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Association of tissue-specific changes in translation elongation after cyclosporin with changes in elongation factor
W C Buss1, J Stepanek, S A Queen
1Department of Pharmacology, University of New Mexico School of Medicine, Albuquerque 87131.
Abstract:
In studies of cyclosporin (CsA) toxicity in Sprague-Dawley rats, CsA administered in vivo produced tissue-specific, dose-dependent changes in microsomal translation throughout the bodies of the animals. The most pronounced translation inhibition was in microsomes from the kidney, the organ in which dose-limiting CsA toxicity occurs. In contrast, translation was stimulated in microsomes from the liver. CsA produced changes at the level of translation elongation, which is regulated by the reversible phosphorylation of elongation factor 2 (EF2). Changes in translation elongation after CsA were found to be associated with, and most likely caused by, changes in EF2 phosphorylation. Reduced renal translation elongation was associated with increased EF2 phosphorylation, and increased hepatic elongation with decreased EF2 phosphorylation. EF2 is phosphorylated by Ca2+ calmodulin-dependent protein kinase III (PKIII). Phosphorylated EF2 is a substrate for protein phosphatase 2A (PP2A), but not calcineurin (protein phosphatase 2B or PP2B), the enzyme inhibited by CsA-cyclophilin complexes in T-cells. When CsA or inhibitors of PKIII (EGTA, trifluoperazine) were added in vitro to assays of EF2 phosphorylation in renal or hepatic cytoplasm, or to assays of renal or hepatic microsomal translation elongation, they were without significant effects. Addition in vitro of the PP2A inhibitor okadaic acid increased EF2 phosphorylation in renal and hepatic cytoplasms, but inconsistently produced an inhibition of microsomal translation. However, in less complex rabbit reticulocyte lysates, addition of okadaic acid inhibited PP2A, increased EF2 phosphorylation, and inhibited translation elongation. Furthermore, addition of EGTA and trifluoperazine to rabbit reticulocyte lysates inhibited Ca2+ calmodulin-dependent PKIII activity, decreased EF2 phosphorylation, and stimulated translation elongation. CsA acting alone or as a complex with cyclophilin could alter EF2 phosphorylation by affecting transcriptional regulation or the enzymatic activity of PKIII, PP2A or EF2. Changes in EF2 phosphorylation and translation in body tissues suggest that CsA causes widespread disturbances in phosphorylation and dephosphorylation pathways regulating cellular processes including transcription and translation factor activity. These disturbances may underlie the broad spectrum of toxicities observed during CsA therapy.
Insights
Cyclosporin A (CsA) causes tissue-specific changes in protein translation by altering elongation factor 2 (EF2) phosphorylation. Kidney translation is inhibited, while liver translation is stimulated, potentially explaining CsA toxicity.
Area of Science:
- Biochemistry
- Pharmacology
- Toxicology
Background:
- Cyclosporin A (CsA) is an immunosuppressant with known toxicities.
- Cellular translation is crucial for protein synthesis and cellular function.
- Elongation factor 2 (EF2) phosphorylation regulates translation elongation.
Purpose of the Study:
- To investigate the effects of CsA on microsomal translation and EF2 phosphorylation in vivo.
- To determine the specific mechanisms by which CsA alters translation elongation.
- To explore the role of protein kinases and phosphatases in CsA-induced translation changes.
Main Methods:
- Administered CsA to Sprague-Dawley rats and analyzed tissue-specific microsomal translation.
- Assessed EF2 phosphorylation status in kidney and liver microsomes.
- Utilized in vitro assays with specific inhibitors (EGTA, trifluoperazine, okadaic acid) and rabbit reticulocyte lysates to probe enzymatic activities.
Main Results:
- CsA induced dose-dependent, tissue-specific changes in microsomal translation; inhibition in kidneys and stimulation in the liver.
- These translation changes correlated with altered EF2 phosphorylation: increased phosphorylation in kidneys and decreased in the liver.
- In vitro studies suggested CsA may disrupt phosphorylation/dephosphorylation balance, impacting EF2 activity and potentially other cellular processes.
Conclusions:
- CsA alters EF2 phosphorylation, leading to tissue-specific changes in translation elongation.
- These disturbances in phosphorylation pathways may underlie the broad spectrum of CsA toxicities.
- Further research into CsA's impact on cellular signaling pathways is warranted.