Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION.

Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM)-dependent member of the unique α-kinase family, impedes protein synthesis by phosphorylating eEF-2. We recently identified Thr-348 and Ser-500 as two key autophosphorylation sites within eEF-2K that regulate its activity. eEF-2K is regulated by Ca2+ ions and multiple upstream signaling pathways, but how it integrates these signals into a coherent output, i.e. phosphorylation of eEF-2, is unclear. This study focuses on understanding how the post-translational phosphorylation of Ser-500 integrates with Ca2+ and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca2+ is shown to enhance the affinity of CaM toward eEF-2K. Ser-500 is found to undergo autophosphorylation in cells treated with ionomycin and is likely also targeted by PKA. In vitro, autophosphorylation of Ser-500 is found to require Ca2+ and CaM and is inhibited by mutations that compromise binding of phosphorylated Thr-348 to an allosteric binding pocket on the kinase domain. A phosphomimetic Ser-500 to aspartic acid mutation (eEF-2K S500D) enhances the rate of activation (Thr-348 autophosphorylation) by 6-fold and lowers the EC50 for Ca2+/CaM binding to activated eEF-2K (Thr-348 phosphorylated) by 20-fold. This is predicted to result in an elevation of the cellular fraction of active eEF-2K. In support of this mechanism, eEF-2K knock-out MCF10A cells reconstituted with eEF-2K S500D display relatively high levels of phospho-eEF-2 under basal conditions. This study reports how phosphorylation of a regulatory site (Ser-500) integrates with Ca2+ and CaM to influence eEF-2K activity.

Synthesis and biological evaluation of pyrido[2,3-d]pyrimidine-2,4-dione derivatives as eEF-2K inhibitors.

A small molecule library of pyrido[2,3-d]pyrimidine-2,4-dione derivatives 6-16 was synthesized from 6-amino-1,3-disubstituted uracils 18, characterized, and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K). To understand the binding pocket of eEF-2K, structural modifications of the pyrido[2,3-d]pyrimidine were made at three regions (R(1), R(2), and R(3)). A homology model of eEF-2K was created, and compound 6 (A-484954, Abbott laboratories) was docked in the catalytic domain of eEF-2K. Compounds 6 (IC50=420nM) and 9 (IC50=930nM) are found to be better molecules in this preliminary series of pyrido[2,3-d]pyrimidine analogs. eEF-2K activity in MDA-MB-231 breast cancer cells is significantly reduced by compound 6, to a lesser extent by compound 9, and is unaffected by compound 12. Similar inhibitory results are observed when eEF-2K activity is stimulated by 2-deoxy-d-glucose (2-DOG) treatment, suggesting that compounds 6 and 9 are able to inhibit AMPK-mediated activation of eEF-2K to a notable extent. The results of this work will shed light on the further design and optimization of novel pyrido[2,3-d]pyrimidine analogs as eEF-2K inhibitors.