Elongation factor-2 kinase (eEF-2K), also known as calmodulin-dependent protein kinase III is a member of specialized class of protein kinases termed α-kinase because of their unique ability to phosphorylate within α-helices. eEF-2K can transiently inhibit protein synthesis and is upregulated in several tumor cell lines as well as human cancer specimens. Furthermore, we have shown that eEF-2K inhibitors, rottlerin and NH-125, cause a G1/S arrest and decrease cell proliferation and viability. However, the precise role of eEF-2K in cancer biology remains poorly understood. Carcinogenesis is a multistep process where alterations in oncogenes and tumor suppressors lead to changes in cell proliferation, mobility, tumor invasion and metastasis. The tumor suppressor, caveolin-1 (Cav-1), inhibits each of these processes. Suppression of Cav-1 can induce cellular transformation as shown by inhibition of anchorage independent growth, resistance to anoikis (detachment-induced apoptosis), and tumor formation in nude mice. Since many of these functions attributed to eEF-2K are antagonistic to those of Cav-1, we investigated the regulation of Cav-1 by this unusual protein kinase. SV-40 transformed wild type (WT) and eEF-2K knockout (KO) fibroblasts, (a gift of Dr. Alexy Ryazanov) were utilized to determine whether or not eEF-2K is involved in Cav-1 regulation. In comparison to SV-40 transformed WT cells, KO cells exhibited increased Cav-1 expression, reduced capacity to form colonies in soft agar, and decreased anchorage-independent growth. This correlated with increased anoikis, as shown by increased Annexin-V staining and a 2-fold decrease in survival measured by MTT. Furthermore, in contrast to WT cells, KO cells showed decreased mobility and invasiveness. Next, we determined if eEF-2K and Cav-1 were mechanistically linked to the changed phenotype and if eEF-2K directly regulated Cav-1 through phosphorylation and protein synthesis. Extracts from WT and KO cells were immunoprecipitated with Cav-1 antibodies. Immunoprecipitates were washed and used in eEF-2K kinase assays. Purified recombinant eEF-2K phosphorylated Cav-1 immunoprecipitates. To determine if eEF-2K would affect Cav-1 protein synthesis, WT and KO cells were methionine starved followed by 35S methionine incubation. Extracts were prepared and immunoprecipitated with Cav-1 antibodies. eEF-2K WT had decreased Cav-1 protein synthesis as compared to the KO cells. Therefore, deletion of eEF-2K in SV-40 transformed fibroblasts decreased cell growth, inhibited anchorage independence, invasion and growth in soft agar. These changes were associated with an increased expression of the tumor suppressor, Cav-1, due to increased protein synthesis. Furthermore, we demonstrate for the first time that Cav-1 can serve as an eEF-2K substrate, suggesting that serine phosphorylation may alter its function.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]