ABSTRACT
The double-stranded RNA-dependent protein kinase (PKR) is one of the key mediators of interferon (IFN) action against certain viruses. PKR also plays an important role in signal transduction and immunomodulation. Understanding the regulation of PKR activity is important for the use of PKR as a tool to discover and develop novel therapeutics for viral infections, cancer and immune dysfunction. We found that phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), decreased the level of autophosphorylated PKR in a dose- and time-dependent manner in IFN-treated mouse fibroblast cells. Polyinosinic-polycytidylic acid (poly I:C) treatment enhanced the activity of PKR induced by IFN, but did not overcome the PMA-induced reduction of PKR autophosphorylation. Western blot analysis with a monoclonal antibody to mouse PKR revealed that the decrease of PKR autophosphorylation in cells by PMA was a result of PKR protein degradation. Selective PKC inhibitors blocked the degradation of PKR stimulated by PMA, indicating that PKC activity was required for the effect. Furthermore, we also found that proteasome inhibitors prevented PMA-induced down regulation of PKR, indicating that an active proteasome is required. Our results identify a novel mechanism for the post-translational regulation of PKR.
Subject(s)
Phorbol Esters/pharmacology , eIF-2 Kinase/antagonists & inhibitors , Animals , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Mice , NIH 3T3 Cells , Phosphorylation/drug effects , Protein Kinase C/metabolism , Protein Processing, Post-Translational , RNA, Double-Stranded/metabolism , Signal Transduction/drug effects , eIF-2 Kinase/metabolismABSTRACT
2'-5' Oligoadenylate (2-5A)-dependent RNase L is one of the key enzymes involved in the molecular mechanisms of interferon (IFN) function. Although the regulation of RNase L by 2-5A has been studied extensively, relatively little is known about how RNase L is controlled by posttranslational processes. Here, we report that phorbol-12-myristate-13-acetate (PMA) treatment of mouse L929 fibroblasts caused rapid degradation of RNase L in a dose-dependent and time-dependent manner. RNase L levels were decreased to 40% of control levels after only 5 min exposure of cells to PMA, suggesting the involvement of protein kinase C (PKC). After PMA treatment for 1 h, RNase L levels decreased to 18% of the pretreatment levels. Decay of RNase L was measured by 2-5A binding assay, ribonuclease activity, and protein levels in Western blots probed with antibody to murine RNase L. PMA treatment caused decreases in the levels of RNase L in both cytoplasm and nucleus. To explore the mechanism of RNase L degradation, we treated cells with the selective proteasome inhibitors, ALLN, MG132, and PSI, prior to PMA treatment. These inhibitors completely blocked the degradation of RNase L caused by PMA. Our results show a novel regulatory pathway for RNase L that could have an impact on its antitumor and antiviral functions.
