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It is imperative to develop novel therapeutic strategies for Alzheimer’s disease (AD) and related dementia syndromes based on solid mechanistic studies. Maintenance of memory and synaptic plasticity relies on de novo protein synthesis, which is partially regulated by phosphorylation of eukaryotic elongation factor 2 (eEF2) via its kinase eEF2K. Abnormally increased eEF2 phosphorylation and impaired mRNA translation have been linked to AD. We recently reported that prenatal genetic suppression of eEF2K is able to prevent aging-related cognitive deficits in AD model mice, suggesting the therapeutic potential of targeting eEF2K/eEF2 signaling in AD. Here, we tested two structurally distinct small-molecule eEF2K inhibitors in two different lines of AD model mice after onset of cognitive impairments. Our data revealed that treatment with eEF2K inhibitors improved AD-associated synaptic plasticity impairments and cognitive dysfunction, without altering brain amyloid β (Aβ) and tau pathology. Furthermore, eEF2K inhibition alleviated AD-associated defects in dendritic spine morphology, postsynaptic density formation, protein synthesis, and dendritic polyribosome assembly. Our results may offer critical therapeutic implications for AD, and the proof-of-principle study indicates translational implication of inhibiting eEF2K for AD and related dementia syndromes.One Sentence SummaryTreatment with eEF2K inhibitors and genetic eEF2K knockout improved cognitive deficits in Alzheimer’s disease model mice.

Molecular signaling mechanisms underlying Alzheimer’s disease (AD) remain unclear. Maintenance of memory and synaptic plasticity depend on de novo protein synthesis, dysregulation of which is implicated in AD. Recent studies showed AD-associated hyperphosphorylation of mRNA translation factor eukaryotic elongation factor 2 (eEF2), which results in inhibition of protein synthesis. We tested to determine whether suppression of eEF2 phosphorylation could improve protein synthesis capacity and AD-associated cognitive and synaptic impairments. Genetic reduction of the eEF2 kinase (eEF2K) in 2 AD mouse models suppressed AD-associated eEF2 hyperphosphorylation and improved memory deficits and hippocampal long-term potentiation (LTP) impairments without altering brain amyloid β (Aβ) pathology. Furthermore, eEF2K reduction alleviated AD-associated defects in dendritic spine morphology, postsynaptic density formation, de novo protein synthesis, and dendritic polyribosome assembly. Our results link eEF2K/eEF2 signaling dysregulation to AD pathophysiology and therefore offer a feasible therapeutic target.

Impaired mRNA translation (protein synthesis) is linked to Alzheimer's disease (AD) pathophysiology. Recent studies revealed the role of increased phosphorylation of eukaryotic elongation factor 2 (eEF2) in AD-associated cognitive deficits. Phosphorylation of eEF2 (at the Thr56 site) by its only known kinase eEF2K leads to inhibition of general protein synthesis. AD is considered as a disease of "synaptic failure" characterized by impairments of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Deficiency of metabotropic glutamate receptor 5-dependent LTD (mGluR-LTD) is indicated in cognitive syndromes associated with various neurological disorders, including AD, but the molecular signaling mechanisms underlying the mGluR-LTD dysregulation in AD remain unclear. In this brief communication, we report genetic repression of eEF2K in aged APP/PS1 AD model mice prevented AD-associated hippocampal mGluR-LTD deficits. Using a pharmacological approach, we further observed that impairments of mGluR-LTD in APP/PS1 mice were rescued by treating hippocampal slices with a small molecule eEF2K antagonist NH125. Our findings, taken together, suggest a critical role of abnormal protein synthesis dysregulation at the elongation phase in AD-associated mGluR-LTD failure, thus providing insights into a mechanistic understanding of synaptic impairments in AD and other related dementia syndromes.

The normal aging process is commonly associated with mild cognitive deficits including memory decline. Previous studies indicate a role of dysregulated messenger ribonucleic acid translation capacity in cognitive defects associated with aging and aging-related diseases, including hyperphosphorylation of eukaryotic elongation factor 2 (eEF2). Phosphorylation of eEF2 by the kinase eEF2K inhibits its activity, hindering general protein synthesis. Here, we sought to determine whether cognitive deficits in aged mice can be improved by genetically deleting eEF2K (eEF2K KO) and consequently reduction of eEF2 phosphorylation. We found that suppression of eEF2K prevented aging-related deficits in novel object recognition memory. Interestingly, deletion of eEF2K did not alter overall protein synthesis in the hippocampus. Ultrastructural analysis revealed increase size and larger active zone lengths of postsynaptic densities in the hippocampus of aged eEF2K KO mice. Biochemical assays showed hippocampal eIF2α hyperphosphorylation in aged eEF2K KO mice, indicating inhibition of translation initiation. Our findings may provide insight into mechanistic understanding and thus development of novel therapeutic strategies for aging-related cognitive decline.

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.

Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg 2+ )/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase–deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg 2+ was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups ( P

Characterization of the molecular signaling pathways underlying protein synthesis-dependent forms of synaptic plasticity, such as late long-term potentiation (L-LTP), can provide insights not only into memory expression/maintenance under physiological conditions but also potential mechanisms associated with the pathogenesis of memory disorders. Here, we report in mice that L-LTP failure induced by the mammalian (mechanistic) target of rapamycin complex 1 (mTORC1) inhibitor rapamycin is reversed by brain-specific genetic deletion of PKR-like ER kinase, PERK (PERK KO), a kinase for eukaryotic initiation factor 2α (eIF2α). In contrast, genetic removal of general control non-derepressible-2, GCN2 (GCN2 KO), another eIF2α kinase, or treatment of hippocampal slices with the PERK inhibitor GSK2606414, does not rescue rapamycin-induced L-LTP failure, suggesting mechanisms independent of eIF2α phosphorylation. Moreover, we demonstrate that phosphorylation of eukaryotic elongation factor 2 (eEF2) is significantly decreased in PERK KO mice but unaltered in GCN2 KO mice or slices treated with the PERK inhibitor. Reduction in eEF2 phosphorylation results in increased general protein synthesis, and thus could contribute to the mTORC1-independent L-LTP in PERK KO mice. We further performed experiments on mutant mice with genetic removal of eEF2K (eEF2K KO), the only known kinase for eEF2, and found that L-LTP in eEF2K KO mice is insensitive to rapamycin. These data, for the first time, connect reduction in PERK activity with the regulation of translation elongation in enabling L-LTP independent of mTORC1. Thus, our findings indicate previously unrecognized levels of complexity in the regulation of protein synthesis-dependent synaptic plasticity. Read the Editorial Highlight for this article on page 119. Cover Image for this issue: doi: 10.1111/jnc.14185.

Inactivation of APC is a strongly predisposing event in the development of colorectal cancer, prompting the search for vulnerabilities specific to cells that have lost APC function. Signalling through the mTOR pathway is known to be required for epithelial cell proliferation and tumour growth, and the current paradigm suggests that a critical function of mTOR activity is to upregulate translational initiation through phosphorylation of 4EBP1 (refs 6, 7). This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 (ref. 8), would be ineffective in limiting cancer progression in APC-deficient lesions. Here we show in mice that mTOR complex 1 (mTORC1) activity is absolutely required for the proliferation of Apc-deficient (but not wild-type) enterocytes, revealing an unexpected opportunity for therapeutic intervention. Although APC-deficient cells show the expected increases in protein synthesis, our study reveals that it is translation elongation, and not initiation, which is the rate-limiting component. Mechanistically, mTORC1-mediated inhibition of eEF2 kinase is required for the proliferation of APC-deficient cells. Importantly, treatment of established APC-deficient adenomas with rapamycin (which can target eEF2 through the mTORC1-S6K-eEF2K axis) causes tumour cells to undergo growth arrest and differentiation. Taken together, our data suggest that inhibition of translation elongation using existing, clinically approved drugs, such as the rapalogs, would provide clear therapeutic benefit for patients at high risk of developing colorectal cancer.

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 is an unusual bifunctional protein consisting of an α-kinase domain fused to a TRP ion channel. Previously, we have identified annexin A1 as a substrate for TRPM7 kinase and found that TRPM7 phosphorylates annexin A1 at Ser5 within the N-terminal α-helix. Annexin A1 is a Ca(2+)-dependent membrane binding protein, which has been implicated in membrane trafficking and reorganization. The N-terminal tail of annexin A1 can interact with either membranes or S100A11 protein, and it adopts the conformation of an amphipathic α-helix upon these interactions. Moreover, the existing evidence indicates that the formation of an α-helix is essential for these interactions. Here we show that phosphorylation at Ser5 prevents the N-terminal peptide of annexin A1 from adopting an α-helical conformation in the presence of membrane-mimetic micelles as well as phospholipid vesicles. We also show that phosphorylation at Ser5 dramatically weakens the binding of the peptide to S100A11. Our data suggest that phosphorylation at Ser5 regulates the interaction of annexin A1 with membranes as well as S100A11 protein.

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 bi-functional protein containing an ion channel covalently linked to a protein kinase domain. TRPM7 is implicated in regulating cellular and systemic magnesium homeostasis. While the biophysical properties of TRPM7 ion channel and its function are relatively well characterized, the function of the TRPM7 enzymatically active kinase domain is not understood yet. To investigate the physiological role of TRPM7 kinase activity, we constructed mice carrying an inactive TRPM7 kinase. We found that these mice were resistant to dietary magnesium deprivation, surviving three times longer than wild type mice; also they displayed decreased chemically induced allergic reaction. Interestingly, mutant mice have lower magnesium bone content compared to wild type mice when fed regular diet; unlike wild type mice, mutant mice placed on magnesium-depleted diet did not alter their bone magnesium content. Furthermore, mouse embryonic fibroblasts isolated from TRPM7 kinase-dead animals exhibited increased resistance to magnesium deprivation and oxidative stress. Finally, electrophysiological data revealed that the activity of the kinase-dead TRPM7 channel was not significantly altered. Together, our results suggest that TRPM7 kinase is a sensor of magnesium status and provides coordination of cellular and systemic responses to magnesium deprivation.

Recently we identified a new class of protein kinases with a novel type of catalytic domain structurally and evolutionarily unrelated to the conventional eukaryotic protein kinases. This new class, which we named alpha-kinases, is represented by eukaryotic elongation factor-2 kinase and the Dictyosteliummyosin heavy chain kinases. Here we cloned, sequenced, and analyzed the tissue distribution of five new putative mammalian α-kinases: melanoma α-kinase, kidney α-kinase, heart α-kinase, skeletal muscle α-kinase, and lymphocyte α-kinase. All five are large proteins of more than 1000 amino acids with an α-kinase catalytic domain located in the carboxyterminal part. We expressed the catalytic domain of melanoma α-kinase in Escherichia coli, and found that it autophosphorylates at threonine residues, demonstrating that it is a genuine protein kinase. Unexpectedly, we found that long aminoterminal portions of melanoma and kidney α-kinases represent new members of the TRP ion channel family, which are thought to mediate the capacitative Ca2+entry in nonexcitable mammalian cells. This suggests that melanoma and kidney α-kinases, which represent a novel type of signaling molecule, are involved in the regulation of Ca2+influx in mammalian cells.

A new class of eukaryotic protein kinases that are not homologous to members of the serine/threonine/tyrosine protein kinase superfamily was recently identified [Futey, L. M., et al. (1995) J. Biol. Chem. 270, 523-529; Ryazanov, A. G., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4884-4889]. This class includes eukaryotic elongation factor-2 kinase, Dictyostelium myosin heavy chain kinases A, B, and C, and several mammalian putative protein kinases that are not yet fully characterized [Ryazanov, A. G., et al. (1999) Curr. Biol. 9, R43-R45]. eEF-2 kinase is a ubiquitous protein kinase that phosphorylates and inactivates eukaryotic translational elongation factor-2, and thus can modulate the rate of polypeptide chain elongation during translation. eEF-2 was the only known substrate for eEF-2 kinase. We demonstrate here that eEF-2 kinase can efficiently phosphorylate a 16-amino acid peptide, MH-1, corresponding to the myosin heavy chain kinase A phosphorylation site in Dictyostelium myosin heavy chains. This enabled us to develop a rapid assay for eEF-2 kinase activity. To localize the functional domains of eEF-2 kinase, we expressed human eEF-2 kinase in Escherichia coli as a GST-tagged fusion protein, and then performed systematic in vitro deletion mutagenesis. We analyzed eEF-2 kinase deletion mutants for the ability to autophosphorylate, and to phosphorylate eEF-2 as well as a peptide substrate, MH-1. Mutants with deletions between amino acids 51 and 335 were unable to autophosphorylate, and were also unable to phosphorylate eEF-2 and MH-1. Mutants with deletions between amino acids 521 and 725 were unable to phosphorylate eEF-2, but were still able to autophosphorylate and to phosphorylate MH-1. The kinases with deletions between amino acids 2 and 50 and 336 and 520 were able to catalyze all three reactions. In addition, the C-terminal domain expressed alone (amino acids 336-725) binds eEF-2 in a coprecipitation assay. These results suggest that eEF-2 kinase consists of two domains connected by a linker region. The amino-terminal domain contains the catalytic domain, while the carboxyl-terminal domain contains the eEF-2 targeting domain. The calmodulin-binding region is located between amino acids 51 and 96. The amino acid sequence of the carboxyl-terminal domain of eEF-2 kinase displays similarity to several proteins, all of which contain repeats of a 36-amino acid motif that we named "motif 36".

A high-resolution map of human phosphorylation networks was constructed by integrating experimentally determined kinase-substrate relationships with other resources, such as in vivo phosphorylation sites., High-quality kinase-substrate relationships (KSRs) were determined using an integrated approach that combines protein microarray technology and bioinformatics analysis. Phosphorylation motifs were predicted for 284 human kinases, representing 55% of the human kinome. A high-resolution map of human phosphorylation networks was constructed that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. A new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling was discovered based on KSRs identified in the phosphorylation networks., The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.

Protein translation is an essential but energetically expensive process, which is carefully regulated in accordance to the cellular nutritional and energy status. Eukaryotic elongation factor 2 (eEF2) is a central regulation point since it mediates ribosomal translocation and can be inhibited by phosphorylation at Thr56. TRPM7 is the unique fusion of an ion channel with a functional Ser/Thr-kinase. While TRPM7's channel function has been implicated in regulating vertebrate Mg2+ uptake required for cell growth, the function of its kinase domain remains unclear. Here, we show that under conditions where cell growth is limited by Mg2+ availability, TRPM7 via its kinase mediates enhanced Thr56 phosphorylation of eEF2. TRPM7-kinase does not appear to directly phosphorylate eEF2, but rather to influence the amount of eEF2's cognate kinase eEF2-k, involving its phosphorylation at Ser77. These findings suggest that TRPM7's structural duality ensures ideal positioning of its kinase in close proximity to channel-mediated Mg2+ uptake, allowing for the adjustment of protein translational rates to the availability of Mg2+.

Abnormal angiogenesis is central to the pathophysiology of diverse disease processes including cancers, ischemic and atherosclerotic heart disease, and visually debilitating eye disease. Resveratrol is a naturally occurring phytoalexin that has been demonstrated to ameliorate and decelerate the aging process as well as blunt end organ damage from obesity. These effects of resveratrol are largely mediated by members of the sirtuin family of proteins. We demonstrate that resveratrol can inhibit pathological angiogenesis in vivo and in vitro by a sirtuin-independent pathway. Resveratrol inhibits the proliferation and migration of vascular endothelial cells by activating eukaryotic elongation factor-2 kinase. The active kinase in turn phosphorylates and inactivates elongation factor-2, a key mediator of ribosomal transfer and protein translation. Functional inhibition of the kinase by gene deletion in vivo or RNA as well as pharmacological inhibition in vitro is able to completely reverse the effects of resveratrol on blood vessel growth. These studies have identified a novel and critical pathway that promotes aberrant vascular proliferation and one that is amenable to modulation by pharmacological means. In addition, these results have uncovered a sirtuin-independent pathway by which resveratrol regulates angiogenesis.

Mg2+ is the second-most abundant cation in animal cells and is an essential cofactor in numerous enzymatic reactions. The molecular mechanisms controlling Mg2+ balance in the organism are not well understood. In this study, we report identification of TRPM7, a bifunctional protein containing a protein kinase fused to an ion channel, as a key regulator of whole body Mg2+ homeostasis in mammals. We generated TRPM7-deficient mice with the deletion of the kinase domain. Homozygous TRPM7Δkinase mice demonstrated early embryonic lethality, whereas heterozygous mice were viable, but developed signs of hypomagnesaemia and revealed a defect in intestinal Mg2+ absorption. Cells derived from heterozygous TRPM7Δkinase mice demonstrated reduced TRPM7 currents that had increased sensitivity to the inhibition by Mg2+. Embryonic stem cells lacking TRPM7 kinase domain displayed a proliferation arrest phenotype that can be rescued by Mg2+ supplementation. Our results demonstrate that TRPM7 is essential for the control of cellular and whole body Mg2+ homeostasis., Magnesium is an essential element of the diet and is a cofactor for many enzymes. In this study, the channel kinase TRPM7 is shown to be essential for magnesium homeostasis, and heterozygous mice lacking the kinase domain show a defect in absorption of magnesium from the diet.

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)

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.

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.

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 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.