Regulation of Elongation Factor 2 Kinase (eEF-2K) by Phosphorylation PDF Download

Author: Clint Damian Jaxon Tavares
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Languages : en
Pages : 528

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Book Description
Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical calcium/calmodulin-dependent protein kinase (CaMK-III). The kinase provides a mechanism by which cells can globally control the rate of the elongation phase of protein synthesis, through inhibition of its only known substrate, elongation factor 2 (eEF-2). eEF-2K has been reported to promote proliferation, migration and survival of cancer cells, and has also been implicated in depression. Understanding the regulation of kinase activity is important for development of inhibitors to treat these disease states. However, the mechanisms through which upstream kinases regulate eEF-2K activity via multisite phosphorylation, and how calcium and calmodulin contribute to this, are still unclear. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. This work reports the purification (a three-step protocol using a calmodulin-affinity column) and characterization of recombinant human eEF-2K expressed in E. coli. Mass spectrometry identified Thr-348 and Ser-500 as major calcium/calmodulin-stimulated autophosphorylation sites. Rapid quench analysis indicates that the rate of Thr-348 autophosphorylation is extremely fast (k = 2.6 s−1, t [subscript 1⁄2] ~ 279 ms), and requires calmodulin. Mutagenesis studies suggest that phosphorylation at Thr-348 is required for eEF-2 phosphorylation both in vitro and in cells. However, while T348A has an overall lower activity than the WT kinase, the extent of stimulation of eEF-2K activity by 2-deoxy-D-glucose, hydrogen peroxide, ionomycin or cellular starvation is the same for both forms of the kinase. Thus, to control the amplitude of eEF-2K activity in cells following a stimulus that promotes calmodulin binding, phosphorylation at Thr-348 could act as an additional allosteric switch. This could potentially occur though interaction with a putative phosphate-binding pocket comprised of Lys-205, Arg-252 and Thr-254. Additionally, we observed that Ser-500 phosphorylation promotes an increase in affinity for calmodulin, and we provide evidence that Ser-500 phosphorylation on the eEF-2K•calmodulin complex significantly enhances the already rapid rate of Thr-348 autophosphorylation. Here we propose a two-step model involving calcium/calmodulin binding and Thr-348 autophosphorylation to modulate eEF-2K activity. Phosphorylation at Ser-500 is able to alter the kinetics at both these levels of regulation.