Lisa M. Petti

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 E5 proteins.

The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating these activities. The primary target of the 44-amino acid BPV1 E5 protein is the PDGF β receptor, whereas the EGF receptor appears to be an important target of the 83-amino acid HPV16 E5 protein. Both E5 proteins also bind to the vacuolar ATPase and affect MHC class I expression and cell-cell communication. Continued studies of the E5 proteins will elucidate important aspects of transmembrane protein-protein interactions, cellular signal transduction, cell biology, virus replication, and tumorigenesis.

Oxidative and calcium stress regulate DSCR1 (Adapt78/MCIP1) protein.

DSCR1 (adapt78) is a stress-inducible gene and cytoprotectant. Its protein product, DSCR1 (Adapt78), also referred to as MCIP1, inhibits intracellular calcineurin, a phosphatase that mediates many cellular responses to calcium. Exposure of human U251 and HeLa cells to hydrogen peroxide led to a rapid hyperphosphorylation of DSCR1 (Adapt78). Inhibitor and agonist studies revealed that a broad range of kinases were not responsible for DSCR1 (Adapt78) hyperphosphorylation, including ERK1/2, although parallel activation of the latter was observed. Phosphorylation of both DSCR1 (Adapt78) and ERK1/2 was attenuated by inhibitors of tyrosine phosphatase, suggesting the common upstream involvement of tyrosine dephosphorylation. The hyperphosphorylation electrophoretic shift in DSCR1 (Adapt78) mobility was also observed with other oxidizing agents (peroxynitrite and menadione) but not nonoxidants. Calcium ionophores strongly induced the levels of both hypo- and hyper-phosphorylated DSCR1 (Adapt78) but did not alter phosphorylation status. Calcium-dependent growth factor- and angiotensin II-stimulation also induced both DSCR1 (Adapt78) species. Phosphorylation of either or both serines in a 13-amino acid peptide made to a calcineurin-interacting conserved region of DSCR1 (Adapt78) attenuated inhibition of calcineurin. These data indicate that DSCR1 (Adapt78) protein is a novel, early stage oxidative stress-activated phosphorylation target and newly identified calcium-inducible protein, and suggest that these response mechanisms may contribute to the known cytoprotective and calcineurin-inhibitory activities of DSCR1 (Adapt78).

Multiple Transmembrane Amino Acid Requirements Suggest a Highly Specific Interaction between the Bovine Papillomavirus E5 Oncoprotein and the Platelet-Derived Growth Factor Beta Receptor

ABSTRACT The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor β receptor (PDGFβR) 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 PDGFβR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFβR-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 PDGFβR 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 PDGFβR 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.

Oncogenic activation of the PDGF β receptor by the transmembrane domain of p185neu

We replaced the transmembrane domain of the wild type murine PDGF β receptor with that of p185neu*, the oncogenic form of p185neu, thereby generating a constitutively activated chimeric receptor PR/neu*. Unlike the wild type PDGF β receptor or a chimeric receptor containing the transmembrane domain of wild type p185neu (PR/neu), PR/neu* induced morphologic transformation, focus formation, and tumorigenicity in mouse C127 fibroblasts. Expression of PR/neu* in mouse Ba/F3 hematopoietic cells, which normally depend on IL-3 for survival and sustained proliferation, induced proliferation in the absence of IL-3. The PR/neu chimera conferred limited IL-3-independent growth of Ba/F3 cells. Only PR/neu* and not PR/neu displayed significantly increased levels of phosphotyrosine compared to the wild type PDGF receptor in C127 and Ba/F3 cells. In addition, PR/neu* immune complexes displayed increased levels of kinase activity in vitro compared to immune complexes of the wild type receptor. Furthermore, novel tyrosine phosphorylated proteins of approximately 60 kDa appeared to specifically complex with PR/neu*, suggesting that PR/neu* may activate distinct signaling pathways. We speculate that the p185neu* transmembrane domain in the context of the PDGF β receptor facilitates receptor homodimerization, thereby inducing tyrosine autophosphorylation followed by association with important signaling substrates and transforming activity. Thus, PR/neu* should be a useful reagent for further characterizing activation and signaling mechanisms of the PDGF β receptor.

Biologically active LIL proteins built with minimal chemical diversity

Significance Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains, whereas transmembrane domains are short membrane-spanning protein segments with mainly hydrophobic amino acids. Here, we have defined the minimal chemical diversity sufficient for a protein to display specific biological activity by isolating artificial 26-aa-long transmembrane proteins consisting of random sequences of only two hydrophobic amino acids, leucine and isoleucine. A small fraction of proteins with this composition interact with the transmembrane domain of a growth factor receptor to specifically activate the receptor, resulting in growth transformation. These findings change our view of what can constitute an active protein and have important implications for protein evolution, protein engineering, and synthetic biology. We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor β-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context.

Detection and quantification of bovine papillomavirus type 2 in urinary bladders and lymph nodes in cases of Bovine Enzootic Hematuria from the endemic region of Azores.

Bovine Enzootic Hematuria (BEH) is a disease with a severe impact on production indexes and characterized by the development of bovine urinary bladder tumors, particularly in the Azores archipelago. The purpose of this study was to investigate and quantify BPV2 tissue distribution in bovine urinary bladder tumors, normal bladders, and iliac lymph nodes of cattle from the Azores. A real-time PCR system targeting the L1 gene was developed and allowed for the specific detection of the virus. BPV2 DNA was detected in a large proportion of the samples tested, both from neoplastic and healthy tissues, indicating that this virus is very prevalent in the bovine population of the Azores. Moreover, all types of tissues tested were positive, confirming a wide viral distribution within the infected animal. Bovine cutaneous papillomas sampled from Portuguese mainland dairy cattle were used as controls. Viral load ranged between 2.2×10(4) copies/cell in the skin papillomas, and 0.0002 copies/cell in the urinary bladders tumors from the Azores. This is the first report presenting quantitative data on BPV2 infection in bovine urinary bladder lesions from the Azores. This approach will provide a useful tool to evaluate the role of BPV2 not only in the pathogenesis BEH but also in cell transformation mechanisms.

De novo selection of oncogenes

Significance Artificial proteins may have improved properties compared with proteins that arose during evolution, but approaches to construct active artificial proteins are cumbersome and often constrained by existing protein structures. Here, we used mouse cells to select proteins that formed tumors from a library of small transmembrane proteins with randomized hydrophobic amino acid sequences. The resulting oncoproteins lack amino acid sequences from any known protein and function by activating a cellular growth factor receptor. This approach can be used to generate structures not observed in nature, create prototypes for research and possibly clinical uses, and provide insight into cell biology, protein–protein interactions, and evolution. All cellular proteins are derived from preexisting ones by natural selection. Because of the random nature of this process, many potentially useful protein structures never arose or were discarded during evolution. Here, we used a single round of genetic selection in mouse cells to isolate chemically simple, biologically active transmembrane proteins that do not contain any amino acid sequences from preexisting proteins. We screened a retroviral library expressing hundreds of thousands of proteins consisting of hydrophobic amino acids in random order to isolate four 29-aa proteins that induced focus formation in mouse and human fibroblasts and tumors in mice. These proteins share no amino acid sequences with known cellular or viral proteins, and the simplest of them contains only seven different amino acids. They transformed cells by forming a stable complex with the platelet-derived growth factor β receptor transmembrane domain and causing ligand-independent receptor activation. We term this approach de novo selection and suggest that it can be used to generate structures and activities not observed in nature, create prototypes for novel research reagents and therapeutics, and provide insight into cell biology, transmembrane protein–protein interactions, and possibly virus evolution and the origin of life.

A single amino acid substitution converts a transmembrane protein activator of the platelet-derived growth factor β receptor into an inhibitor

Background: Certain artificial small transmembrane proteins can activate the PDGFβ receptor to promote cellular growth transformation. Results: A point mutant of one such protein binds to this receptor and inhibits activation induced by different ligands. Conclusion: Small transmembrane proteins can be engineered to inhibit PDGFβ receptor activity. Significance: This strategy could be generalized for designing novel inhibitors of growth factor receptors, which may have therapeutic implications. Receptors for PDGF play an important role in cell proliferation and migration and have been implicated in certain cancers. The 44-amino acid E5 protein of bovine papillomavirus binds to and activates the PDGFβ receptor (PDGFβR), resulting in oncogenic transformation of cultured fibroblasts. Previously, we isolated an artificial 36-amino acid transmembrane protein, pTM36-4, which transforms cells because of its ability to activate the PDGFβR despite limited sequence similarity to E5. Here, we demonstrated complex formation between the PDGFβR and three pTM36-4 mutants: T21E, T21Q, and T21N. T21Q retained wild type transforming activity and activated the PDGFβR in a ligand-independent manner as a consequence of binding to the transmembrane domain of the PDGFβR, but T21E and T21N were severely defective. In fact, T21N substantially inhibited E5-induced PDGFβR activation and transformation in both mouse and human fibroblasts. T21N did not prevent E5 from binding to the receptor, and genetic evidence suggested that T21N and E5 bind to nonidentical sites in the transmembrane domain of the receptor. T21N also inhibited transformation and PDGFβR activation induced by v-Sis, a viral homologue of PDGF-BB, as well as PDGF-induced mitogenesis and signaling by preventing phosphorylation of the PDGFβR at particular tyrosine residues. These results demonstrated that T21N acts as a novel inhibitor of the PDGFβR and validated a new strategy for designing highly specific short transmembrane protein inhibitors of growth factor receptors and possibly other transmembrane proteins.

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

Significance Highly specific protein–protein interactions between transmembrane domains play crucial roles in many biological processes, but are difficult to study because they occur within membranes. The E5 protein of bovine papillomavirus is a 44-residue transmembrane protein that transforms cells by binding the transmembrane domain of the PDGF receptor, resulting in receptor activation. By combining computational modeling, genetic analysis, and biochemical studies, we propose a quaternary structure of the complex between the E5 protein and the PDGF receptor, in which two dimers of the E5 protein clamp two molecules of the receptor transmembrane domain into an active dimeric conformation. These studies reveal the molecular mechanism of action of an unusual oncogene and provide a pathway to study biologically interesting transmembrane complexes. The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex. In this model, an active dimer of the PDGFβR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFβR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFβR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.