BB. Regions of the molecule specifically involved in PDGF receptor binding and activation have yet to be elucidated.…”

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confidence: 99%

Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.

1989

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A monoclonal antibody (mAb), sis 1, generated against human c-sis-encoded platelet-derived growth factor (PDGF) BB, was shown by enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis to recognize human PDGF BB and human platelet PDGF AB but not the human PDGF AA. This monoclonal antibody potently inhibited PDGF receptor-binding and mitogenic activities of both human PDGF BB and PDGF AB but had no effect on PDGF AA. Finally, we demonstrated that an immunoaffinity-purified anti-c-sis peptide antibody (anti-V4) which also blocked binding of PDGF BB to its cognate receptor and competed with mAb sis 1 for binding to PDGF BB. All of these results suggest that mAb sis 1 recognizes an epitope of the c-sis gene product, PDGF BB, that spatially overlaps the V4 surface domain of PDGF BB, immunochemically localizing a region of PDGF BB critical for PDGF receptor binding and activation.

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Deletions in the C-terminal coding region of the v-sis gene: dimerization is required for transformation.

Cited by 36 publications

References 30 publications

Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.

Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.

Transactivation of gene expression by nuclear and cytoplasmic rel proteins.

Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.

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