Artificial transmembrane oncoproteins smaller than the bovine papillomavirus E5 protein redefine sequence requirements for activation of the platelet-derived growth factor beta receptor

Abstract

The bovine papillomavirus E5 protein (BPV E5) is a 44-amino-acid homodimeric transmembrane protein that binds directly to the transmembrane domain of the platelet-derived growth factor (PDGF) beta receptor and induces ligand-independent receptor activation. Three specific features of BPV E5 are considered important for its ability to activate the PDGF beta receptor and transform mouse fibroblasts: a pair of C-terminal cysteines, a transmembrane glutamine, and a juxtamembrane aspartic acid. By using a new genetic technique to screen libraries expressing artificial transmembrane proteins for activators of the PDGF beta receptor, we isolated much smaller proteins, from 32 to 36 residues, that lack all three of these features yet still dimerize noncovalently, specifically activate the PDGF beta receptor via its transmembrane domain, and transform cells efficiently. The primary amino acid sequence of BPV E5 is virtually unrecognizable in some of these proteins, which share as few as seven consecutive amino acids with the viral protein. Thus, small artificial proteins that bear little resemblance to a viral oncoprotein can nevertheless productively interact with the same cellular target. We speculate that similar cellular proteins may exist but have been overlooked due to their small size and hydrophobicity.

FIG. 1.

Amino acid sequences of small transmembrane proteins and libraries. The wild-type BPV E5 protein sequence is shown in bold, as are the BPV E5 residues deliberately retained in the other small transmembrane proteins. All sequences are aligned to the fixed tryptophan at position 5 in BPV E5. Epitopes for antibody recognition are underlined. Residues randomized in small transmembrane protein libraries are represented by X's.

FIG. 2.

pTM32-1 specifically activates the PDGF β receptor. (A) BaF3 cells without exogenous receptor (dashed lines, open shapes) or with PDGF β receptor (solid lines, closed shapes) expressing empty retroviral vector (circle), BPV E5 (triangle), or pTM32-1 (square) were incubated in medium lacking IL-3. Live cells were counted on the indicated days. (B) BaF3-βR cells expressing BPV E5 or pTM32-1 were grown in the presence or absence of IL-3 as indicated and were treated with either DMSO (filled bars) or the PDGF β receptor kinase inhibitor AG1295 (open bars). Live cells were counted on day 4. (C) PDGF β receptor was immunoprecipitated (IP) from BaF3-βR lysates expressing either empty vector, pTM32-1, or the E19L mutant. Western blots (WB) were probed for PDGF β receptor expression (βR) or phosphorylated tyrosine (PY). The mature (M) and precursor (P) forms of the PDGF β receptor are indicated. (D) BaF3-βR cells expressing BPV E5 (triangle), pTM32-1 (square), or the pTM32-1 E19L mutant (circle) were incubated in medium lacking IL-3. Live cells were counted on the indicated days. Results shown in all panels are representative of at least three independent experiments.

FIG. 3.

Induction of IL-3 independence by pTM32-1 requires the transmembrane domain of the PDGF β receptor. Parental BaF3 cells (open bars) or BaF3-βR (filled bars), BaF3-βαβ (hatched bars), or BaF3-βKitβ (gray bars) cells were infected with the empty retroviral vector or with viruses expressing wild-type BPV E5, pTM32-1, or the v-sis oncogene. Cells were plated in medium lacking IL-3, and live cells were counted after 5 days. Results shown are representative of multiple independent experiments.

FIG. 4.

Dimerization and molecular modeling of pTM32-1. (A) The graph shows normalized CAT activity induced by fusion proteins with the indicated wild-type and mutant transmembrane domains in a TOXCAT assay, which is proportional to the strength of the transmembrane interactions. Means and standard deviations of two independent experiments, done in triplicate, are shown. G83I, glycophorin A glycine to isoleucine mutation at position 83. wt, wild type. (B) (left) Helical backbone of three models of the pTM32-1 homodimer predicted by CHI molecular dynamics simulation. Glutamic acid at position 19 is shown in orange. (Right) The plots show the interaction energy of the amino acids predicted to form the homodimer interface for each model. The sequence of the predicted transmembrane domain of pTM32-1 is shown at the bottom. (C) RMSD of the backbone atoms from the initial coordinates for the three homodimer models during NAMD molecular dynamics simulations. Model 1, black; model 2, red; model 3, green.

FIG. 5.

Ha/E5₃₃ activates the PDGF β receptor in BaF3 cells. (A) Parental BaF3 cells (open bars), BaF3-βR cells (filled bars), or BaF3-βKitβ cells (gray bars) stably expressing empty retroviral vector, wild-type BPV E5, or HA/E5₃₃ were plated in medium lacking IL-3, and live cells were counted after 5 days. Results shown are representative of three independent experiments. (B) PDGF β receptor was immunoprecipitated (IP) from BaF3-βR lysates expressing either empty vector, BPV E5, or HA/E5₃₃. Western blots (WB) were probed for PDGF β receptor expression (βR) or phosphorylated tyrosine (PY). The mature (M) and precursor (P) forms of the PDGF β receptor are indicated.

FIG. 6.

Truncated E5 protein induces low-level focus formation, forms a stable complex with the PDGF β receptor in C127 cells, and induces receptor phosphorylation. (A) C127 cells were infected with high-titer stocks of the empty retroviral vector or viruses expressing the indicated small transmembrane protein and incubated for 11 days. (B) PDGF β receptor was immunoprecipitated (IP) from lysates of C127 cells expressing empty vector, BPV E5, HA/E5₃₃, or pTM32-1. Western blots (WB) were probed for the receptor (βR) or phosphorylated tyrosine (PY). The mature (M) and precursor (P) forms of the PDGF β receptor are indicated. (C) Lysates of C127 cells expressing empty vector, BPV E5, or HA/E5₃₃ were immunoprecipitated with antibody recognizing the HA epitope and probed for the PDGF β receptor. A molecular mass marker (in kilodaltons) is shown on the right for reference.

FIG. 7.

pTM36-3 and pTM36-4 specifically activate the PDGF β receptor. (A) BaF3-βR (solid lines with closed symbols) and BaF3-βKitβ (dashed lines with open symbols) expressing BPV E5 (squares), pTM36-3 (triangles), or pTM36-4 (circles) were incubated in medium lacking IL-3. Live cells were counted on indicated days. Results shown are representative of three independent experiments. (B) C127 cells stably transformed with the E5 protein or pTM36-4 were incubated for 4 days in medium containing DMSO (top) or AG1295 (bottom) and photographed by phase-contrast microscopy. (C) PDGF β receptor was immunoprecipitated (IP) from lysates of C127 cells expressing empty vector, BPV E5, pTM36-3, or pTM36-4. Western blots (WB) were probed for the receptor (βR) or phosphorylated tyrosine (PY). The mature (M) and precursor (P) forms of the PDGF β receptor are indicated.

FIG. 8.

Dimerization of pTM36-4 peptide. Synthetic peptides corresponding to the transmembrane domains of pTM36-4 and BPV E5 were solubilized in 10 mM SDS, subjected to cross-linking with bis(sulfosuccinimidyl)suberate, as indicated, and electrophoresed in bis-Tris gels. The position of the pTM36-4 peptide dimer is indicated by the arrow. Molecular mass markers (in kilodaltons) are shown on the right.