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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.

Cancer cells predominantly metabolize glucose by glycolysis to produce energy in order to meet their metabolic requirement, a phenomenon known as Warburg effect. Although Warburg effect is considered a peculiarity critical for survival and proliferation of cancer cells, the regulatory mechanisms behind this phenomenon remain incompletely understood. We report here that eukaryotic elongation factor-2 kinase (eEF-2K), a negative regulator of protein synthesis, has a critical role in promoting glycolysis in cancer cells. We showed that deficiency in eEF-2K significantly reduced the uptake of glucose and decreased the productions of lactate and adenosine triphosphate in tumor cells and in the Ras-transformed mouse embryonic fibroblasts. We further demonstrated that the promotive effect of eEF-2K on glycolysis resulted from the kinase-mediated restriction of synthesis of the protein phosphatase 2A-A (PP2A-A), a key factor that facilitates the ubiquitin-proteasomal degradation of c-Myc protein, as knockdown of eEF-2K expression led to a significant increase in PP2A-A protein synthesis and remarkable downregulation of c-Myc and pyruvate kinase M2 isoform, the key glycolytic enzyme transcriptionally activated by c-Myc. In addition, depletion of eEF-2K reduced the ability of the transformed cells to proliferate and enhanced the sensitivity of tumor cells to chemotherapy both in vitro and in vivo. These results, which uncover a role of the eEF-2K-mediated control of PP2A-A in tumor cell glycolysis, provide new insights into the regulation of the Warburg effect.

Protein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A–cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylation in vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2K in vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity.

Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV‐PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti‐viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir‐resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir‐mediated anti‐tumoral activity is severely compromised in eEF2K‐deficient engrafted tumors in vivo. Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti‐tumoral properties of HIV‐PIs.

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.

TRPM7 is an essential and ubiquitous channel-kinase regulating cellular influx of Mg2+. Although TRPM7 mRNA is highly abundant, very small amount of the protein is detected in cells, suggesting post-transcriptional regulation of trpm7 gene expression. We found that TRPM7 mRNA 5′-leader contains two evolutionarily conserved upstream open reading frames that act together to drastically inhibit translation of the TRPM7 reading frame at high magnesium levels and ensure its optimal translation at low magnesium levels, when the activity of the channel-kinase is most required. The study provides the first example of magnesium channel synthesis being controlled by Mg2+ in higher eukaryotes.

SummaryThe control of germline quality is critical to reproductive success and survival of a species; however, the mechanisms underlying this process remain unknown. Here, we demonstrate that elongation factor 2 kinase (eEF2K), an evolutionarily conserved regulator of protein synthesis, functions to maintain germline quality and eliminate defective oocytes. We show that disruption of eEF2K in mice reduces ovarian apoptosis and results in the accumulation of aberrant follicles and defective oocytes at advanced reproductive age. Furthermore, the loss of eEF2K in Caenorhabditis elegans results in a reduction of germ cell death and significant decline in oocyte quality and embryonic viability. Examination of the mechanisms by which eEF2K regulates apoptosis shows that eEF2K senses oxidative stress and quickly downregulates short-lived antiapoptotic proteins, XIAP and c-FLIPL by inhibiting global protein synthesis. These results suggest that eEF2K-mediated inhibition of protein synthesis renders cells susceptible to apoptosis and functions to eliminate suboptimal germ cells.

Macrophages are central effectors of innate immune responses to bacteria. We have investigated how activation of the abundant macrophage lysosomal protease, cathepsin D, regulates the macrophage proteome during killing of Streptococcus pneumoniae. Using the cathepsin D inhibitor pepstatin A, we demonstrate that cathepsin D differentially regulates multiple targets out of 679 proteins identified and quantified by eight-plex isobaric tag for relative and absolute quantitation. Our statistical analysis identified 18 differentially expressed proteins that passed all paired t-tests (alpha = 0.05). This dataset was enriched for proteins regulating the mitochondrial pathway of apoptosis or inhibiting competing death programs. Five proteins were selected for further analysis. Western blotting, followed by pharmacological inhibition or genetic manipulation of cathepsin D, verified cathepsin D-dependent regulation of these proteins, after exposure to S. pneumoniae. Superoxide dismutase-2 up-regulation was temporally related to increased reactive oxygen species generation. Gelsolin, a known regulator of mitochondrial outer membrane permeabilization, was down-regulated in association with cytochrome c release from mitochondria. Eukaryotic elongation factor (eEF2), a regulator of protein translation, was also down-regulated by cathepsin D. Using absence of the negative regulator of eEF2, eEF2 kinase, we confirm that eEF2 function is required to maintain expression of the anti-apoptotic protein Mcl-1, delaying macrophage apoptosis and confirm using a murine model that maintaining eEF2 function is associated with impaired macrophage apoptosis-associated killing of Streptococcus pneumoniae. These findings demonstrate that cathepsin D regulates multiple proteins controlling the mitochondrial pathway of macrophage apoptosis or competing death processes, facilitating intracellular bacterial killing. Molecular & Cellular Proteomics 10: 10.1074/mcp.M111.008193, 1-14, 2011.

TRPM6 and TRPM7 encode channel-kinases. While these channels share electrophysiological properties and cellular functions, TRPM6 and TRPM7 are non-redundant genes raising the possibility that the kinases have distinct substrates. Here, we demonstrate that TRPM6 and TRPM7 phosphorylate the assembly domain of myosin IIA, IIB and IIC on identical residues. Whereas phosphorylation of myosin IIA is restricted to the coiled-coil domain, TRPM6 and TRPM7 also phosphorylate the non-helical tails of myosin IIB and IIC. TRPM7 does not phosphorylate eukaryotic elongation factor-2 (eEF-2) and myosin II is a poor substrate for eEF-2 kinase. In conclusion, TRPM6 and TRPM7 share exogenous substrates among themselves but not with functionally distant α-kinases.Structured summaryMINT-6700314:GNA1 (uniprotkb:Q96EK6) and GNA1 (uniprotkb:Q96EK6) bind (MI:0407) by X-ray crystallography (MI:0114)

Contains fulltext : 70900.pdf (Publisher’s version ) (Closed access) Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian alpha-kinase TRPM7 inhibits myosin II-based contractility in a Ca(2+)- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains--the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the alpha-helical tail of the myosin IIA heavy chain.

SummaryGroup I metabotropic glutamate receptors (mGluR) induce long-term depression (LTD) that requires protein synthesis. Here, we demonstrate that Arc/Arg3.1 is translationally induced within 5 min of mGluR activation, and this response is essential for mGluR-dependent LTD. The increase in Arc/Arg3.1 translation requires eEF2K, a Ca2+/calmodulin-dependent kinase that binds mGluR and dissociates upon mGluR activation, whereupon it phosphorylates eEF2. Phospho-eEF2 acts to slow the elongation step of translation and inhibits general protein synthesis but simultaneously increases Arc/Arg3.1 translation. Genetic deletion of eEF2K results in a selective deficit of rapid mGluR-dependent Arc/Arg3.1 translation and mGluR-LTD. This rapid translational mechanism is disrupted in the fragile X disease mouse (Fmr1 KO) in which mGluR-LTD does not require de novo protein synthesis but does require Arc/Arg3.1. We propose a model in which eEF2K-eEF2 and FMRP coordinately control the dynamic translation of Arc/Arg3.1 mRNA in dendrites that is critical for synapse-specific LTD.

The role of autophagy in ageing regulation has been suggested based on studies in C. elegans, in which knockdown of the expression of bec-1 (ortholog of the yeast and mammalian autophagy genes ATG6/VPS30 and beclin 1, respectively) shortens lifespan of the daf-2(e1370) mutant C. elegans. However, Beclin1/ATG6 is also known to be involved in other cellular functions in addition to autophagy. In the current study, we knocked down two other autophagy genes, atg-7 and atg-12, in C. elegans using RNAi. We showed that RNAi shortened the lifespan of both wild type and daf-2 mutant C. elegans, providing strong support for a role of autophagy in ageing regulation.

Contains fulltext : 51410.pdf (Publisher’s version ) (Closed access) Actomyosin contractility regulates various cell biological processes including cytokinesis, adhesion and migration. While in lower eukaryotes, alpha-kinases control actomyosin relaxation, a similar role for mammalian alpha-kinases has yet to be established. Here, we examined whether TRPM7, a cation channel fused to an alpha-kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by bradykinin leads to a Ca(2+)- and kinase-dependent interaction with the actomyosin cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain. Accordingly, low overexpression of TRPM7 increases intracellular Ca2+ levels accompanied by cell spreading, adhesion and the formation of focal adhesions. Activation of TRPM7 induces the transformation of these focal adhesions into podosomes by a kinase-dependent mechanism, an effect that can be mimicked by pharmacological inhibition of myosin II. Collectively, our results demonstrate that regulation of cell adhesion by TRPM7 is the combined effect of kinase-dependent and -independent pathways on actomyosin contractility.

Guamanian amyotrophic lateral sclerosis (ALS-G) and parkinsonism dementia (PD-G) have been epidemiologically linked to an environment severely deficient in calcium (Ca 2+ ) and magnesium (Mg 2+ ). Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein containing both channel and kinase domains that has been proposed to be involved in the homeostatic regulation of intracellular Ca 2+ , Mg 2+ , and trace metal ion concentration. There is evidence that TRPM7 is constitutively active and that the number of available channels is dependent on intracellular free Mg 2+ levels. We found a TRPM7 variant in a subset of ALS-G and PD-G patients that produces a protein with a missense mutation, T1482I. Recombinant T1482I TRPM7 exhibits the same kinase catalytic activity as WT TRPM7. However, heterologously expressed T1482I TRPM7 produces functional channels that show an increased sensitivity to inhibition by intracellular Mg 2+ . Because the incidence of ALS-G and PD-G has been associated with prolonged exposure to an environment severely deficient in Ca 2+ and Mg 2+ , we propose that this variant TRPM7 allele confers a susceptibility genotype in such an environment. This study represents an initial attempt to address the important issue of gene–environment interactions in the etiology of these diseases.

A key aspect in the structure of epithelial cells is the maintenance of a polarized organization based on a highly specific sorting machinery for cargo destined for the apical or the basolateral membrane domain at the exit site of the trans-Golgi network. We could recently identify two distinct post-trans-Golgi network vesicle populations that travel along separate routes to the plasma membrane, a lipid raft-dependent and a lipid raft-independent pathway. A new component of raft-carrying apical vesicles is alpha-kinase 1 (ALPK1), which was identified in immunoisolated vesicles carrying raft-associated sucrase-isomaltase (SI). This kinase was absent from vesicles carrying raft-non-associated lactase-phlorizin hydrolase. The expression of ALPK1 increases by the time of epithelial cell differentiation, whereas the intracellular localization of ALPK1 on apical transport vesicles was confirmed by confocal analysis. A phosphorylation assay on isolated SI-carrying vesicles revealed the phosphorylation of a protein band of about 105 kDa, which could be identified as the motor protein myosin I. Finally, a specific reduction of ALPK1-expression by RNA interference results in a significant decrease in the apical delivery of SI. Taken together, our data suggest that the phosphorylation of myosin I by ALPK1 is an essential process in the apical trafficking of raft-associated SI.

Alpha-kinases are a recently discovered family of protein kinases that have no detectable sequence homology to conventional protein kinases (CPKs). They include elongation factor 2 kinase, Dictyostelium myosin heavy chain kinases and many other protein kinases from diverse organisms, as revealed by various genome sequencing projects. Mammals have six alpha-kinases, including two channel-kinases—novel signaling molecules that contain an alpha-kinase domain fused to an ion-channel. Analysis of all known alpha-kinase sequences reveals the presence of several highly conserved motifs. Despite the fact that alpha-kinases have no detectable sequence identity with CPKs, the recently determined three-dimensional structure of the channel-kinase TRPM7/ChaK1 kinase domain reveals that alpha-kinases have a fold very similar to CPKs. Using the structural alignment of channel-kinase TRPM7/ChaK1 with cyclic-AMP dependent kinase, the consensus motifs of alpha-kinases and CPKs were aligned and compared. Remarkably, the majority of structural elements, sequence motifs, and the position of key amino acid residues important for catalysis appear to be very similar in alpha-kinases and CPKs. Differences between alpha-kinases and CPKs, and their possible impact on substrate recognition are discussed.

Channel-kinase TRPM7/ChaK1 is a member of a recently discovered family of protein kinases called alpha-kinases that display no sequence homology to conventional protein kinases. It is an unusual bifunctional protein that contains an alpha-kinase domain fused to an ion channel. The TRPM7/ChaK1 channel has been characterized using electrophysiological techniques, and recent evidence suggests that it may play a key role in the regulation of magnesium homeostasis. However, little is known about its protein kinase activity. To characterize the kinase activity of TRPM7/ChaK1, we expressed the kinase catalytic domain in bacteria. ChaK1-cat is able to undergo autophosphorylation and to phosphorylate myelin basic protein and histone H3 on serine and threonine residues. The kinase is specific for ATP and cannot use GTP as a substrate. ChaK1-cat is insensitive to staurosporine (up to 0.1 mM) but can be inhibited by rottlerin. Because the kinase domain is physically linked to an ion channel, we investigated the effect of ions on ChaK1-cat activity. The kinase requires Mg(2+) (optimum at 4-10 mM) or Mn(2+) (optimum at 3-5 mM), with activity in the presence of Mn(2+) being 2 orders of magnitude higher than in the presence of Mg(2+). Zn(2+) and Co(2+) inhibited ChaK1-cat kinase activity. Ca(2+) at concentrations up to 1 mM did not affect kinase activity. Considering intracellular ion concentrations, our results suggest that, among divalent metal ions, only Mg(2+) can directly modulate TRPM7/ChaK1 kinase activity in vivo.

Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells. Recently, Vega et al. proposed that exogenous prothymosin alpha can specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernstedt, C., and Domínguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response. A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha. However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate. Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2.

Mobilization of Ca 2+ sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca 2+ ([Ca 2+ ] i ) due to plasmalemmal Ca 2+ influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca 2+ stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca 2+ ] i per se causes slowing of peptide chain elongation is lacking. L-type Ca 2+ channel activity of GH 3 pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca 2+ ] i on eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca 2+ , resting [Ca 2+ ] i values were high (154–255 nM) and eEF-2 was phosphorylated. The Ca 2+ channel antagonist, nisoldipine, lowered [Ca 2+ ] i and reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca 2+ channel agonist, Bay K 8644, produced sustained elevations of [Ca 2+ ] i that were associated with 25–50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca 2+ influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50–100% above control) and inhibited leucine incorporation by 20–70% but ATP content was reduced by 25–50% and total cell-associated Ca 2+ contents rose by 3- to 13-fold. eIF-2α was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca 2+ ] i that resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2α, not eEF-2. It is concluded that changes in [Ca 2+ ] i occurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.

Elongation factor 2 was identified in the two-dimensional gel patterns of asynchronous human amnion cells (AMA) by comigration with purified rabbit reticulocyte elongation factor 2 and by two-dimensional gel immunoblot analysis using a specific rabbit polyclonal antibody. In all, four polypeptides were identified corresponding to isoelectric focusing polypeptides 2713 (95.0 kDa), 2714 (94.8 kDa), 3727 (94.8 kDa), and 3702 (93.6 kDa) (listed in order of decreasing pI values) in the computerized comprehensive two-dimensional gel data base of human AMA proteins. The relative proportion of two of these variants (isoelectric focusing polypeptides 3727 and 3702), which are phosphorylated, increased dramatically during mitosis. Since phosphorylation is known to render elongation factor 2 inactive in translation, this observation may partly explain the decline in the rate of protein synthesis observed during cell division.

Previously we have found that elongation factor 2 (EF-2) from mammalian cells can be phosphorylated by a special Ca2+/calmodulin-dependent protein kinase (EF-2 kinase). Phosphorylation results in complete inactivation of EF-2 in the poly(U)-directed cell-free translation system. However, the partial function of EF-2 affected by phosphorylation remained unknown. Here we show that phosphorylated EF-2, unlike non-phosphorylated EF-2, is unable to switch ribosomes carrying poly(U) and Phe-tRNA in the A site to a puromycin-reactive state. Thus, phosphorylation of EF-2 seems to block its ability to promote a shift of the aminoacyl(peptidyl)-tRNA from the A site to the P site i.e. translocation itself.

The addition of 5 mM cAMP to a cell-free translation system from rabbit reticulocytes increases the rate of protein synthesis 3–5-fold. Lower concentrations of cAMP (0.005, 0.05 and 0.5 mM) have no effect on translation in this system. cAMP at all the concentrations tested stimulates the phosphorylation of the same pattern of polypeptides, while 5 mM cAMP additionally stimulates dephosphorylation of the 95 kDa polypeptide identified as elongation factor 2 (EF-2). Testing of the preparations of EF-2 with a different content of the phosphorylated form in poly(U)-directed poly(Phe) synthesis reveals that the EF-2 activity correlates with the fraction of non-phosphorylated EF-2. Thus cAMP-dependent activation of protein synthesis seems to be due to dephosphorylation of EF-2.

There are many reports which demonstrate that the rate of protein biosynthesis at the elongation stage is actively regulated in eukaryotic cells. Possible physiological roles for this type of regulation are: the coordination of translation of mRNA with different initiation rate constants; regulation of transition between different physiological states of a cell, such as transition between stages or the cell cycle; and in general, any situation where the maintenance of a particular physiological state is dependent on continuous protein synthesis. A number of covalent modifications of elongation factors offer potential mechanisms for such regulation. Among the various modifications of elongation factors, phosphorylation of eEF-2 by the specific Ca2+/calmodulin-dependent eEF-2 kinase is the best studied and perhaps the most important mechanism for regulation of elongation rate. Since this phosphorylation is strictly Ca2+-dependent, and makes eEF-2 inactive in translation, this mechanism could explain how changes in the intracellular free Ca2+ concentration may regulate elongation rate. We also discuss some recent findings concerning elongation factors, such as the discovery of developmental stage-specific elongation factors and the regulated binding of eEF-1α to cytoskeletal elements. Together, these observations underline the importance of the elongation stage of translation in the regulation of the cellular processes essential for normal cell life.

Incubation of a ribosome-free extract of rabbit reticulocytes or rat liver with [gamma-32P]ATP and Ca2+ results in incorporation of 32P predominantly into a single polypeptide of Mr approximately 100,000. This polypeptide is identified as elongation factor 2 (EF-2). Phosphorylation of EF-2 is strictly Ca2+-dependent and can be inhibited by the calmodulin antagonist trifluoperazine. It is suggested that the Ca2+/calmodulin-dependent phosphorylation of EF-2 is involved in regulation of protein biosynthesis.

eEF-2 kinase is a ubiquitous Ca2+/calmodulin-dependent protein kinase that is specific for protein synthesis elongation factor-2 (eEF-2). This study describes an improved procedure for the purification of eEF-2 kinase from rabbit reticulocyte lysate. The eEF-2 kinase preparation was used to raise polyclonal antibodies, which immunoprecipitated eEF-2 kinase protein and activity from rabbit reticulocyte lysate. The antibodies recognized a single 103 kDa band in extracts from several cell lines including NIH 3T3, PC12, C6 glioma, HeLa, and MCF-7 breast carcinoma. However, there was no immunoreactivity in extracts of rabbit or bovine liver or rabbit kidney despite the presence of abundant eEF-2 kinase activity in these tissues. Exposure of PC12 cells to nerve growth factor (NGF) resulted in rapid down-regulation of eEF-2 kinase activity and a decrease in immunoreactivity. After 24 h of incubation with NGF, the activity of the kinase recovered to 80% of initial values. In contrast, the immunoreactivity of eEF-2 kinase continued to decrease. These data suggest that tissue-specific isoforms of eEF-2 kinase may exist and that these isoforms may be regulated by growth factors.

A model is proposed uniting two groups of facts: The adsorption of enzymes on subcellular structures and the direct (‘from hand to hand’) transfer of metabolites between enzymes. The basic idea is that the binding of metabolites (substrates and/or products) results in desorption of the enzymes from subcellular structures during each catalytic act. This makes the enzymes mobile and capable of directly (from hand to hand) transferring metabolites to other enzymes adsorbed on subcellular structures. The model leads to a mechanism by means of which soluble enzymes can be compartmentalized in defined regions of the cytoplasm.

Several aminoacyl-tRNA synthetases of higher eukaryotes have always been found as multienzyme complexes. There are indications that these complexes can be associated with some tRNA-modifying enzymes. The function of such complexes is unclear. I have noticed that 6 out of 7 aminoacyl-tRNA synthetases most commonly occurring in complexes correspond to a group of tRNAs which must always contain a modified U in the first position of their anticodons. A hypothesis is proposed according to which association of 6 aminoacyl-tRNA synthetases with U-modifying enzymes can protect a cell from miscoding.

The nerve growth factor-sensitive phosphoprotein from PC12 cells, previously designated Nsp100, has been shown to be elongation factor 2 (EF-2). The criteria used for this identification include: (i) similarity of N-terminal sequence; (ii) phosphorylation by the same kinase; (iii) ADP-ribosylation mediated by diphtheria toxin; (iv) comparable function in cell-free protein synthesis. According to these criteria, Nsp 100 and EF-2 are identical and the kinase that phosphorylates Nsp100 in PC12 cells is calcium/calmodulin kinase III.