Localization of a molecular form of interferon-regulated RNase L in the cytoskeleton.
RNase L (also termed 2-5A-dependent RNase) is a crucial enzyme involved in the molecular mechanism of interferon (IFN) action. Activated by 2',5'-oligoadenylate oligomers (2-5A), this enzyme controls the regulation of RNA stability in IFN-treated or virus-infected mammalian cells. Knowledge of RNase location within cells may provide additional information about its function. Previous work located RNase as a detergent-soluble molecule in nuclei and cytoplasm. In this study, we demonstrate that this enzyme was also present in a detergent-insoluble fraction associated with proteins of the cytoskeleton. A cellular fractionation procedure was used to prepare the cytoskeleton, which was shown to contain 2-5A binding activity not due to cytoplasmic contaminants. In contrast to the cytoplasmic fraction, which contained RNase L with a 2-5A-accessible site, the insoluble RNase molecular form of the cytoskeleton could not be assayed by the classic radiobinding method or the covalent UV cross-linking procedure, which only detects the 2-5A binding site in an open position, that is, free of 2-5A or with an unmasked 2-5A site. The 2-5A binding site present in the cytoskeleton was completely masked and not directly accessible to its 2-5A activator. This particular molecular form of RNase can be detected after a specific denaturing-renaturing treatment of the cytoskeleton, which separates the RNase from cytoskeletal proteins, unmasking the 2-5A site. The cytoskeletal RNase was no longer present at this site when cells were stimulated for a short time with 12-O-tetradecanoylphorbol-13-acetate (TPA). Our data suggest the existence of a pathway that targets the RNase to another subcellular location. To explore the issue further, we examined in vitro the ability of calcium and phospholipid-dependent protein kinase C (PKC) to catalyze significant phosphorylation of the RNase.
Tnani
M
M
CNRS, UMR 5539, Université Montpellier II, France.
Aliau
S
S
Bayard
B
B
D000227:Adenine Nucleotides / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004722:Endoribonucleases / Q000032:analysis*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007372:Interferons / Q000378:metabolism; D051379:Mice; D009843:Oligoribonucleotides / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000227:Adenine Nucleotides; D003598:Cytoskeletal Proteins; D009843:Oligoribonucleotides; D011500:Protein Synthesis Inhibitors; C023505:2',5'-oligoadenylate; D007372:Interferons; D011493:Protein Kinase C; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; D013755:Tetradecanoylphorbol Acetate