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.     

Mutational analysis of the interaction between the bovine papillomavirus E5 transforming protein and the endogenous beta receptor for platelet-derived growth factor in mouse C127 cells.

The bovine papillomavirus E5 protein is a 44-amino-acid membrane-associated protein that forms a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor in rodent and bovine fibroblasts, resulting in sustained receptor activation and cell transformation. We report here that high-level expression of the E5 protein caused a reduction in the level of the mature form of the PDGF beta receptor in acutely and stably transformed mouse C127 cells. To explore in more detail the interaction of the E5 protein and the PDGF beta receptor, we tested the abilities of various E5 point mutants to bind the PDGF receptor, to induce PDGF receptor down-regulation and tyrosine phosphorylation, and to transform cells. A transformation-competent mutant, like the wild-type E5 protein, bound the receptor and induced receptor tyrosine phosphorylation and down-regulation. Transformation-defective E5 proteins either failed to interact with the endogenous PDGF beta receptor in mouse fibroblasts or underwent an aberrant interaction with the receptor. Mutation of glutamine at position 17, aspartic acid at position 33, or both carboxyl-terminal cysteine residues required for E5 homodimerization interfered with stable complex formation with the PDGF receptor, tyrosine phosphorylation and down-regulation of the receptor, and cell transformation. Point mutations at several other carboxyl-terminal positions generated transformation-defective E5 proteins that formed a complex with the PDGF receptor and induced receptor tyrosine phosphorylation but did not induce PDGF receptor down-regulation. Either PDGF receptor activation is not sufficient for transformation of C127 cells or the receptors that are tyrosine phosphorylated in response to these mutant E5 proteins are not fully activated and therefore are not able to deliver a mitogenic signal.     

The bovine papillomavirus type 1 E5 transforming protein specifically binds and activates the beta-type receptor for the platelet-derived growth factor but not other related tyrosine kinase-containing receptors to induce cellular transformation.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is a highly hydrophobic protein which appears to transform cells through the activation of growth factor receptors. To investigate the specificity of E5-growth factor receptor interactions required for mitogenic signaling, we utilized a nontumorigenic, murine myeloid cell line (32D) which is strictly dependent on interleukin-3 (IL-3) for sustained proliferation in culture. This IL-3 dependence can be functionally substituted by the expression of a variety of surrogate growth factor receptors and the addition of the corresponding ligand. Several receptor cDNAs for the alpha- and beta-type platelet-derived growth factor receptors [alpha PDGFR and beta PDGFR], the epidermal growth factor receptor, and the colony-stimulating factor 1 receptor) were transfected into 32D cells constitutively expressing the E5 protein to test for IL-3-independent growth. Only beta PDGFR was capable of abrogating the IL-3 dependence of 32D cells. The proliferative signal induced by the coexpression of beta PDGFR and E5 was accompanied by stable complex formation between these proteins, constitutive tyrosine phosphorylation of the receptor, and tumorigenicity in nude mice. The lack of cooperative interaction between E5 and the epidermal growth factor receptor, the colony-stimulating factor 1 receptor, and the highly related alpha PDGFR was paralleled by the inability of E5 to bind to these receptors and failure to increase receptor tyrosine phosphorylation. Thus, these data indicate that the ability of E5 to induce sustained proliferation and transformation of 32D cells is a direct consequence of specific interaction between the E5 protein and the beta PDGFR signaling complex and the subsequent stimulation of receptor tyrosine phosphorylation.     

Productive interaction between transmembrane mutants of the bovine papillomavirus E5 protein and the platelet-derived growth factor beta receptor

The bovine papillomavirus E5 protein is a 44-amino-acid transmembrane protein that transforms cells by binding to the transmembrane region of the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in sustained receptor signaling. However, there are published reports that certain mutants with amino acid substitutions in the membrane-spanning segment of the E5 protein transform cells without activating the PDGF beta receptor. We re-examined several of these transmembrane mutants, and here we present five lines of evidence that these mutants do in fact activate the PDGF beta receptor, resulting in cellular signaling and transformation.     

Specific interaction between the bovine papillomavirus E5 transforming protein and the beta receptor for platelet-derived growth factor in stably transformed and acutely transfected cells.

The E5 protein of bovine papillomavirus is a 44-amino-acid membrane protein which induces morphologic and tumorigenic transformation of fibroblasts. We previously showed that the E5 protein activates and forms a complex with the endogenous beta receptor for platelet-derived growth factor (PDGF) in transformed rodent fibroblasts and that the PDGF beta receptor can mediate tumorigenic transformation by the E5 protein in a heterologous cell system. Other workers have identified the receptor for epidermal growth factor (EGF) as a potential target of the E5 protein in NIH 3T3 cells. Here, we investigate the specificity of the interaction of the E5 protein with various growth factor receptors, with particular emphasis on the PDGF beta receptor and the EGF receptor. Under conditions where both the PDGF beta receptor and the EGF receptor are stably expressed in E5-transformed mouse and bovine fibroblasts and in E5-transformed epithelial cells, the E5 protein specifically forms a complex with and activates the PDGF receptor and not the EGF receptor. Under conditions of transient overexpression in COS cells, the E5 protein has the potential to associate with several growth factor receptors, including the EGF receptor. However, upon coexpression of PDGF beta receptors and EGF receptors in COS cells, the E5 protein preferentially forms a complex with the PDGF receptor. Therefore, we conclude that the PDGF beta receptor is the primary target for the E5 protein in a variety of cell types, including bovine fibroblasts.     

The platelet-derived growth factor beta receptor as a target of the bovine papillomavirus E5 protein

The 44-amino acid E5 protein of bovine papillomavirus is a homo-dimeric, transmembrane protein that transforms cells by activating the platelet-derived growth factor ß receptor in a ligand-independent fashion. The E5 protein induces receptor activation by forming a stable complex with the receptor, thereby inducing receptor dimerization, trans-phosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, and recruitment of cellular SH2 domain-containing proteins into a signal transduction complex. Direct interactions between specific transmembrane and juxtamembrane amino acids in the E5 protein and the PDGF ß receptor appear to drive complex formation and dimerization of the receptor. Further analysis of this unique mechanism of viral transformation promises to yield new insight into the regulation of growth factor receptor activity and cellular signal transduction pathways.     

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.     

Molecular examination of the transmembrane requirements of the platelet-derived growth factor beta receptor for a productive interaction with the bovine papillomavirus E5 oncoprotein

The small transmembrane E5 protein of bovine papillomavirus (BPV) transforms cells by forming a stable complex with and activating the platelet-derived growth factor beta receptor (PDGFbetaR). The E5/PDGFbetaR interaction is thought to involve specific physical contacts between the transmembrane domains of the two proteins. Lys(499) at the extracellular juxtamembrane position and Thr(513) within the transmembrane domain of the PDGFbetaR are required for the interaction and are predicted to contact analogously positioned residues in the E5 protein. Here, mutagenic analysis of the transmembrane region of the PDGFbetaR was performed to further characterize the nature of the E5/PDGFbetaR interaction. We show that the receptor transmembrane domain, with minimal extracellular and intracellular sequence, is sufficient for the interaction. In addition, we provide evidence that the polar nature of Thr(513) as well as its positioning along the transmembrane alpha-helix is important for the interaction. We also identify the receptor transmembrane amino acids Ile(506) and Leu(520) as additional requirements for the interaction. Because Lys(499), Thr(513), Ile(506), and Leu(520) all align along the same face of the predicted PDGFbetaR transmembrane alpha-helix, our data support the model that the PDGFbetaR contacts the E5 protein via multiple amino acids along a single alpha-helical interface.     

Platelet-derived growth factor receptor can mediate tumorigenic transformation by the bovine papillomavirus E5 protein.

We showed previously that the beta receptor for platelet-derived growth factor (PDGF) is constitutively activated in fibroblasts transformed by the 44-amino-acid bovine papillomavirus type 1 (BPV) E5 protein and that the E5 protein and the PDGF receptor exist in a stable complex in E5-transformed fibroblasts. On the basis of these results, we proposed that activation of the PDGF receptor by the BPV E5 protein generates a sustained proliferative signal, resulting in fibroblast transformation. In this study, we used a gene transfer approach to provide functional evidence that the PDGF receptor can mediate transformation by the E5 protein. We show that normal mouse mammary gland (NMuMG) cells, a murine mammary epithelial cell line that does not express PDGF receptors, are not susceptible to transformation by the E5 protein. Coexpression of the PDGF beta receptor and E5 genes in these cells results in markedly increased tyrosine phosphorylation of an immature PDGF receptor species and the formation of a stable complex between the E5 protein and this immature PDGF receptor form. Importantly, introduction of the PDGF receptor gene into NMuMG cells renders them highly susceptible to E5-mediated tumorigenic transformation. In contrast, the E5 protein does not induce transformation via the endogenous epidermal growth factor receptor pathway in these cells. These results demonstrate that the PDGF receptor, a cellular protein with a well-characterized role in the positive control of cell proliferation, can mediate transformation by a DNA virus transforming protein.     

Ligand-independent activation of the platelet-derived growth factor beta receptor: requirements for bovine papillomavirus E5-induced mitogenic signaling.

The E5 protein of bovine papillomavirus type 1 binds to and activates the endogenous platelet-derived growth factor (PDGF) beta receptor in fibroblasts, resulting in cell transformation. We have developed a functional assay to test the ability of PDGF beta receptor mutants to mediate a mitogenic signal initiated by the E5 protein. Lymphoid Ba/F3 cells are strictly dependent on interleukin-3 for growth, but coexpression of the wild-type PDGF beta receptor and the E5 or v-sis-encoded protein generated a mitogenic signal which allowed Ba/F3-derived cells to proliferate in the absence of interleukin-3. In these cells, the E5 protein bound to and caused increased tyrosine phosphorylation of both the mature and the precursor forms of the wild-type PDGF beta receptor. The tyrosine kinase activity of the receptor was necessary for E5-induced receptor tyrosine phosphorylation and mitogenic activity but not for complex formation with the E5 protein. In contrast, the PDGF-binding domain of the receptor was not required for complex formation with the E5 protein, E5-induced tyrosine phosphorylation or mitogenic activity, demonstrating that E5-mediated receptor activation is ligand independent. Analysis of receptor mutants lacking various combinations of tyrosine phosphorylation sites revealed that the E5 and v-sis-encoded proteins display similar requirements for signaling and suggested that the wild-type PDGF beta receptor can generate multiple independent mitogenic signals. Importantly, these mutants dissociated two activities of the PDGF beta receptor tyrosine kinase, both of which are required for sustained mitogenic signaling: (i) receptor autophosphorylation and creation of binding sites for SH2 domain-containing proteins and (ii) phosphorylation of substrates other than the receptor itself.     

Mutational analysis of open reading frame E4 of bovine papillomavirus type 1.

Open reading frame (ORF) E4 is a 353-base-pair ORF of bovine papillomavirus type 1. To determine the biological activities of this ORF in mouse C127 cells, we analyzed the effects of two constructed mutations which are predicted to prevent synthesis of ORF E4 proteins while leaving the amino acid sequence encoded by the overlapping ORF E2 unchanged. Neither mutation interfered with the abilities of the mutants to efficiently induce focus formation, induce growth in soft agarose, or transactivate an inducible bovine papillomavirus type 1 enhancer. Also, neither mutation prevented establishment of the viral DNA as an extrachromosomal plasmid in transformed cells. These results suggest that ORF E4 proteins are not required for these biological activities, and they are consistent with the observation of others (J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986) that the ORF E4 protein of a human papillomavirus is associated with late gene expression during papilloma formation.     

Multiple transmembrane amino acid requirements suggest a highly specific interaction between the bovine papillomavirus E5 oncoprotein and the platelet-derived growth factor beta receptor

The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor beta receptor (PDGFbetaR) tyrosine kinase in a ligand-independent manner. Evidence suggests that the small transmembrane E5 protein homodimerizes and physically interacts with the transmembrane domain of the PDGFbetaR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFbetaR-E5 interaction are a transmembrane Thr513 and a juxtamembrane Lys499. Here, we sought to determine if these are the only two receptor amino acids required for an interaction with the E5 protein. Substitution of large portions of the PDGFbetaR transmembrane domain indicated that additional amino acids in both the amino and carboxyl halves of the receptor transmembrane domain are required for a productive interaction with the E5 protein. Indeed, individual amino acid substitutions in the receptor transmembrane domain identified roles for the extracellular proximal transmembrane residues in the interaction. These data suggest that multiple amino acids within the transmembrane domain of the PDGFbetaR are required for a stable interaction with the E5 protein. These may be involved in direct protein-protein contacts or may support the proper transmembrane alpha-helical conformation for optimal positioning of the primary amino acid requirements.     

Genetic and biochemical definition of the bovine papillomavirus E5 transforming protein.

Mutations surrounding the first methionine codon of the E5 transforming gene of bovine papillomavirus (type 1) were analyzed for their effect on cellular transformation and on the synthesis of the 7-kd E5 polypeptide. Frameshift mutations upstream of this methionine codon (bp 3879) affect neither transforming activity nor the ability to synthesize full-size E5 protein. In contrast, frameshift mutations distal to this position result in the inhibition of cell transformation and prevent synthesis or accumulation of E5 protein in cells containing the mutant viral genomes. Several in-frame mutations distal to the first methionine codon have a minimal effect on transforming activity but alter the electrophoretic mobility of the E5 protein in a manner consistent with the generated genetic alteration (deletion, insertion or substitution). In all cases where the protein is detected, it fractionates with cellular membranes and forms dimers. These studies indicate that (i) the methionine codon at bp 3879 serves as the initiation codon for the mature E5 protein, (ii) changing the charge of the E5 amino-terminus (from neutral to positive) does not prevent the association of this hydrophobic polypeptide with cellular membranes, and (iii) E5 amino-terminal mutations do not interfere with the ability of this polypeptide to form homodimers. We conclude that the major focus-inducing activity of the intact BPV genome is due to the function of the small polypeptide encoded in the 3' half of the E5 ORF.     

E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene.

We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low-molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not.     

Mutational analysis of bovine papillomavirus type 1 E5 peptide domains involved in induction of cellular DNA synthesis.

Early gene E5 of bovine papillomavirus type 1 encodes a 44-amino-acid protein whose expression can transform immortalized mouse cell lines. We have previously reported that a chemically synthesized E5 peptide functions to induce cellular DNA synthesis upon microinjection into growth-arrested mouse cells. We further defined the two E5 domains essential for the full DNA synthesis induction activity by the analysis of E5 deletion and amino acid substitution mutant peptides. The first domain is the C-terminal 13-amino-acid core which is sufficient to activate DNA synthesis at high peptide concentration and contains two essential, highly conserved cysteine residues. The second domain is the 7-amino-acid hydrophobic sequence contiguous to the core domain which is sufficient to confer a 1,000-fold higher molar specific activity to the E5 peptide. A random hydrophobic sequence, but not charged amino acids, fulfills the function of the second domain.     

Mutational analysis of bovine papillomavirus E6 gene.

The bovine papillomavirus E6 gene can independently transform mouse C127 cells. To characterize E6 in greater detail, we created 16 site-directed mutations in E6, including substitution mutations in the cysteine codons of the four Cys-X-X-Cys motifs that are conserved in all papillomavirus E6 proteins. Proteins mutated in six of the seven cysteines tested, as well as those lacking the nonconserved C-terminus, were stable in transfected cells but were unable to induce morphological transformation, indicating that these amino acids play an important role in the function of E6.     

Mutational analysis of human papillomavirus type 11 E5a oncoprotein.

In this study, we investigated the structural basis of human papillomavirus type 11 (HPV-11) E5a transforming activity at the amino acid level. The effects of insertion, deletion , and substitution mutations on teh E5a transforming activity were determined by the assay of anchorage-independent growth. In the conserved Cys-X-Cys structure, substitution of Ser for Cys-73 resulted in indistinguishable transforming activity, whereas substitution of Ser for Cys-75 or Ser for both Cys-73 and Cys-75 retained 50 and 42% transformation, respectively. This suggests that Cys at position 75 may be important for transformation. Charge and structural changes at teh COOH termini of several mutants impaired transformation significantly, but those at the middle region did so only mildly. In addition, the 16,000-molecular-weight pore-forming protein (16K protein) is known to associate with BPV-1, HPV-6, and HPV-16 E5 proteins. In this study, we investigated the correlation between E5a-16K binding affinity and the transforming activity of E5a by the use of 11 E5a mutants. Results show that E5a and these 11 E5a mutants could bind to the 16K protein when these proteins were coexpressed in COS cells, suggesting that simple binding of the 16K protein by E5a may not be sufficient for cell transformation.     

The bovine papillomavirus E5 transforming protein can stimulate the transforming activity of EGF and CSF-1 receptors

The bovine papillomavirus E5 transforming gene encodes a 44 amino acid protein product that is localized to cytoplasmic membranes, including the plasma membrane. We now report that E5 can cooperate with human EGF receptors and with human CSF-1 receptors to induce cellular transformation of NIH 3T3 cells. Cooperation occurred in the absence of receptor stimulation by ligand, and it was further augmented by treatment with ligand. Cooperation was not seen between E5 and either c-fes or c-src. The cooperation between E5 and high levels of EGF receptors was associated with inhibition of receptor degradation and persistence of activated receptors on the cell surface. We conclude that E5 may enhance the receptor activity via inhibition of receptor down-modulation.     

The bovine papillomavirus E5 protein requires a juxtamembrane negative charge for activation of the platelet-derived growth factor beta receptor and transformation of C127 cells

The bovine papillomavirus E5 gene encodes a 44-amino-acid, homodimeric transmembrane protein that is the smallest known transforming protein. The E5 protein transforms cultured fibroblasts by forming a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, leading to sustained receptor activation. Aspartic acid 33 in the extracellular juxtamembrane region of the E5 protein is important for cell transformation and interaction with the PDGF beta receptor. A. N. Meyer et al. (Proc. Natl. Acad. Sci USA 91:4634-4638, 1994) speculated that this residue interacted with lysine 499 on the receptor. We constructed E5 mutants containing all possible substitutions at position 33, as well as several double mutants containing substitutions at aspartic acid 33 and at glutamic acid 36, and we examined the ability of these mutants to transform C127 mouse fibroblasts and to bind to and induce activation of the PDGF beta receptor. There was an excellent correlation between the transformation activities of the various mutants and their ability to bind to and activate the PDGF beta receptor. Analysis of the mutants demonstrated that a juxtamembrane negative charge on the E5 protein was required for cell transformation and for productive interaction with the PDGF beta receptor and indicated that aspartic acid 33 was more important for these activities than was glutamic acid 36. These results are consistent with the existence of an essential juxtamembrane salt bridge between lysine 499 on the PDGF beta receptor and an acidic residue in the C terminus of the E5 protein and lend support to our proposed model for the complex between the E5 dimer and the PDGF beta receptor.