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D DiMaio

Publications and source records attributed to D DiMaio.

At least 19 recordsLinked to original sources
PubMed2000

Bovine papillomavirus E5 protein induces the formation of signal transduction complexes containing dimeric activated platelet-derived growth factor beta receptor and associated signaling proteins.

The bovine papillomavirus E5 protein binds to the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in constitutive activation of the receptor and cell growth transformation. By subjecting extracts from E5-transformed or PDGF-treated cells to velocity sedimentation in sucrose gradients, activated PDGF beta receptor complexes were separated from monomeric, inactive receptor. Rapidly sedimenting activated complexes contained oligomeric (apparently dimeric), tyrosine-phosphorylated PDGF beta receptor, the E5 protein, and associated cellular signaling proteins including the p85 subunit of phosphoinositol 3'-kinase, phospholipase Cgamma, and Ras-GTPase activating protein. These signaling proteins made the major contribution to the increased sedimentation rate of the activated receptor complexes. Pairwise analysis of components of these complexes indicated that multiple signaling proteins and the E5 protein were simultaneously present in the activated complexes. Our results also showed that the E5 protein and PDGF activated only a small fraction of the total PDGF beta receptor, that not all receptor molecules associated with the E5 protein were tyrosine-phosphorylated, and that signaling proteins could bind to hemiphosphorylated receptor dimers. On the basis of these results, we propose a model for the assembly of multiprotein, activated PDGF beta receptor complexes in response to the E5 protein.

Bovine papillomavirus 1↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

Bovine papillomavirus E2 protein activates a complex growth-inhibitory program in p53-negative HT-3 cervical carcinoma cells that includes repression of cyclin A and cdc25A phosphatase genes and accumulation of hypophosphorylated retinoblastoma protein.

The bovine papillomavirus E2 protein can inhibit the proliferation of HT-3 cells, a p53-negative cervical carcinoma cell line containing integrated human papillomavirus type 30 DNA. Here, we analyzed HT-3 cells to explore the mechanism of p53-independent E2-mediated growth inhibition. Expression of the E2 protein repressed expression of the endogenous human papillomavirus type 30 E6/E7 genes. This was accompanied by hypophosphorylation and increased accumulation of p105Rb and repression of E2F1 expression. The E2 protein also caused reduced cyclin-dependent kinase (cdk) 2 activity, but this did not appear to be due to increased expression of cdk inhibitors. Rather, expression of cyclin A, which regulates cdk2 activity, and the cdc25A and cdc25B phosphatases, which are thought to activate cdk2, was significantly reduced at both the RNA and protein levels in response to E2 expression. The E2 protein reduced expression of cdc25A and cdc25B in both HT-3 and HeLa cells, but not in cells that were not growth-inhibited by the E2 protein. E2 point mutants unable to inhibit cell growth did not repress cdc25A and cdc25B expression, nor did the cell cycle inhibitors hydroxyurea and mimosine. Based on these results and the known properties of cell cycle components, we propose a model to account for E2-induced growth inhibition of cervical carcinoma cell lines.

Animals↗
PubMed1998

Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor beta receptor.

The bovine papillomavirus E5 protein is a 44-aa transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor and induces constitutive tyrosine phosphorylation and activation of the receptor, resulting in cell transformation. The E5 protein does not resemble PDGF, but rather activates the receptor in a ligand-independent fashion, thus providing a unique system to examine activation of receptor tyrosine kinases. Here, we used a variety of approaches to explore the mechanism of receptor activation by the E5 protein. Chemical cross-linking experiments revealed that the E5 protein activated only a small fraction of the endogenous PDGF beta receptor in transformed fibroblasts and suggested that this fraction was constitutively dimerized. Coimmunoprecipitation experiments using extracts of cells engineered to coexpress full-length and truncated PDGF beta receptors confirmed that the E5 protein induced oligomerization of the receptor. Furthermore, in cells expressing the E5 protein, a kinase-active receptor was able to trans-phosphorylate a kinase-negative mutant receptor but was unable to catalyze intramolecular autophosphorylation. These results indicated that the E5 protein induced PDGF beta receptor activation by forming a stable complex with the receptor, resulting in receptor dimerization and trans-phosphorylation.

Amino Acid Substitution↗
PubMed1998

A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation.

Platelet-derived growth factor beta receptor (PDGFbetaR) is a transmembrane receptor tyrosine kinase involved in a variety of cellular functions. We have generated a constitutively activated murine PDGFbetaR containing a valine to alanine substitution at residue 536, located in the cytoplasmic juxtamembrane domain. When this mutant receptor (PR-V536A) was expressed in Ba/F3 cells, it allowed the cells to survive and proliferate in the absence of IL-3 or PDGF, and tyrosine phosphorylation of PR-V536A was increased markedly compared with that of the wild-type PDGFbetaR in the absence of ligand and similar to that observed in ligand-activated PDGFbetaR. PR-V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFbetaR mutant unable to bind PDGF. Alanine substitutions at positions homologous to V536 of the murine PDGFbetaR also activated other members of the PDGF receptor subfamily. The amino acid sequence of this region revealed a strong similarity to WW domains present in other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFR specifically associated with peptides containing the WW domain consensus recognition sequence PPXY. The results suggest that the cytoplasmic juxtamembrane domain plays a role in the regulation of receptor activity and function, perhaps by participating in protein-protein interactions.

Alanine↗
PubMed1998

Structural models of the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein is thought to be a type II integral membrane protein that exists as a disulfide-linked homodimer in transformed cells. Polarized-infrared measurements show that the E5 dimer in membrane bilayers is largely alpha-helical and has a transmembrane orientation. Computational searches of helix-helix conformations reveal two possible low-energy dimer structures. Correlation of these results with previous mutagenesis studies on the E5 protein suggests how the E5 dimer may serve as a molecular scaffold for dimerization and ligand-independent activation of the PDGF-beta receptor. We propose that on each face of the E5 dimer a PDGF-beta receptor molecule interacts directly with Gln17 from one E5 monomer and with Asp33 from the other E5 monomer. This model accounts for the requirement of Gln17 and Asp33 for complex formation and explains genetic results that dimerization of the E5 protein is essential for cell transformation.

Algorithms↗
PubMed1998

Oncogenic activation of the PDGF beta receptor by the transmembrane domain of p185neu*.

We replaced the transmembrane domain of the wild type murine PDGF beta receptor with that of p185neu*, the oncogenic form of p185neu, thereby generating a constitutively activated chimeric receptor PR/neu*. Unlike the wild type PDGF beta 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 beta 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 beta receptor.

Amino Acid Sequence↗
PubMed1998

Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation.

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF beta receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF beta receptor.

Animals↗
PubMed1998

Transactivation-competent bovine papillomavirus E2 protein is specifically required for efficient repression of human papillomavirus oncogene expression and for acute growth inhibition of cervical carcinoma cell lines.

The papillomavirus E2 proteins can function as sequence-specific transactivators or transrepressors of transcription and as cofactors in viral DNA replication. We previously demonstrated that acute expression of the bovine papillomavirus type 1 (BPV1) E2 protein in HeLa and HT-3 cervical carcinoma cell lines greatly reduced cellular proliferation by imposing a specific G1/S phase growth arrest. In this report, we analyzed the effects of a panel of point mutations in the BPV1 E2 protein to identify the functional requirements for acute growth inhibition. Disruption of E2-specific transactivation by mutations within either the transactivation domain or the DNA binding domain severely impaired E2-mediated growth inhibition in HeLa and HT-3 cells, even though these mutants retain various other E2 activities. This result indicates that functional transactivation activity is required for acute E2-mediated growth inhibition. HeLa cells, which contain a wild-type p53 gene, and HT-3 cells, which contain a transactivation-defective p53 gene, exhibited similar responses to the E2 mutants, suggesting that identical functions of the E2 protein were required for growth arrest regardless of p53 status. Replacement of the E2 transactivation domain with that of the herpes simplex virus VP16 generated a chimeric transactivator that efficiently stimulated expression of an E2-responsive reporter plasmid yet was completely defective for growth inhibition, suggesting that an E2-specific transactivation function is required for growth arrest. Surprisingly, the transactivation-defective E2 mutants were also markedly defective in their ability to repress transcription of the native human papillomavirus type 18 (HPV18) E6/E7 oncogenes in HeLa cells and of the HPV18 promoter present in a transfected reporter plasmid. These mutants were also defective in their ability to increase p53 levels. Therefore, efficient repression of the HPV18 promoter in HeLa cells is not merely a consequence of the binding of an E2 protein to appropriately situated binding sites in the promoter.

Animals↗
PubMed1998

Virocrine transformation: the intersection between viral transforming proteins and cellular signal transduction pathways.

This review describes a mechanism of viral transformation involving activation of cellular signaling pathways. We focus on four viral oncoproteins: the E5 protein of bovine papillomavirus, which activates the platelet-derived growth factor beta receptor; gp55 of spleen focus forming virus, which activates the erythropoietin receptor; polyoma virus middle T antigen, which resembles an activated receptor tyrosine kinase; and LMP-1 of Epstein-Barr virus, which mimics an activated tumor necrosis factor receptor. These examples indicate that diverse viruses induce cell transformation by activating cellular signal transduction pathways. Study of this mechanism of viral transformation will provide new insights into viral tumorigenesis and cellular signal transduction.

Animals↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1996

Activation of the endogenous p53 growth inhibitory pathway in HeLa cervical carcinoma cells by expression of the bovine papillomavirus E2 gene.

We previously showed that expression of the bovine papillomavirus (BPV) E2 gene results in a dramatic inhibition of the proliferation of several human cervical carcinoma cell lines, including HeLa cells which contain human papillomavirus (HPV) type 18 DNA. We have assessed the status of endogenous G1 cell cycle regulatory proteins, including the tumor suppressor proteins, p53 and p105Rb, in order to investigate growth regulatory pathways in HeLa cells following E2 expression. The p53 tumor suppressor protein is stabilized following the introduction of the E2 gene into HeLa cells. This results in the induction of the p53-responsive gene encoding the cyclin dependent kinase (cdk) inhibitor, p21/WAF1, complex formation between p21/WAF1 and cdk2 and reduction of in vitro cdk2/cyclin E kinase activity. The reduced cdk kinase activity is accompanied by the accumulation of the growth inhibitory hypophosphorylated form of the tumor suppressor protein, p105Rb. The level of the p105Rb-regulated transcription factor, E2F1, is reduced, as is transcription of a variety of E2F1-regulated genes, including B-myb. Thus, the p53 growth inhibitory pathway has evidently not accumulated mutations in HeLa cells but rather appears intact. However, this pathway remains dormant, until it is mobilized by appropriate manipulations, such as the expression of the BPV E2 protein.

Bovine papillomavirus 1↗
PubMed1995

An intact PDGF signaling pathway is required for efficient growth transformation of mouse C127 cells by the bovine papillomavirus E5 protein.

The bovine papillomavirus type 1 (BPV) E5 protein is a 44 amino acid, membrane-associated protein that induces growth transformation of cultured rodent and bovine fibroblasts. In transformed fibroblasts, the BPV E5 protein activates the endogenous platelet-derived growth factor (PDGF) beta receptor, and the introduction of the PDGF beta receptor gene into heterologous cell types normally lacking PDGF beta receptor expression permits transformation by the E5 protein. However, neither the endogenous PDGF beta receptor nor its signaling pathway has been shown to be required for efficient growth transformation of fibroblasts by the BPV E5 gene. Here we have tested whether the endogenous PDGF beta receptor serves as a target for the BPV E5 protein in mouse C127 fibroblasts. We isolated variant C127 cell lines that displayed reduced DNA synthesis in response to PDGF but a normal response to other mitogens, suggesting that they harbor specific defects in the PDGF signaling pathway. The variant lines also exhibited a specific reduction in the level of DNA synthesis induced by the acute expression of the BPV E5 gene, and a variant cell line containing a reduced level of PDGF beta receptor also displayed reduced stable growth transformation by the BPV E5 and v-sis oncogenes. These results provide genetic support for the model that the PDGF beta receptor signaling pathway is required for efficient growth transformation of C127 cells by the BPV E5 gene.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

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.

Animals↗