Abstract 2222: Analysis of the autophosphorylation-induced calcium-independent activity of human elongation factor 2 kinase (eEF-2K) - a therapeutic target for breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Breast cancer is the most common malignancy in women in the Western world, with over 40,000 deaths attributed to this disease. Studies have demonstrated that breast cancer mitogens regulate the activity of eukaryotic elongation factor 2 kinase (eEF-2K). The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. We have recently shown for the first time that the targeting of eEF-2K in an in vivo orthotopic model inhibits growth of established breast cancer tumors and sensitizes the tumors to doxorubicin, an important agent in a number of chemotherapy regimens. Our data suggest that eEF-2K may enhance tumorigenesis through the up-regulation of pro-tumorigenic proteins and pathways including cyclin D1, c-Myc, c-Src/FAK and Akt, whose dysregulation are associated with a poor prognosis in breast cancer. Thus, the targeting of eEF-2K in breast cancer cells has potential as a therapeutic approach for the treatment of breast cancer. In order to develop inhibitors of eEF-2K, an understanding of the regulation of enzyme activity is essential. eEF-2K is an atypical calcium/calmodulin-dependent protein kinase (CaMK-III), that through its phosphorylation of elongation factor 2 (eEF-2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The purpose of our study was to analyze the role of calcium/calmodulin-stimulated autophosphorylation in the generation of a calcium-independent form of eEF-2K. Analysis of the kinase activity using [γ-32P]ATP indicates that autophosphorylation of eEF-2K induces calcium-independent activity. Site-directed mutagenesis of the autophosphorylation sites to alanine and aspartate suggests that the site responsible for this calcium-independent activity is Ser-500. Moreover, using a phospho-specific antibody we have determined that the time course of incorporation of phosphate at Ser-500 correlates with the induction of calcium-independent activity. Surprisingly, calmodulin appears to be essential for this calcium-independent activity of eEF-2K. Mechanistic studies indicate that phosphorylation of eEF-2K on Ser-500 increases its affinity for calmodulin in the absence of calcium, potentially by decreasing its rate of dissociation. We have also found that eEF-2K is indeed phosphorylated on Ser-500 in cells, and we are currently studying the dynamic nature of phosphorylation at this site, and its effect on stability of the enzyme and its affinity for calmodulin. Citation Format: Clint D. J. Tavares, Scarlett B. Ferguson, Rebecca M. Wellmann, Tamer S. Kaoud, John P. O'Brien, Bulent Ozpolat, Jennifer S. Brodbelt, Kevin N. Dalby. Analysis of the autophosphorylation-induced calcium-independent activity of human elongation factor 2 kinase (eEF-2K) - a therapeutic target for breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2222. doi:10.1158/1538-7445.AM2013-2222