scholarly journals Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation, and proteolytic processing.

1986 ◽  
Vol 6 (4) ◽  
pp. 1343-1348 ◽  
Author(s):  
M Hannink ◽  
D J Donoghue
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Gene Product ◽  
Signal Sequence ◽  
Intracellular Localization ◽  
Growth Factor Receptor ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Platelet Derived Growth Factor ◽  
Directed Mutagenesis

The v-sis oncogene and its cellular homolog c-sis encode chain B of platelet-derived growth factor. Cells transformed by v-sis produce a platelet-derived growth factor-related molecule which is able to stimulate the platelet-derived growth factor receptor in an autocrine fashion. Site-directed mutagenesis was used to construct several mutations which substitute charged residues for hydrophobic residues in the proposed signal sequence of the v-sis gene product. Two of these mutations resulted in the synthesis of altered v-sis gene products with an unexpected nuclear location and a loss of biological activity. We also report here the intracellular localization of the v-sis gene product to the endoplasmic reticulum-Golgi compartment, where signal sequence cleavage and N-linked glycosylation occur. The v-sis gene product contains no transmembrane regions, as it is completely protected within isolated microsomes from trypsin proteolysis. Site-directed mutagenesis was also used to alter a proposed proteolytic processing site in the v-sis gene product. This mutant v-sis gene, which encodes Asn-Ser in place of Lys-Arg at residues 110 to 111, was found to retain full biological activity.

Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation, and proteolytic processing

1986 ◽  
Vol 6 (4) ◽  
pp. 1343-1348
Author(s):  
M Hannink ◽  
D J Donoghue
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Gene Product ◽  
Signal Sequence ◽  
Intracellular Localization ◽  
Growth Factor Receptor ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Platelet Derived Growth Factor ◽  
Directed Mutagenesis

The v-sis oncogene and its cellular homolog c-sis encode chain B of platelet-derived growth factor. Cells transformed by v-sis produce a platelet-derived growth factor-related molecule which is able to stimulate the platelet-derived growth factor receptor in an autocrine fashion. Site-directed mutagenesis was used to construct several mutations which substitute charged residues for hydrophobic residues in the proposed signal sequence of the v-sis gene product. Two of these mutations resulted in the synthesis of altered v-sis gene products with an unexpected nuclear location and a loss of biological activity. We also report here the intracellular localization of the v-sis gene product to the endoplasmic reticulum-Golgi compartment, where signal sequence cleavage and N-linked glycosylation occur. The v-sis gene product contains no transmembrane regions, as it is completely protected within isolated microsomes from trypsin proteolysis. Site-directed mutagenesis was also used to alter a proposed proteolytic processing site in the v-sis gene product. This mutant v-sis gene, which encodes Asn-Ser in place of Lys-Arg at residues 110 to 111, was found to retain full biological activity.

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

1987 ◽  
Vol 7 (10) ◽  
pp. 3527-3537
Author(s):  
B A Lee ◽  
D W Maher ◽  
M Hannink ◽  
D J Donoghue
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Amino Acid Sequence ◽  
Gene Product ◽  
Signal Sequence ◽  
Nuclear Transport ◽  
Proteolytic Processing ◽  
Platelet Derived Growth Factor ◽  
Cell Fractionation ◽  
Mutant Proteins

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.

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

1987 ◽  
Vol 7 (10) ◽  
pp. 3527-3537 ◽  
Author(s):  
B A Lee ◽  
D W Maher ◽  
M Hannink ◽  
D J Donoghue
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Amino Acid Sequence ◽  
Gene Product ◽  
Signal Sequence ◽  
Nuclear Transport ◽  
Proteolytic Processing ◽  
Platelet Derived Growth Factor ◽  
Cell Fractionation ◽  
Mutant Proteins

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.

scholarly journals Cell surface expression of membrane-anchored v-sis gene products: glycosylation is not required for cell surface transport.

1986 ◽  
Vol 103 (6) ◽  
pp. 2311-2322 ◽  
Author(s):  
M Hannink ◽  
D J Donoghue
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Gene Product ◽  
Surface Expression ◽  
Proteolytic Processing ◽  
Biologically Active ◽  
Platelet Derived Growth Factor ◽  
Cell Surface Expression ◽  
Gene Products ◽  
Surface Transport

The v-sis gene is able to transform cells by production of a growth factor that is structurally related to platelet-derived growth factor. This growth factor has been detected in the conditioned media of v-sis transformed cells, and is able to stimulate the autophosphorylation of the platelet-derived growth factor receptor. We have used the v-sis gene product to analyze the role of protein-encoded signals in cell surface transport. We constructed several gene fusions that encode transmembrane forms of the v-sis gene product. These membrane-anchored forms of the v-sis gene product are properly folded into a native structure, as indicated by their dimerization, glycosylation, and NH2-terminal proteolytic processing. Indirect immunofluorescence demonstrated that several of these membrane-anchored gene products are transported to the cell surface. Removal of the N-linked glycosylation site from the v-sis gene product did not prevent cell surface transport. Several of these mutant genes are able to induce focus formation in NIH3T3 cells, providing further evidence that the membrane-anchored proteins are properly folded. These results demonstrate that N-linked glycosylation is not required for the cell surface transport of a protein that is in a native, biologically active conformation. These results provide a correlation between cell surface expression of the membrane-anchored v-sis gene products and transformation.

scholarly journals Two conserved cysteine residues are critical for the enzymic function of the human platelet-derived growth factor receptor-β: evidence for different roles of Cys-822 and Cys-940 in the kinase activity

2004 ◽  
Vol 382 (2) ◽  
pp. 631-639 ◽  
Author(s):  
Joon-Won LEE ◽  
Jee-Eun KIM ◽  
Eun-Jung PARK ◽  
Jin-Hyun KIM ◽  
Chang-Hun LEE ◽  
...  
Keyword(s):  
Growth Factor ◽  
Enzyme Catalysis ◽  
Kinase Activity ◽  
Growth Factor Receptor ◽  
Site Directed Mutagenesis ◽  
Platelet Derived Growth Factor ◽  
Cysteine Residues ◽  
Disulphide Bond Formation ◽  
Catalytic Loop ◽  
The Individual

The platelet-derived growth factor receptor-β (PDGFR-β) has a number of conserved cysteine residues on its cytoplasmic domain. We have examined whether the cysteine residues play a role in the enzymic function of PDGFR-β. We found that N-ethylmaleimide, which selectively alkylates free thiol groups of cysteine residues, completely inhibited the kinase activity of PDGFR-β. We then identified, through site-directed mutagenesis, two conserved cysteine residues critical for the enzymic function of PDGFR-β. Cys to Ser mutations for either Cys-822, positioned in the catalytic loop, or Cys-940, located in the C-terminal kinase subdomain, significantly reduced the activities of autophosphorylation and phosphorylation towards exogenous substrates. The non-reducing gel analysis indicated that neither of these cysteine residues contributes to the kinase activity by disulphide-bond formation. In addition, the individual mutation of Cys-822 and Cys-940 had no effect on protein stability or the binding of substrates or ATP, implying that these cysteine residues are involved in enzyme catalysis. Finally, proteolytic cleavage assays showed that the mutation of Cys-940, but not Cys-822, induced a protein conformational change. Taken together, these results suggest that Cys-940 contributes to the catalytic activity of PDGFR-β by playing a structural role, whereas Cys-822 contributes through a different mechanism.

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