Cellular transformation by a transmembrane peptide: structural requirements for the bovine papillomavirus E5 oncoprotein.

Abstract

The ES oncoprotein of bovine papillomavirus, only 44 amino adds long, occurs as a disulfide-bonded transmembrane dimer. This remarkable oncoprotein stimulates signal transduction.through activation of the plateletderived growth factor (PDGF) receptor, and E5 exhibits limited amino acid sequence similarit with PDGF. Results presented here sugest that a key feature of the hydrophobic transmembrane domain is an amino acid side chain that participates in interhelical hydrogen bond formation. These data are reminiscent of the activated neu oncogene, in which a point mutation in the membrane domain leads to lipndindependent dimerization and activation of a receptor tyrosine kinase. Signfcantly, the tranmembrane domain of E5 can be largely replaced by the transmembrane domain from the activated neu receptor tyrosine kinase. Extensive mutageneis defines the minimal structural features required for transformation by the ES oncoprotein as, first, the ability to dimerize and, second, presentation ofa negatively charged residue at the extralular side of the membrane. The biological activity of ES mutants that lack most amino acid residues similar to PDGF snggests that ES and PDGF activate the PDGF receptor by disnct mechanisms.Bovine papillomavirus type I (BPV) typifies the fibropapilloma viruses, which transform fibroblasts in vitro and lead to development of fibrosarcomas in their host. In contrast, the human papillomaviruses infect epithelial cells and are strongly implicated in carcinomas of the cervix and respiratory tract (1). The fibroblast-transforming function of BPV is due to a small open reading frame, designated E5, encoding a 44-residue protein that forms membrane-associated disulfide-bonded dimers (2-6). Recent work has demonstrated that the E5 oncoprotein activates the platelet-derived growth factor (PDGF) P-receptor (7,8) and may display functional similarity with the human T-lymphotropic virus type I p12' protein (9).Inspection of the amino acid sequence of the E5 oncoprotein reveals two distinct domains (2-6): an extremely hydrophobic N-terminal domain, residues 1-32, and a hydrophilic C-terminal domain, residues 33-44. Several studies have led to the conclusion that the E5 oncoprotein is membraneanchored with a type II orientation, with the N terminus intracellular and the C terminus extracellular (10). In addition, the hydrophobic domain of E5 can function as a signal-anchor domain, indistinguishable from signal-anchor domains of well-characterized type II proteins such as neuraminidase, transferrin receptor, or asialoglycoprotein receptor (11).In this work, we describe the minimal structural features required for biological transforming activity by a transmembrane peptide. The results presented here suggest that E5 and PDGF activate the PDGF receptor by fundamentally distinct mechanisms. ES/neu Substitution Mutant. Degenerate oligonucleotides were synthesized that would encode a pool of E5/neu transmembrane recombinants in which the 44 residues would be derived as follows: 1-5 from E5, 6-10 from...

Modulation of cell function by small transmembrane proteins modeled on the bovine papillomavirus E5 protein

“…Inspection of the sequences of the active proteins recovered from transformed cells revealed that many different sequences were able to induce transformation (Figure 4). This result is consistent with the construction of a limited number of active E5-like proteins in earlier experiments (Horwitz et al, 1989;Kulke et al, 1992;Meyer et al, 1994).…”

“…Furthermore, dimerization of the E5 protein is required for stable complex formation with the PDGF b receptor and receptor activation (Horwitz et al, 1988;Meyer et al, 1994;Mattoon et al, 2001). Finally, because the E5 protein and the PDGF receptor are thought to traverse the membrane in opposite orientations, the transmembrane domains of these two proteins evidently align in an antiparallel orientation.…”

“…On the basis of these results and extensive biochemical and mutational analysis, we proposed a model in which dimerization of the E5 protein creates binding sites for the transmembrane domains of two molecules of the PDGF b receptor, one on each face of the E5 dimer, resulting in receptor dimerization and activation ( Figure 2) . Productive interaction between the E5 protein and the PDGF b receptor requires the integrity of Gln17 and Asp33 in the E5 protein as well as juxtamembrane Lys499 and transmembrane Thr513 in the PDGF b receptor (Horwitz et al, 1988;Meyer et al, 1994;Sparkowski et al, 1994;Nilson et al, 1995;Petti et al, 1997;Klein et al, 1998Klein et al, , 1999, suggesting the existence of essential hydrophilic interactions between Gln17 and Thr513 and between Asp33 and Lys499. There also appear to be additional packing contacts between hydrophobic side chains in the transmembrane domains ( ; L Ely and D DiMaio, unpublished).…”

“…In transformed cells, the E5 protein exists as a disulfidelinked type II transmembrane homodimer localized largely to the membranes of the endoplasmic reticulum and Golgi apparatus (Schlegel et al, 1986;Burkhardt et al, 1987Burkhardt et al, , 1989Surti et al, 1998). Transformation requires Gln17, Asp33, and two conserved cysteine residues in the carboxy-terminus that mediate disulfide bond formation, but activity is not impaired by a variety of hydrophobic substitution mutations in the central hydrophobic domain of the E5 protein (Horwitz et al, 1988;Meyer et al, 1994). The E5 protein induces transformation by activating the PDGF b receptor, a type I transmembrane receptor tyrosine kinase (Petti et al, 1991;Nilson and DiMaio, 1993;Lai et al, 2005).…”

Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF β receptor in an active dimeric conformation

“…Unlike PDGF, which binds to the extracellular domain of the PDGFβR, the E5 protein binds to the TMD of the receptor, thereby causing receptor dimerization (16)(17)(18)(19)(20). Genetic studies imply that the E5 protein and the PDGFβR TMDs align side by side in the membrane and contact one another directly, a conclusion supported by biophysical studies with purified E5 and PDGFβR TMD peptides (4,5,9,12,(21)(22)(23)(24)(25). Because the E5 protein and the PDGFβR adopt opposite transmembrane orientations (type II for E5 and type I-i.e., N terminus in the luminal space-for the PDGFβR), their TMDs are antiparallel.…”