Identification of the Ubiquitin-Proteasome Pathway in the Regulation of the Stability of Eukaryotic Elongation Factor-2 Kinase Sonia Arora, 1,2 Jin-Ming Yang, 1,2 and William N. Hait 1,2 Departments of 1 Pharmacology and 2 Medicine, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, New Brunswick, New Jersey Abstract Eukaryotic elongation factor-2 kinase (eEF-2 kinase) is a highly conserved calcium/calmodulin-dependent enzyme in- volved 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 trans- duction 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 coimmu- noprecipitation 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 polyubiquiti- nated 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. (Cancer Res 2005; 65(9): 3806-10) Introduction Eukaryotic elongation factor-2 kinase (eEF-2 kinase; calmodulin- dependent kinase III) is a calcium- and calmodulin-dependent enzyme that catalyzes the phosphorylation of eukaryotic elonga- tion factor-2. Cloning and sequencing of the kinase by our laboratory (1) and others (2) suggest that eEF-2 kinase may represent a new class of protein kinases now known to include myosin heavy chain kinase A, B, and C (1, 3, 4) and several proteins with both ion channel and kinase features (5). The activity of eEF-2 kinase is believed to regulate protein translation (6). For example, phosphorylation of elongation factor-2 terminates peptide elonga- tion by decreasing the affinity of the elongated chain for the ribosome (7). In addition, the activity of this kinase seems to be involved in several cellular processes. For example, increased eEF-2 kinase activity is linked to augmented fibroblast growth (8), myoblast fusion and differentiation (8, 9), fluctuations in oogenesis (10), cell cycle progression of Xenopus eggs and human amnion cells (11), cellular differentiation (12–14), and response of cells to growth factors and serum (8, 15, 16). Growing evidence suggest that the modulation of eEF-2 by eEF-2 kinase may also be important in the development of neuronal functions (17–19). eEF-2 kinase has also been shown to be involved in the generation of synaptic connections and synaptic plasticity (18, 19). Our laboratory was the first to recognize an increased activity of eEF-2 kinase in malignant cell lines (20) and in human cancers (15, 21). The marked increase in activity in rat glioblastoma led us to investigate the activity of eEF-2 kinase in normal rat glia. These experiments showed that kinase activity was up-regulated in rapidly proliferating normal and malignant glia. In the study of eEF-2 kinase regulation, we observed that the protein content of the kinase, not its regulators such as calcium and calmodulin, changed rapidly on growth stimulation or serum deprivation (16). Furthermore, in recent studies we found that disruption of heat shock protein 90 (Hsp90) chaperoning of eEF-2 kinase also produced rapid disappearance of the enzyme (22). Yet, the mechanisms regulating the stability of eEF-2 kinase remain unknown. Protein modification via covalent attachment of ubiquitin has emerged as one of the most common pathways for targeting protein for degradation by the 26S proteasome (23). Ubiquitina- tion has also been implicated in other regulatory mechanisms, ranging from protein kinase activation to control of protein translation (24). The ubiquitin-proteasome pathway is composed of the ubiquitin conjugating system and the 26S proteasome; the latter contains the multicatalytic protease complex. Coordinated function of the ubiquitin-conjugating system involves several classes of enzymes including ubiquitin-activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). The activity of this pathway may result in mono- or polyubiquiti- nation on the target protein, each of which serves as a different signaling tag (25–27). Whereas polyubiquitin tags the protein for degradation through proteasome pathway, monoubiquitination serves as a signaling marker (23, 28, 29). Because of the role of the ubiquitin-proteasome pathway in regulating the degradation Requests for reprints: William N. Hait and Jin-Ming Yang, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08901. Phone: 732-235- 8075; Fax: 732-235-8094; E-mail: jyang@umdnj.edu; haitwn@umdnj.edu. #2005 American Association for Cancer Research. Cancer Res 2005; 65: (9). 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