ABSTRACT
For the first time it has been shown that RNase activity is induced under the influence of EGF on epidermoid carcinoma cell line A431. Proteasomes from EGF-treated A431 cells destabilize the 3'-untranslated regions of non-muscle beta actin mRNA, creating a specific cleavage pattern. In addition, these particles have been shown to specifically cleave Alu-containing informational RNA. The enzymatic activity under study has been shown to be dependent on phosphorylation of proteasomal subunits and specifically and selectively regulated by Ca and Mg ions. Proteasome involvement in the coordinated control of stability of specific messenger RNA molecules is suggested. The endoribonuclease activity of 26S proteasomes can constitute a link between EGF signaling pathways and RNA stability.
Subject(s)
Endoribonucleases/metabolism , Epidermal Growth Factor/pharmacology , Peptide Hydrolases/drug effects , Proteasome Endopeptidase Complex , RNA, Messenger/metabolism , 3' Untranslated Regions/metabolism , Actins/genetics , Aluminum , Calcium Chloride , Cell Line, Tumor , Humans , Magnesium Chloride , Peptide Hydrolases/metabolism , Phosphorylation , RNA, Messenger/chemistry , Signal TransductionABSTRACT
A comparative study was made of reactive oxygen species (ROS) in rat embryo fibroblasts and their transformants. Primary rat embryo fibroblasts (REF), REF transformed by the complementing oncogenes E1A plus cHa-ras (cell line E1A + Ras), and REF transformed by E1A plus E1B-19 kDa (cell line E1A + E1B) were studied. ROS generation was measured with microfluorometric assay using fluorescent probe 2',7'-dichlorofluorescin diacetate. It has been shown that the block of REF and E1A + 1B cells in the G1/S under serum-starved conditions (0.5% serum) for 24-48 h was paralleled by a decrease in ROS generation. Activation of serum-starved REF and E1A + 1B cells with 10% serum resulted in reactivation of cell cycle and gradual increase in ROS generation. The maximum intracellular level of ROS correlated in time with the phase of DNA synthesis. Serum-starved E1A + Ras cells were not stopped in the G1/S and ROS production of these cells was not dependent on serum growth factors. The prolonged cultivation of E1A + Ras cells in the medium with low serum content (0.5%) caused a sharp increase in ROS generation, which was accompanied by apoptotic death.
Subject(s)
Endoribonucleases/metabolism , Epidermal Growth Factor/genetics , Ribonucleoproteins, Small Nuclear/metabolism , 3' Untranslated Regions , Epidermal Growth Factor/metabolism , Humans , Molecular Weight , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Tumor Cells, CulturedABSTRACT
Our analysis detected in 26S proteasomes of human A-431 cells a strong endoribonuclease activity, degrading cytoplasmic high-molecular-mass RNA, particularly, specific mRNAs. Enzymatic nature of this activity has been confirmed, and the optimal conditions studied. This endonuclease activity of proteasomes has not been earlier observed. Proteasome involvement in the stability control of specific messenger RNA molecules is suggested, and proteasome participation in the coordinated control of various stages of gene expression is discussed.
