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Biomedical subjects

Peter K Vogt

Publications and source records attributed to Peter K Vogt.

30 records · Page 2Linked to original sources
PubMed2004

Inhibition of the proteolytic activity of anthrax lethal factor by aminoglycosides.

The anthrax lethal factor (LF), a Zn-dependent endopeptidase, is considered the dominant virulence factor of anthrax. Because pharmacological inhibition of the catalytic activity of LF is considered a plausible mechanism for preventing the lethality of anthrax, a high-throughput screening experiment based on LF-catalyzed cleavage of a fluorescent substrate was performed to identify novel inhibitors of LF. The RNA-targeting antibiotics, neomycin B and some synthetic dimeric aminoglycosides, were found to be nanomolar active-site inhibitors of LF.

Antigens, Bacterial↗
PubMed2004

An essential role for protein synthesis in oncogenic cellular transformation.

The induction and maintenance of oncogenic transformation requires interference with the controls that regulate translation and transcription. The PI 3-kinase pathway, which shows gain of function in numerous and diverse human cancers, generates signals that have a positive effect on the initiation of protein synthesis. Here we review the components of the PI 3-kinase signaling pathway and the mRNA-binding protein YB-1, exploring their roles in protein synthesis and oncogenic cell transformation.

Animals↗
PubMed2003

Y box-binding protein 1 induces resistance to oncogenic transformation by the phosphatidylinositol 3-kinase pathway.

Y box-binding protein 1 (YB-1) is a multifunctional protein that can act as a regulator of transcription and of translation. In chicken embryo fibroblasts transformed by the oncoproteins P3k (phosphatidylinositol 3-kinase) or Akt, YB-1 is transcriptionally down-regulated. Expression of YB-1 from a retroviral vector induces a strong cellular resistance to transformation by P3k or Akt but does not affect sensitivity to transformation by other oncoproteins, such as Src, Jun, or Qin. The YB-1-expressing cells assume a tightly adherent, flat phenotype, with YB-1 localized in the cytoplasm, and show a greatly reduced saturation density. Both cap-dependent and cap-independent translation is inhibited in these cells, but the activity of Akt remains unaffected, suggesting that YB-1 functions downstream of Akt. A YB-1 protein with a loss-of-function mutation in the RNA-binding motif no longer binds to the mRNA cap structure, is localized in the cell nucleus, does not induce the flat cellular phenotype, and fails to interfere with P3k- or Akt-induced oncogenic transformation. This mutant also does not inhibit cap-dependent or cap-independent translation. These results suggest that YB-1 acts like a rapamycin mimic, inhibiting translational events that are required in phosphatidylinositol 3-kinase-driven oncogenic transformation.

Animals↗
PubMed2003

Genome-scale functional profiling of the mammalian AP-1 signaling pathway.

Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of approximately 20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin alpha1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis.

Animals↗
PubMed2003

MafA has strong cell transforming ability but is a weak transactivator.

The maf oncogene of the avian oncogenic retrovirus AS42 encodes a nuclear bZip protein, v-Maf, that recognizes sequences related to the AP-1 target site. The corresponding cellular protein, c-Maf belongs to a family of related bZip proteins together with MafA and MafB. In this paper, we compare the transactivation and cell transforming abilities of MafA and MafB along with two forms of the c-Maf protein. These proteins induce cellular transformation when expressed in chicken embryo fibroblasts. In reporter assays, MafA is a much less effective transactivator than the other Maf proteins, but unexpectedly shows the strongest activity in cell transformation. Chimeras of MafA and MafB correlate the strong cell transforming ability of MafA with its DNA-binding domain. The DNA-binding domain of MafA is also correlated with weak transactivation. Additional mutagenesis experiments show that transactivation and transformation by MafA are also controlled by phosphorylation of two conserved serine residues in the transactivation domain. Finally, we constructed MafA-estrogen receptor fusion molecules that show tightly hormone-dependent cell transforming ability. These regulatable constructs permit a kinetic characterization of target gene responses and facilitate discrimination between direct and indirect targets.

Amino Acid Sequence↗
PubMed2003

Artificial oncoproteins: modified versions of the yeast bZip protein GCN4 induce cellular transformation.

We have constructed artificial AP-1 proteins containing elements derived from yeast GCN4 and from the herpes simplex virus activator VP16. These proteins can only homodimerize but do not heterodimerize, and lacking significant homology to Jun outside the DNA-binding domain, they are largely unaffected by proteins that modulate Jun. Constructs in which the transactivation domain of GCN4 is replaced by that of VP16 induce oncogenic transformation in cultures of chicken embryo fibroblasts. The availability of transforming VP16-GCN4 fusion proteins permits an evaluation of downstream target genes, based on the hypothesis that transformation-relevant targets should be common between Jun and the artificial AP-1 proteins. In a pilot study, we examined the expression of several Jun target genes in cells transformed by the VP16-GCN4 fusions and found that some of the Jun targets are not upregulated by the GCN4-derived transforming construct, suggesting that their upregulation in Jun-transformed cells is not essential for cell transformation. We have further constructed a regulatable GCN4-VP16 protein that will permit a kinetic characterization of target gene responses and will facilitate discrimination between direct and indirect targets.

Animals↗
PubMed2003

Partial oncogenic transformation of chicken embryo fibroblasts by Jun dimerization protein 2, a negative regulator of TRE- and CRE-dependent transcription.

Jun dimerization protein 2 (JDP2) was identified as a bZIP protein that forms dimers with Jun proteins. JDP2 represses transcriptional activation of reporter constructs containing 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive elements (TRE) or cyclic AMP responsive elements (CRE). JDP2, overexpressed by the avian retroviral vector RCAS, induces partial oncogenic transformation of chicken embryo fibroblasts. JDP2-expressing cells form multilayered foci in monolayer cultures but do not show anchorage-independent growth. Both the carboxyl and the amino terminus of JDP2 are required for the transforming activity. Chimeric constructs of JDP2 carrying the leucine zipper domain of Fos, GCN4 or EB1 fail to transform CEF. The leucine zipper of Fos mediates only heterodimerization; it cannot homodimerize. In contrast, the leucine zippers of GCN4 and of EB1 exclusively homodimerize and do not form dimers with other bZip proteins. The results with the JDP2 chimeras suggest that the JDP2 homodimer and the JDP2/Jun heterodimer (or other bZip heterodimers formed with the Fos leucine zipper) are nontransforming, leaving as possible transforming combination the JDP2/Fos heterodimer. The unexpected transforming activity of a negative regulator of TRE- and CRE-dependent transcription raises an important question concerning the mechanisms of transformation by the related bZIP proteins Jun and Fos that address the same target sequences.

Activating Transcription Factor 2↗
PubMed2003

The C-terminal region of cellular Qin oligomerizes: correlation with oncogenic transformation and transcriptional repression.

Expression of the oncoprotein Qin induces tumors in chickens and oncogenic transformation of chicken embryo fibroblasts in culture. We performed a detailed deletion analysis of the C-terminal region of Qin (amino acids 246-451, extending from the winged helix domain to the C-terminus) and identified amino acids 246-379 as important for transformation. The same region mediates homo-oligomerization of Qin as documented in vitro by GST pulldowns and in vivo by coimmunoprecipitation. A 60 amino-acid region within the oligomerization domain is necessary and sufficient for transcriptional repression induced by Qin.

Animals↗
PubMed2003

Binding of the corepressor TLE1 to Qin enhances Qin-mediated transformation of chicken embryo fibroblasts.

The oncoprotein Qin is a member of the winged helix family of transcriptional regulators. The region C-terminal to its winged helix DNA-binding domain is required for transformation of chicken embryo fibroblasts. We isolated the corepressor TLE1 as a binding partner for Qin in a yeast two-hybrid screen and localized the TLE1-binding region to a 60 amino-acid stretch directly C-terminal of the winged helix domain of Qin. We show in vivo interaction of full-length Qin and TLE1 in a mammalian two-hybrid system. Coexpression of TLE1-binding Qin and TLE1 induces phosphorylation of TLE1. The DNA-binding activity of Qin is not required for this function. Binding of Qin to TLE1 correlates with Qin-induced transformation. Addition of the TLE1-binding motif WRPW to the C-terminus of a transformation-defective Qin deletion mutant restores binding to TLE1 and significantly enhances transformation. Expression of TLE1 in CEF by the retroviral vector RCAS enhances cell growth and induces formation of agar colonies.

Animals↗
PubMed2002

Oncogenic transformation by beta-catenin: deletion analysis and characterization of selected target genes.

Genetic analysis of beta-catenin-induced oncogenic transformation in chicken embryo fibroblasts (CEF) revealed the following prerequisites for oncogenicity: (1) removal of the N terminal phosphorylation sites targeted by glycogen synthase kinase 3beta (GSK3beta), (2) retention of the N terminal transactivation domain, and (3) retention of the armadillo repeats. The C terminal transactivation domain played an ancillary role in the transformation of CEF. There was a rough correlation between the transforming activity of various beta-catenin constructs and their transactivation of the TOPFLASH reporter. Expression levels of the candidate target genes of beta-catenin-LEF, cyclin D1 and myc were not correlated with each other or with the transforming activity of beta-catenin constructs. A new target gene, coding for inositol hexakisphosphate kinase 2 (IP6K2) was identified. Its expression showed concordance with the transforming activity of beta-catenin constructs.

Animals↗
PubMed2002

Small-molecule antagonists of Myc/Max dimerization inhibit Myc-induced transformation of chicken embryo fibroblasts.

Myc is a transcriptional regulator of the basic helix-loop-helix leucine zipper protein family. It has strong oncogenic potential, mutated or virally transduced forms of Myc induce lymphoid tumors in animals, and deregulated expression of Myc is associated with numerous types of human cancers. For its oncogenic activity, Myc must dimerize with the ubiquitously expressed basic helix-loop-helix leucine zipper protein Max. This requirement for dimerization may allow control of Myc activity with small molecules that interfere with Myc/Max dimerization. We have measured Myc/Max dimerization with fluorescence resonance energy transfer and have screened combinatorial chemical libraries for inhibitors of dimerization. Candidate inhibitors were isolated from a peptidomimetics library. Inhibition of Myc/Max interaction was validated by ELISA and electrophoretic mobility-shift assay. Two of the candidate inhibitors also interfere with Myc-induced oncogenic transformation in chicken embryo fibroblast cultures. Our work provides proof of principle for the identification of small molecule inhibitors of protein-protein interactions by using high-throughput screens of combinatorial chemical libraries.

Animals↗