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Protein synthesis inhibition is an immediate response during stress to switch the composition of protein pool in order to adapt to the new environment. It was reported that this response could be either protective or deleterious. However, how cells choose to live or die upon protein synthesis inhibition is largely unknown. Previously, we have shown that elongation factor-2 kinase (eEF2K), a protein kinase that suppresses protein synthesis during elongation phase, is a positive regulator of apoptosis both in vivo and in vitro Consistently, here we report that knock-out of eEF2K protects mice from a lethal dose of whole-body ionizing radiation at 8 Gy by reducing apoptosis levels in both bone marrow and gastrointestinal tracts. Surprisingly, similar to the loss of p53, eEF2K deficiency results in more severe damage to the gastrointestinal tract at 20 Gy with the increased mitotic cell death in small intestinal stem cells. Furthermore, using epithelial cell lines, we showed that eEF2K is required for G2/M arrest induced by radiation to prevent mitotic catastrophe in a p53-independent manner. Specifically, we observed the elevation of Akt/ERK activity as well as the reduction of p21 expression in Eef2k(-/-) cells. Therefore, eEF2K also provides a protective strategy to maintain genomic integrity by arresting cell cycle in response to stress. Our results suggest that protective versus pro-apoptotic roles of eEF2K depend on the type of cells: eEF2K is protective in highly proliferative cells, such as small intestinal stem cells and cancer cells, which are more susceptible to mitotic catastrophe.

The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function, and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange, and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His(6)-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ∼85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme.

TRPM7 and its closest homologue, TRPM6, are the only known fusions of an ion channel pore with a kinase domain. Deletion of TRPM7 in DT40 B-lymphocytes causes growth arrest, Mg(2+) deficiency, and cell death within 24-48 h. Amazingly, in analogy to TRPM6-deficient patients who can live a normal life if provided with a Mg(2+)-rich diet, TRPM7-deficient DT40 B-lymphocytes show wild type cell growth if supplied with 5-10 mm Mg(2+) concentrations in their extracellular medium. Here we have investigated the functional relationship between TRPM6 and TRPM7. We show that TRPM7 deficiency in DT40 cells cannot be complemented by heterologously expressed TRPM6. Nevertheless, both channels can influence each other's biological activity. Our data demonstrate that TRPM6 requires TRPM7 for surface expression in HEK-293 cells and also that TRPM6 is capable of cross-phosphorylating TRPM7 as assessed using a phosphothreonine-specific antibody but not vice versa. TRPM6 and TRPM7 coexpression studies in DT40 B-cells indicate that TRPM6 can modulate TRPM7 function. In conclusion, although TRPM6 and TRPM7 are closely related and deficiency in either one of these molecules severely affects Mg(2+) homeostasis regulation, TRPM6 and TRPM7 do not appear to be functionally redundant but rather two unique and essential components of vertebrate ion homeostasis regulation.

TRPM7 is an unusual bifunctional molecule consisting of a TRP ion channel fused to a protein kinase domain. It has been shown that TRPM7 plays a key role in the regulation of intracellular magnesium homeostasis as well as in anoxic neuronal death. TRPM7 channel has been characterized using electrophysiological techniques; however, the function of the kinase domain is not known and endogenous substrates for the kinase have not been reported previously. Here we have identified annexin 1 as a substrate for TRPM7 kinase. Phosphorylation of annexin 1 by TRPM7 kinase is stimulated by Ca2+ and is dramatically increased in extracts from cells overexpressing TRPM7. Phosphorylation of annexin 1 by TRPM7 kinase occurs at a conserved serine residue (Ser5) located within the N-terminal amphipathic alpha-helix of annexin 1. The N-terminal region plays a crucial role in interaction of annexin 1 with other proteins and membranes, and therefore, phosphorylation of annexin 1 at Ser5 by TRPM7 kinase may modulate function of annexin 1.

Though the mechanisms of protein biosynthesis are similar in the cells of prokaryotes and eukaryotes, the eukaryotic translational machinery in the cell is arranged more intricately. One of the most striking characteristic features of the eukaryotic translational machinery is that the eukaryotic proteins involved in the translational process, such as initiation factors, elongation factors and aminoacyl-tRNA synthetases, in contrast to their prokaryotic analogs, possess a non-specific affinity for RNA. Due to the RNA-binding ability, these eukaryotic proteins can be compartmentalized on polyribosomes. In addition to the proteins of the translational apparatus, several other eukaryotic RNA-binding proteins can be also compartmentalized on polyribosomes; these proteins include glycolytic enzymes, steroid hormone receptors and intermediate filament proteins. Thus, the eukaryotic polyribosome is an element of the cytoplasmic labile structure on which various proteins can be compartmentalized and, consequently, different biochemical pathways can be integrated.