Characterisation of NF-kappa B complexes in Theileria parva-transformedT cells.
Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-kappa B degradation and NF-kappa B activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-kappa B-mediated responses are regulated by selective combinations of NF-kappa B proteins as homo- or heterodimers and by distinct kappa B motifs. We characterised the NF-kappa B complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-kappa B/Rel family of proteins translocate to the nucleus of infected cells. Using two different kappa B oligonucleotides (kappa B-1 and kappa B-2), both containing the decameric consensus kappa B motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to kappa B-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived kappa B-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-kappa B activation pathways had no inhibitory effect on the activation of NF-kappa B DNA binding in TpM(803) T cells.
Machado
J
J
Laboratory of Molecular Pathology, Institute of Animal Pathology, University of Berne, Länggass-Strasse 122, 3012, Berne, Switzerland.
Fernandez
P C
PC
Baumann
I
I
Dobbelaere
D A
DA
D051698:Activating Transcription Factor 2; D021581:Active Transport, Cell Nucleus; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D008213:Lymphocyte Activation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D020569:Proto-Oncogene Proteins c-rel / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism* / Q000469:parasitology*; D016797:Theileria parva / Q000502:physiology*; D051996:Transcription Factor RelA; D051841:Transcription Factor RelB; D014157:Transcription Factors / Q000378:metabolism; D014466:Ultraviolet Rays
D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D020569:Proto-Oncogene Proteins c-rel; D051996:Transcription Factor RelA; D014157:Transcription Factors; D051841:Transcription Factor RelB; D004247:DNA