Deletions in the N-terminal coding region of the v-sis gene: determination of the minimal transforming region.

The gene product of the v-sis gene is closely related to the B chain of platelet-derived growth factor (PDGF). However, v-sis also encodes additional amino acids at its N and C termini, which are not represented in the sequence data of PDGF. We have constructed a series of N-terminal deletion mutants in the v-sis gene to define the minimum region required for transformation. These mutants were assayed for biological activity by using retroviral expression vectors which donate a signal sequence, required for translocation across the rough endoplasmic reticulum, to the mutant gene product. The minimal transforming region of the v-sis gene product defined by this analysis has 15 residues missing at the N terminus when compared with the PDGF-B chain. There are only two residues separating the closest transforming and nontransforming gene products. Mutant gene products lacking both the basic dipeptide processing site and the N-linked glycosylation site were found to be biologically active, indicating the dispensability of those processing steps. These results delimit the minimal transforming region of the v-sis gene product to residues 127 through 214, a total of 21 residues smaller than the PDGF-B chain.     

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

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.     

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

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.     

The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor.

The relationship between platelet-derived growth factor (PDGF) and the proto-oncogene c-sis has been determined by amino acid sequence analysis of PDGF and nucleotide sequence analysis of c-sis genomic clones. The nucleotide sequences of five regions of the human c-sis gene which are homologous to sequences of the transforming region (v-sis) of simian sarcoma virus (SSV) were determined. By alignment of the c-sis and v-sis nucleotide sequences the predicted amino acid sequence of a polypeptide homologous to the putative transforming protein p28sis of SSV was deduced. Both predicted sequences use the same termination codon and additional coding sequences may lie 5' to the homologous regions. Amino acid sequence analysis of the PDGF B chain shows identity to the amino acid sequence predicted from the c-sis sequences over 109 amino acid residues. Polymorphism may exist at two amino acid residues. These results suggest that c-sis encodes a polypeptide precursor of the B chain. A partial amino acid sequence of the PDGF A chain is also described. This chain is 60% homologous to the B chain and cannot be encoded by that part of c-sis which has been sequenced but could be encoded by sequences which lie 5' to the five regions of v-sis homology in c-sis, or at a separate locus.     

Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.     

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

A small v-sis/platelet-derived growth factor (PDGF) B-protein domain in which subtle conformational changes abrogate PDGF receptor interaction and transforming activity.

Deletion scanning mutagenesis within the transforming region of the v-sis oncogene was used to dissect structure-function relationships. Mutations affecting codons within a domain encoding amino acids 136 through 148 had no effect upon homodimer formation or recognition by antisera which detect determinants dependent upon native intrachain disulfide linkages, yet the same mutations completely abolished transforming activity. A platelet-derived growth factor B (PDGF B) monoclonal antibody that prevents its interaction with PDGF receptors recognized v-sis, delta 142 (deletion of codon 142), and delta 148 but not delta 136, delta 137, or delta 139 mutants. These findings mapped the epitope recognized by this monoclonal antibody to include amino acid residues 136 to 139. Furthermore, mutations in the codon 136 to 148 domain caused markedly impaired ability to induce PDGF receptor tyrosine phosphorylation. Thus, subtle conformational alterations in this small domain critically affect PDGF receptor recognition and/or functional activation.     

Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli

Preparations of the mitogen platelet-derived growth factor (PDGF) from human platelets contain two related polypeptides termed A chain and B chain. PDGF-B is highly homologous to a portion of p28v-sis, the transforming protein of simian sarcoma virus. We have studied the mitogenic potential of a PDGF-BB-like homodimer by expressing the sequence coding for the mature part of PDGF-B in Escherichia coli. Expression was achieved as cro-beta-gal-PDGF-B fusion protein which was exclusively found in the "inclusion bodies". A monomeric PDGF-B fragment shortened by 12 amino acid residues from the NH2 terminus was excised from the fusion protein by CNBr cleavage. After protection of thiols by S-sulfonation, this fragment was purified by gel permeation chromatography and reversed-phase high-performance liquid chromatography. This monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active rPDGF-BB with an overall yield of approximately 0.7 mg of rPDGF-BB/L of culture. Escherichia coli rPDGF-BB stimulated [3H]thymidine incorporation into AKR2B fibroblast at concentrations of about 1 ng/mL.     

Nucleotide sequence analysis identifies the human c-sis proto-oncogene as a structural gene for platelet-derived growth factor

The simian sarcoma virus transforming gene, v-sis, encodes a protein, p28sis , that is closely related to human platelet-derived growth factor (PDGF). The human locus related to v-sis was cloned and shown to contain at least five exons corresponding to the v-sis coding region. Nucleotide sequence analysis of these exons revealed that the predicted amino acid sequence of human c-sis differed by 6% from that of the woolly monkey-derived v-sis. These findings imply that the sis proto-oncogene has been well conserved during primate evolution. By comparison of the known amino acid sequences of PDGF peptides with the predicted human c-sis protein, it was possible to demonstrate that this human proto-oncogene is the structural gene encoding one of the two major polypeptides of this potent mitogen for connective tissue cells.     

Structure of the murine c-sis proto-oncogene (Sis, PDGFB) encoding the B chain of platelet-derived growth factor

The murine proto-oncogene c-sis (Sis, PDGFB), encoding the B chain of platelet-derived growth factor, has been cloned. Its structure, with seven exons spanning approximately 20 kb, closely resembles that of the human and feline homologs. The predicted amino acid sequence of murine PDGF-B has residues 89% identical to those of human PDGF-B. A noncoding region at the start of exon 7, which is deleted by alternative splicing during the generation of the viral v-sis oncogene, is highly conserved in human, mouse, and cat and may represent an important regulatory element.     

Analysis of E1A-mediated growth regulation functions: binding of the 300-kilodalton cellular product correlates with E1A enhancer repression function and DNA synthesis-inducing activity.

Adenovirus E1A transforming function requires two distinct regions of the protein. Transforming activity is closely linked with the presence of a region designated conserved domain 2 and the ability of this region to bind the product of the cellular retinoblastoma tumor suppressor gene. We have investigated the biological properties of the second transforming region of E1A, which is located near the N terminus. Transformation-defective mutants containing deletions in the N terminus (deletion of residues between amino acids 2 and 36) were deficient in the ability to induce DNA synthesis and repress insulin enhancer-stimulated activity. The function of the N-terminal region correlated closely with binding of the 300-kilodalton E1A-associated protein and not with binding of the retinoblastoma protein. These results indicate that transformation by E1A is mediated by two functionally independent regions of the protein which interact with different specific cellular proteins and suggest that the 300-kilodalton E1A-associated protein plays a major role in E1A-mediated cell growth control mechanisms.     

Intracellular turnover, novel secretion, and mitogenically active intracellular forms of v-sis gene product in simian sarcoma virus-transformed cells. Implications for intracellular loop autocrine transformation

In simian sarcoma virus (SSV)-transformed cells (SSV-NRK, SSV-NIH 3T3, and SSV-NP1 cells), the v-sis gene product was synthesized as a 36-kDa glycopolypeptide with one endoglycosidase (Endo) H-sensitive oligosaccharide chain and formed a dimer (p72) with a half-time of less than 5 min. p72 was proteolytically processed to generate sequentially p68 and p58 in the endoplasmic reticulum/Golgi complex, p44 in the post-Golgi complex compartments, and p27 in an endosomal/lysosomal compartment. A portion (20-30%) of p72 and p68 later became Endo H-resistant but Endo F-sensitive. During processing, the v-sis gene products exhibited rapid turnover, possibly in the endoplasmic reticulum and/or Golgi complex. The rate of turnover correlated with the tumorigenicity previously reported in these SSV-transformed cells. All three SSV-transformed cells secreted v-sis gene product (p44). p44 was secreted but remained tightly associated with the cell surface. This novel secretion provided an efficient system for the interaction of p44 with the cell surface platelet-derived growth factor receptor which resulted in the intracellular formation of p27. A fraction of secreted p44 was converted extracellularly to a 27-kDa product (extracellular p27) after a longer time in culture. The identical N-terminal amino acid sequence of p44 and extracellular p27 (H2N-SLGSLSVAEPAMIA) indicated a preferential site (Lys110-Arg111) for the proteolytic processing. The intracellular turnover of the v-sis gene product and its correlation with tumorigenicity as well as the demonstration of mitogenically active intracellular forms of v-sis gene product support the hypothesis of intracellular loop autocrine transformation.     

The v-sis oncogene product but not platelet-derived growth factor (PDGF) A homodimers activate PDGF alpha and beta receptors intracellularly and initiate cellular transformation.

The v-sis oncogene product p28v-sis and the platelet-derived growth factor (PDGF) B chain share 92% homology with each other and over 50% homology with the PDGF A chain. Exogenously added homodimers of PDGF A and PDGF B and of p28v-sis are potent mitogens but only PDGF B and p28v-sis induce transformation when endogenously expressed with a strong promoter. Because exogenous PDGF AA and PDGF BB both initiate a full mitogenic response, understanding the mechanisms underlying the difference in their transforming potential may clarify how growth factor genes act as oncogenes. In this work, we compared cells expressing high levels of PDGF A and v-sis. We observed that transformation by v-sis correlated directly with the rapid degradation (t1/2 approximately 20 min) of the alpha and beta PDGF receptors, with a failure of either the alpha or beta receptor to be fully processed and with the association of high levels of phosphatidylinositol (PI) 3-kinase with immunoprecipitates of the PDGF receptors. In contrast, in cells expressing essentially equal levels of PDGF A, transformation was not detected, alpha and beta PDGF receptor processing was normal, and association of PI 3-kinase with receptors in immunoprecipitates was not found above control values. The ability of v-sis to autoactivate PDGF receptors within processing compartments and to initiate activation of the PI 3-kinase signaling pathway coupled with the failure of PDGF A to activate its receptor intracellularly and to induce transformation when endogenously expressed at high levels suggests that the internal autoactivation of PDGF receptors may be essential for transformation by v-sis.     

The v-sis protein retains biological activity as a type II membrane protein when anchored by various signal-anchor domains, including the hydrophobic domain of the bovine papilloma virus E5 oncoprotein

Membrane-anchored forms of the v-sis oncoprotein have been previously described which are oriented as type I transmembrane proteins and which efficiently induce autocrine transformation. Several examples of naturally occurring membrane-anchored growth factors have been identified, but all exhibit a type I orientation. In this work, we wished to construct and characterize membrane-anchored growth factors with a type II orientation. These experiments were designed to determine whether type II membrane-anchored growth factors would in fact exhibit biological activity. Additionally, we wished to determine whether the hydrophobic domain of the E5 oncoprotein of bovine papilloma virus (BPV) can function as a signal-anchor domain to direct type II membrane insertion. Type II derivatives of the v-sis oncoprotein were constructed, with the NH2 terminus intracellular and the COOH terminus extracellular, by substituting the NH2 terminal signal sequence with the signal-anchor domain of a known type II membrane protein. The signal-anchor domains of neuraminidase (NA), asialoglycoprotein receptor (ASGPR) and transferrin receptor (TR) all yielded biologically active type II derivatives of the v-sis oncoprotein. Although transforming all of the type II signal/anchor-sis proteins exhibited a very short half-life. The short half-life exhibited by the signal/anchor-sis constructs suggests that, in some cases, cellular transformation may result from the synthesis of growth factors so labile that they activate undetectable autocrine loops. The E5 oncoprotein encoded by BPV exhibits amino acid sequence similarity with PDGF, activates the PDGF beta-receptor, and thus resembles a miniature membrane-anchored growth factor with a putative type II orientation. The hydrophobic domain of the E5 oncoprotein, when substituted in place of the signal sequence of v-sis, was indistinguishable compared with the signal-anchor domains of NA, TR, and ASGPR, demonstrating its ability to function as a signal-anchor domain. NIH 3T3 cells transformed by the signal/anchor-sis constructs exhibited morphological reversion upon treatment with suramin, indicating a requirement for ligand/receptor interactions in a suramin- sensitive compartment, most likely the cell surface. In contrast, NIH 3T3 cells transformed by the E5 oncoprotein did not exhibit morphological reversion in response to suramin.     

Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.     

Cloning,expression, characterization,and role in autocrine cell growth of cell surface retention sequence binding protein-1

Cell surface retention sequence binding protein-1 (CRSBP-1) is a cell surface binding protein for the cell surface retention sequence (CRS) motif of the v-sis gene product (platelet-derived growth factor-BB). It has been shown to be responsible for cell surface retention of the v-sis gene product in v-sis-transformed cells (fibroblasts) and has been hypothesized to play a role in autocrine growth and transformation of these cells. Here we demonstrate that the CRSBP-1 cDNA cloned from bovine liver libraries encodes a 322-residue type I membrane protein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain. CRSBP-1 expressed in transfected cells is an approximately 120-kDa disulfide-linked homodimeric glycoprotein and exhibits dual ligand (CRS-containing growth regulators (v-sis gene product and insulin-like growth factor binding protein-3, IGFBP-3) and hyaluronic acid) binding activity. CRSBP-1 overexpression (by stable transfection of cells with CRSBP-1 cDNA) enhances autocrine loop signaling, cell growth, and tumorigenicity (in mice) of v-sis-transformed cells. CRSBP-1 expression also enhances autocrine cell growth mediated by IGFBP-3 in human lung carcinoma cells (H1299 cells), which express very little, if any, endogenous CRSBP-1 and exhibits a mitogenic response to exogenous IGFBP-3, stably transfected with IGFBP-3 cDNA. However, CRSBP-1 overexpression does not affect growth of normal and transformed cells that do not produce these CRS-containing growth regulators. These results suggest that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by growth regulators containing CRS.     

Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transforming protein.

The bovine papillomavirus E5 gene encodes a 44 amino acid membrane-associated protein that can induce tumorigenic transformation of rodent fibroblast cell lines. Genetic studies suggest that the E5 protein may transform cells by influencing the activity of cellular proteins involved in growth regulation. We report here that the endogenous cellular beta type receptor for the platelet-derived growth factor (PDGF) is constitutively activated in C127 and FR3T3 cells stably transformed by the E5 protein, but not in these cell types transformed by a variety of other oncogenes. In C127 cells, a metabolic precursor as well as the mature form of the receptor is activated by E5 transformation. Activation of the receptor also occurs upon acute E5-mediated transformation of these cells and precedes mitogenic stimulation in this system. Moreover, activation of the receptor by addition of PDGF or the v-sis gene to untransformed cells is sufficient to induce DNA synthesis and stable growth transformation. We propose that the PDGF receptor is an important cellular intermediate in the transforming activity of the bovine papillomavirus E5 protein. There is a short region of sequence similarity between the fibropapillomavirus E5 proteins and PDGF, suggesting that the E5 proteins may activate the PDGF receptor by binding directly to it.     

The phenotypic characteristics of simian sarcoma virus-transformed human fibroblasts suggest that the v-sis gene product acts solely as a PDGF receptor agonist in cell transformation.

Previous studies have indicated that the oncogene v-sis of simian sarcoma virus (SSV) encodes a growth factor that is structurally and functionally similar to platelet-derived growth factor (PDGF). In the present investigation we have analysed the phenotypic characteristics of human foreskin fibroblasts transformed by SSV. It was found that the PDGF receptors were extensively down-regulated. This finding is consistent with a high, local, extracellular concentration of a PDGF-like factor, synthesized by the transformed cell. The receptors were up-regulated by suramin, a drug that is known to dissociate PDGF and the v-sis product from the PDGF receptors. A cell-associated v-sis product of mol. wt 24,000 was identified by immunoprecipitation with PDGF antibodies; release of this component was induced by a high concentration of exogenous PDGF, indicating that a fraction of the product is associated with the PDGF receptors. SSV was not found to be an immortalizing virus; when serially passaged, SSV-transformed cells had essentially the same life-span as their non-transformed counterparts. Moreover, SSV did not induce growth in soft agar beyond the level afforded by exogenously added PDGF. Thus, the present study favors the notion that SSV transformation is mediated by a growth factor that mimics PDGF but has no further cellular effects.     

Basic fibroblast-like growth factor is present in the conditioned medium of simian sarcoma virus transformed NRK cells

The conditioned medium of Simian sarcoma virus (SSV)-transformed NRK cells contains at least two activities that down regulate the epidermal growth factor receptor. To identify these activities, we analyzed the medium for the presence of factors both related to and distinct from the v-sis oncogene product. Fractionation of the conditioned medium from SSV-transformed NRK cells by chromatography on heparin-Sepharose yielded two active fractions capable of inhibiting EGF binding. The first component, which eluted at 0.8 M NaCl, is able to induce autophosphorylation of the platelet-derived growth factor (PDGF) receptor, is a mitogen for Swiss 3T3 cells and corresponds to the PDGF B chain product of the v-sis oncogene. The second component requires 2 M NaCl for elution, is mitogenic for Swiss 3T3 cells and inhibits high affinity EGF binding through a protein kinase C-independent pathway, all properties of basic FGF. These results suggest that the conditioned medium of v-sis-transformed cells contains at least two factors that can act in an autocrine capacity, one derived from v-sis and one corresponding to basic FGF.