Identification of the Ubiquitin-Proteasome Pathway in the Regulation of the Stability of Eukaryotic Elongation Factor-2 Kinase
Open Access
- 1 May 2005
- journal article
- Published by American Association for Cancer Research (AACR) in Cancer Research
- Vol. 65 (9), 3806-3810
- https://doi.org/10.1158/0008-5472.can-04-4036
Abstract
Eukaryotic elongation factor-2 kinase (eEF-2 kinase) is a highly conserved calcium/calmodulin-dependent enzyme involved in the regulation of protein translation and cell proliferation. Rapid changes in the activity and abundance of eEF-2 kinase have been observed on growth stimulation, and increased enzyme activity is characteristic of malignant cell growth. Yet the mechanism for controlling the turnover of this kinase is unknown. The ubiquitin-proteasome pathway regulates the degradation of many cellular proteins, including transcription factors, cell cycle regulators, and signal transduction proteins. Therefore, we determined whether the ubiquitin-proteasome pathway regulates the turnover of eEF-2 kinase. We found that eEF-2 kinase was a relatively short-lived protein with a half-life of less than 6 hours. eEF-2 kinase was ubiquitinated in vivo as determined by coimmunoprecipitation and polyubiquitin affinity matrix. Incubation of purified eEF-2 kinase with a source of ubiquitination enzymes (rabbit reticulocyte lysate), purified ubiquitin, and ATP revealed the presence of increasing molecular weight species of ubiquitinated eEF-2 kinase. Treatment of cells with MG132, a proteasome inhibitor, inhibited eEF-2 kinase degradation and induced the accumulation of polyubiquitinated forms of the enzyme, resulting in an increase in its half-life. These results suggest involvement of the proteasome in the turnover of the ubiquitinated kinase. Because eEF-2 kinase is chaperoned by heat shock protein 90 (Hsp90), we next determined if disruption of the Hsp90-eEF-2 kinase complex promoted degradation of the kinase. Treatment of cells with geldanamycin, an Hsp90 inhibitor, enhanced ubiquitination of eEF-2 kinase and decreased the half-life of the kinase to less than 2 hours. These results indicate that cellular levels of eEF-2 kinase are maintained by a balance between association with Hsp90 and degradation by the ubiquitin-proteasome pathway. In conclusion, these data show that the turnover of eEF-2 kinase is regulated by the ubiquitin-proteasome pathway and, therefore, modulating the ubiquitination of eEF-2 kinase might control the abundance of this enzyme and have implications in the treatment of certain forms of cancer.Keywords
This publication has 29 references indexed in Scilit:
- Mono- Versus Polyubiquitination: Differential Control of p53 Fate by Mdm2Science, 2003
- The ubiquitin proteolytic system and pathogenesis of human diseases: a novel platform for mechanism-based drug targetingBiochemical Society Transactions, 2003
- Stress-induced regulation of eukaryotic elongation factor 2 kinase by SB 203580-sensitive and −insensitive pathwaysBiochemical Journal, 2002
- Ubiquitin-mediated proteolysis: biological regulation via destructionBioEssays, 2000
- Mapping of the Novel Protein Kinase Catalytic Domain of Dictyostelium Myosin II Heavy Chain Kinase APublished by Elsevier ,1997
- Elongation factor-2 kinase: immunological evidence for the existence of tissue-specific isoformsFEBS Letters, 1996
- Regulation of protein synthesis at the elongation stage New insights into the control of gene expression in eukaryotesFEBS Letters, 1991
- Nerve Growth Factor‐Induced Down‐Regulation of Calmodulin‐Dependent Protein Kinase III in PC12 Cells Involves Cyclic AMP‐Dependent Protein KinaseJournal of Neurochemistry, 1990
- Identification of Nsp100 as elongation factor 2 (EF‐2)FEBS Letters, 1989
- Phosphorylation of elongation factor 2 by EF-2 kinase affects rate of translationNature, 1988