ABSTRACT
The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1--70K and SR proteins and therefore may acquire new functions.
Subject(s)
Apoptosis/physiology , Cell Cycle/physiology , Cell Nucleus/metabolism , RNA Splicing/physiology , RNA-Binding Proteins/metabolism , Active Transport, Cell Nucleus/physiology , Amino Acid Sequence , Animals , Base Sequence , Bone Marrow Cells/metabolism , Cell Cycle/genetics , Cell Fractionation , Cells, Cultured , Cloning, Molecular , Dactinomycin/pharmacology , Female , Genes, Reporter , Humans , Immunoblotting , Immunohistochemistry , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Nucleic Acid Synthesis Inhibitors/pharmacology , PTB-Associated Splicing Factor , Phosphorylation , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/immunology , Recombinant Fusion Proteins/metabolism , Sequence AlignmentABSTRACT
Activin A, a member of the transforming growth factor beta (TGFbeta) superfamily, blocks interleukin (IL)-6 biological functions. The molecular basis of the influence of this TGFbeta signaling on the IL-6 receptor triggered cascade is unknown. We studied IL-6-induced secretion of the acute phase protein haptoglobin by hepatoma cells. Overexpression of the C/EBPbeta gene, a downstream effector in the IL-6 pathway, activated transcription from the haptoglobin promoter. This was abolished by either a constitutively active form of activin A type IB receptor (CAactRIB) or by a combination of Smad3 and Smad4. Similarly, Smads abolished transcriptional activation by co-stimulation with IL-6 and STAT3. The transcription co-activator p300 partially overcame the suppressive effect of Smads. Electrophoretic mobility shift assays indicated that C/EBPbeta binding to haptoglobin promoter DNA was reduced by over-expression of CAactRIB and Smad4. We thus show that Smad proteins operate as transcription inhibitors on target genes of the IL-6 induced pathway. The effect of Smads is exerted on components of the transcription activation complex and may also involve interference with DNA binding. This study thus depicts molecular sites of interaction between the TGFbeta superfamily and the IL-6 signaling cascades.
Subject(s)
CCAAT-Enhancer-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/pharmacology , Haptoglobins/genetics , Promoter Regions, Genetic , Trans-Activators/antagonists & inhibitors , Trans-Activators/pharmacology , Transcriptional Activation/drug effects , Activins , Blotting, Western , Cell Division/drug effects , DNA/metabolism , Humans , Inhibins/pharmacology , Interleukin-6 , Receptors, Interleukin-6/metabolism , STAT3 Transcription Factor , Smad3 Protein , Smad4 Protein , Transcription, Genetic/drug effects , Tumor Cells, CulturedABSTRACT
Authors present an original approach to the cephalometric measuring by obtaining absolute coordinates of the cephalometric points in a coordinate system fixed on the skull. An instrument especially constructed for that purpose is shown, and also the way of using it in vivo.
