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Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

Abstract In this study, we conducted a comparative analysis of the structure of agonists and antagonists of transmembrane (TM) β-adrenoceptors (β-ARs) and their interactions with the β-ARs and proposed the mechanism of receptor activation. A characteristic feature of agonist and antagonist molecules is the presence of a hydrophobic head (most often, one or two aromatic rings) and a tail with a positively charged amino group. All β-adrenergic agonists have two carbon atoms between the aromatic ring of the head and the nitrogen atom of the amino group. In antagonist molecules, this fragment can be either reduced or increased to four atoms due to the additional carbon and oxygen atoms. The agonist head, as a rule, has two H-bond donors or acceptors in the para- and meta-positions of the aromatic rings, while in the antagonist heads, these donors/acceptors are absent or located in other positions. Analysis of known three-dimensional structures of β-AR complexes with agonists showed that the agonist head forms two H-bonds with the TM5 helix, and the tail forms an ionic bond with the D3.32 residue of the TM3 helix and one or two H-bonds with the TM7 helix. The tail of the antagonist can form similar bonds, but the interaction between the head and the TM5 helix is much weaker. As a result of these interactions, the agonist molecule acquires an extended “strained string” conformation, in contrast to the antagonist molecule, which has a longer, bended, and flexible tail. The “strained string” of the agonist interacts with the TM6 helix (primarily with the W6.48 residue) and turns it, which leads to the opening of the G protein-binding site on the intracellular side of the receptor, while flexible and larger antagonist molecules do not have the same effect on the receptor.

Hyperaldosteronism causes cardiovascular disease as well as hypomagnesemia. Mechanisms are ill-defined but dysregulation of TRPM7, a Mg2+-permeable channel/α-kinase, may be important. We examined the role of TRPM7 in aldosterone-dependent cardiovascular and renal injury by studying aldosterone-salt treated TRPM7-deficient (TRPM7+/Δkinase) mice. Plasma/tissue [Mg2+] and TRPM7 phosphorylation were reduced in vehicle-treated TRPM7+/Δkinase mice, effects recapitulated in aldosterone-salt-treated wild-type mice. Aldosterone-salt treatment exaggerated vascular dysfunction and amplified cardiovascular and renal fibrosis, with associated increased blood pressure in TRPM7+/Δkinase mice. Tissue expression of Mg2+-regulated phosphatases (PPM1A, PTEN) was downregulated and phosphorylation of Smad3, ERK1/2, and Stat1 was upregulated in aldosterone-salt TRPM7-deficient mice. Aldosterone-induced phosphorylation of pro-fibrotic signaling was increased in TRPM7+/Δkinase fibroblasts, effects ameliorated by Mg2+ supplementation. TRPM7 deficiency amplifies aldosterone-salt-induced cardiovascular remodeling and damage. We identify TRPM7 downregulation and associated hypomagnesemia as putative molecular mechanisms underlying deleterious cardiovascular and renal effects of hyperaldosteronism.

Maintenance of memory and synaptic plasticity depends on de novo protein synthesis, and accumulating evidence implicates a role of dysregulated mRNA translation in cognitive impairments associated with Alzheimer’s disease (AD). Accumulating evidence demonstrates hyper-phosphorylation of translation factor eukaryotic elongation factor 2 (eEF2) in the hippocampi of human AD patients as well as transgenic AD model mice. Phosphorylation of eEF2 (at the Thr 56 site) by its only known kinase, eEF2K, leads to inhibition of general protein synthesis. A recent study suggests that amyloid β (Aβ)-induced neurotoxicity could be associated with an interaction between eEF2 phosphorylation and the transcription factor nuclear erythroid 2-related factor (NRF2)-mediated antioxidant response. In this brief communication, we report that global homozygous knockout of the eEF2K gene alleviates deficits of long-term recognition and spatial learning in a mouse model of AD (APP/PS1). Moreover, eEF2K knockout does not alter brain Aβ pathology in APP/PS1 mice. The hippocampal NRF2 antioxidant response in the APP/PS1 mice, measured by expression levels of nicotinamide adenine dinucleotide plus hydrogen (NADPH) quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), is ameliorated by suppression of eEF2K signaling. Together, the findings may contribute to our understanding of the molecular mechanisms underlying AD pathogenesis, indicating that suppression of eEF2K activity could be a beneficial therapeutic option for this devastating neurodegenerative disease.

Protein phosphorylation is one of the most widespread post-translational modifications in biology. With the advent of mass spectrometry-based phosphoproteomics, more than 200,000 sites of serine and threonine phosphorylation have been reported, of which several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein Ser/Thr kinases encoded in the human genome is responsible. Here, we utilize synthetic peptide libraries to profile the substrate sequence specificity of nearly every functional human Ser/Thr kinase. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. Our kinome-wide dataset was used to computationally annotate and identify the most likely protein kinases for every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites where the protein kinases involved have been previously identified, our predictions were in excellent agreement. When this approach was applied to examine the signaling response of tissues and cell lines to hormones, growth factors, targeted inhibitors, and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the full extent of substrate specificity of the human Ser/Thr kinome, illuminate cellular signaling responses, and provide a rich resource to link unannotated phosphorylation events to biological pathways.

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 the 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, post-synaptic 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. Cover Image for this issue: https://doi.org/10.1111/jnc.15392.

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

Key points The Mg2+ and Ca2+ conducting transient receptor potential melastatin 7 (TRPM7) channel–enzyme (chanzyme) has been implicated in immune cell function. Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not. As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release. Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function. Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca2+ and extracellular Mg2+ sensitivity of mast cell degranulation. Abstract Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C-terminally located α-kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7+/∆K) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7KR), are not hyperallergic and are resistant to systemic magnesium (Mg2+) deprivation. Since allergic reactions are triggered by mast cell-mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild-type (TRPM7+/+), TRPM7+/∆K and TRPM7KR mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg2+ assured unperturbed IgE-DNP-dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE-DNP-dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca2+) in G protein-induced exocytosis. In addition, G protein-coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg2+ caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca2+ and affecting granular mobility and/or histamine contents.

Transient Receptor Potential Melastatin 7 (TRPM7) cation channel is a chanzyme (channel + kinase) that influences cellular Mg2+ homeostasis and vascular signalling. However, the pathophysiological significance of TRPM7 in the cardiovascular system is unclear. The aim of this study was to investigate the role of this chanzyme in the cardiovascular system focusing on inflammation and fibrosis.TRPM7-deficient mice with deletion of the kinase domain (TRPM7+/Δkinase) were studied and molecular mechanisms investigated in TRPM7+/Δkinase bone marrow-derived macrophages (BMDM) and co-culture systems with cardiac fibroblasts. TRPM7-deficient mice had significant cardiac hypertrophy, fibrosis, and inflammation. Cardiac collagen and fibronectin content, expression of pro-inflammatory mediators (SMAD3, TGFβ) and cytokines [interleukin (IL)-6, IL-10, IL-12, tumour necrosis factor-α] and phosphorylation of the pro-inflammatory signalling molecule Stat1, were increased in TRPM7+/Δkinase mice. These processes were associated with infiltration of inflammatory cells (F4/80+CD206+ cardiac macrophages) and increased galectin-3 expression. Cardiac [Mg2+]i, but not [Ca2+]i, was reduced in TRPM7+/Δkinase mice. Calpain, a downstream TRPM7 target, was upregulated (increased expression and activation) in TRPM7+/Δkinase hearts. Vascular functional and inflammatory responses, assessed in vivo by intra-vital microscopy, demonstrated impaired neutrophil rolling, increased neutrophil: endothelial attachment and transmigration of leucocytes in TRPM7+/Δkinase mice. TRPM7+/Δkinase BMDMs had increased levels of galectin-3, IL-10, and IL-6. In co-culture systems, TRPM7+/Δkinase macrophages increased expression of fibronectin, proliferating cell nuclear antigen, and TGFβ in cardiac fibroblasts from wild-type mice, effects ameliorated by MgCl2 treatment.We identify a novel anti-inflammatory and anti-fibrotic role for TRPM7 and suggest that its protective effects are mediated, in part, through Mg2+-sensitive processes.

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.

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.

Mg2+ regulates many physiological processes and signalling pathways. However, little is known about the mechanisms underlying the organismal balance of Mg2+. Capitalizing on a set of newly generated mouse models, we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6. We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development. In adult mice, TRPM6 is required in the intestine to maintain organismal Mg2+ balance, but is dispensable in the kidney. Trpm6 inactivation in adult mice leads to a shortened lifespan, growth deficit and metabolic alterations indicative of impaired energy balance. Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice, but also may extend the lifespan of wildtype mice. Hence, maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood. DOI: http://dx.doi.org/10.7554/eLife.20914.001, eLife digest A balanced diet contains a variety of minerals such as magnesium ions, which are required for many chemical reactions in our body. A shortage of magnesium ions is linked to many diseases and is thought to be especially harmful to babies in the womb and shortly after birth. Magnesium ion deficiency is widespread in human populations and in the US is thought to affect up to 68% of people. Despite its prominent role in human health, our understanding of how the body maintains the right balance of magnesium ions remains extremely vague. Magnesium ions can enter and leave a cell by passing through specific types of proteins that form channels in the membrane surrounding the cell. There are thought to be around ten types of these magnesium ion channels in human cells, but we do not know what roles any of them perform in the body. One such channel called TRPM6 may be particularly important because mutations in the gene that encodes this channel can cause magnesium ion deficiency in human infants. However, the loss of TRPM6 in mice disrupts how mouse embryos develop, suggesting that our current view on the role that TRPM6 plays in regulating the magnesium ion balance in humans may be too simplistic. To address this question, Chubanov et al. studied mice with mutations that disrupted the production of TRPM6 in specific tissues only. The experiments show that TRPM6 primarily operates in the placenta and intestine to regulate the balance of magnesium ions in the body. Further experiments show that the loss of TRPM6 in adult mice leads to reduced lifespan, growth defects and poor health by disrupting important biochemical reactions. Supplying the mutant mice with magnesium ion supplements improved their health and could extend lifespans of normal animals. The findings of Chubanov et al. demonstrate that TRPM6 plays a crucial role in regulating the levels of magnesium ions in mice before birth and into adulthood. The next step is to carry out large-scale experiments to investigate the effects of altering the levels of magnesium ions in human diets. DOI: http://dx.doi.org/10.7554/eLife.20914.002

Magnesium ion (Mg(2+)) is the fourth most common cation in the human body, and has a crucial role in many physiological functions. Mg(2+) homeostasis is an important contributor to bone development, however, its roles in the development of dental mineralized tissues have not yet been well known. We identified that transient receptor potential cation channel, subfamily M, member 7 (TRPM7), was significantly upregulated in the mature ameloblasts as compared to other ameloblasts through our whole transcript microarray analyses of the ameloblasts. TRPM7, an ion channel for divalent metal cations with an intrinsic serine/threonine protein kinase activity, has been characterized as a key regulator of whole body Mg(2+) homeostasis. Semi-quantitative PCR and immunostaining for TRMP7 confirmed its upregulation during the maturation stage of enamel formation, at which ameloblasts direct rapid mineralization of the enamel matrix. The significantly hypomineralized craniofacial structures, including incisors, molars, and cranial bones were demonstrated by microCT analysis, von Kossa and trichrome staining in Trpm7 (Δkinase∕+) mice. A previously generated heterozygous mouse model with the deletion of the TRPM7 kinase domain. Interestingly, the skeletal phenotype of Trpm7 (Δkinase∕+) mice resembled those found in the tissue-nonspecific alkaline phosphatase (Alpl) KO mice, thus we further examined whether ALPL protein content and alkaline phosphatase (ALPase) activity in ameloblasts, odontoblasts and osteoblasts were affected in those mice. While ALPL protein in Trpm7 (Δkinase∕+) mice remained at the similar level as that in wt mice, ALPase activities in the Trpm7 (Δkinase∕+) mice were almost nonexistent. Supplemented magnesium successfully rescued the activities of ALPase in ameloblasts, odontoblasts and osteoblasts of Trpm7 (Δkinase∕+) mice. These results suggested that TRPM7 is essential for mineralization of enamel as well as dentin and bone by providing sufficient Mg(2+) for the ALPL activity, underlining the key importance of ALPL for biomineralization.

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.

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.

The Mg(2+) and Ca(2+) conducting transient receptor potential melastatin 7 (TRPM7) channel-enzyme (chanzyme) has been implicated in immune cell function. Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not. As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release. Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function. Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca(2+) and extracellular Mg(2+) sensitivity of mast cell degranulation.Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C-terminally located α-kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7(+/∆K) ) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7(KR) ), are not hyperallergic and are resistant to systemic magnesium (Mg(2+) ) deprivation. Since allergic reactions are triggered by mast cell-mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild-type (TRPM7(+/+) ), TRPM7(+/∆K) and TRPM7(KR) mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg(2+) assured unperturbed IgE-DNP-dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE-DNP-dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca(2+) ) in G protein-induced exocytosis. In addition, G protein-coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg(2+) caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca(2+) and affecting granular mobility and/or histamine contents.

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 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.

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.

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".

Double-stranded RNA interference (RNAi) is an effective method for disrupting expression of specific genes in Caenorhabditis elegans and other organisms. Applications of this reverse-genetics tool, however, are somewhat restricted in nematodes because introduced dsRNA is not stably inherited. Another difficulty is that RNAi disruption of late-acting genes has been generally less consistent than that of embryonically expressed genes, perhaps because the concentration of dsRNA becomes lower as cellular division proceeds or as developmental time advances. In particular, some neuronally expressed genes appear refractory to dsRNA-mediated interference. We sought to extend the applicability of RNAi by in vivo expression of heritable inverted-repeat (IR) genes. We assayed the efficacy of in vivo-driven RNAi in three situations for which heritable, inducible RNAi would be advantageous: (i) production of large numbers of animals deficient for gene activities required for viability or reproduction; (ii) generation of large populations of phenocopy mutants for biochemical analysis; and (iii) effective gene inactivation in the nervous system. We report that heritable IR genes confer potent and specific gene inactivation for each of these applications. We suggest that a similar strategy might be used to test for dsRNA interference effects in higher organisms in which it is feasible to construct transgenic animals, but impossible to directly or transiently introduce high concentrations of dsRNA.

The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of ≈200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.

Transient receptor potential melastatin 7 (TRPM7) cation channel is a unique protein that has the dual ability to act as a channel to regulate transmembrane Mg 2+ transport and also as a kinase to promote cellular signaling. Despite increasing awareness of the importance of Mg 2+ in cardiovascular biology nothing is known about TRPM7 and its kinase domain in the pathophysiology of hypertension. We previously demonstrated that Ang II regulates TRPM7 in vitro . Here we studied TRPM7 kinase-deficient mice to explore the role of the TRPM7 kinase domain in Ang II-induced hypertension. TRPM7 kinase deficient mice (TRPM7 +/- ) and wild type (WT) counterparts were infused with Ang II (400 ng/kg/min; minipumps) for 4 weeks. Blood pressure (BP) was measured by tail cuff. Vascular reactivity and structure studies were performed by myography in mesenteric arteries. Although baseline BP tended to be higher in TRPM7 +/- versus WT mice (127 ± 6.0 vs 119 ± 2.2 mmHg), significance was not achieved. TRPM7 +/- mice displayed earlier onset of BP increase by Ang II (2 weeks; WT-Ang II: 145 ± 5 vs TRPM7 +/- Ang II: 178 ± 9; mmHg). After 4 weeks, BP was significantly higher in TRPM7 +/- (174 ± 10 mmHg) than in WT mice (147 ± 8 mmHg). Ang II-induced hypertension was associated with cardiac hypertrophy, an effect that was exaggerated in TRPM7 +/- mice (WT: 4.4 ± 0.1; WT-Ang II: 5.0 ± 0.2; TRPM7 +/- : 4.5 ± 0.1; TRPM7 +/- Ang II: 5.7 ± 0.1 g/body weight). Mesenteric arteries from Ang II-infused TRPM7 +/- mice exhibited decreased sensitivity to acetylcholine (pD2; WT-Ang II: 7.6 ± 0.3 vs. TRPM7 +/- Ang II: 6.7 ± 0.4), and reduced maximal relaxation compared to WT mice (WT-Ang II: 88 ± 8% vs TRPM7 +/- Ang II: 59 ± 10%). Ang II induced a leftward shift in the stress-strain relationship for both WT and TRPM7 +/- mice in a similar fashion. Plasma analysis revealed that TRPM7 +/- mice were hypomagnesemic, and that Ang II increased Mg 2+ levels to a greater extent in WT than in TRPM7 +/- mice (WT: 0.65 ± 0.02; WT-Ang II: 0.74 ± 0.04; TRPM7 +/- : 0.60 ± 0.01; TRPM7 +/- Ang II: 0.64 ± 0.02; mmol/L). In conclusion, our findings demonstrate that hypertension, cardiac hypertrophy and endothelial dysfunction are exaggerated by Ang II in TRPM7 +/- hypomagnesemic mice, suggesting a novel role for TRPM7 kinase domain in cardiovascular pathophysiology.

Transient receptor potential melastatin 7 (TRPM7) cation channel is a unique protein, which regulates transmembrane magnesium (Mg2+) transport through its channel domain, and intracellular signaling through its kinase domain. We previously demonstrated that TRPM7, is expressed on vascular smooth muscle cells (VSMC), controls Mg2+ influx, and is regulated by angiotensin II (Ang II). Low Mg2+ levels are among the many factors leading to hypertension, where intracellular Mg2+ deficiency and TRPM7 downregulation is observed. Despite the advancement in the understanding of Mg2+ and TRPM7 biology, the role of TRPM7, and its kinase, in hypertension remains elusive. In this study, we investigated whether deletion of TRPM7 kinase domain alters Ang II-induced Mg2+ influx and signaling in VSMCs. Cultured VSMCs from mesenteric arteries obtained from heterozygous mice for the TRPM7 kinase deletion (+/-) and WT mice were used. Mg2+ influx was assessed by fluorescence microscopy. Activation of MAP kinases was evaluated by immunoblotting. Ang II (10-7M) increase in Mg2+ influx was similar in VSMC from both, WT and TRPM7+/- mice (2.5-fold; p

Proteins in cells are constantly undergoing damage. Nevertheless, the concentration of damaged proteins in young organisms is maintained at a low level owing to the continual protein turnover: breakdown of damaged proteins and synthesis of new ones. During aging, the concentration of damaged proteins increases because of decelerating protein turnover, the cause of which is unknown; however, it may be related to the decrease in ribosome concentration.

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.

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.

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.

Introduction The loss of Apc, causing Wnt-mediated epithelial proliferation, is an early event in colorectal cancer (CRC) development. This hyperproliferative state requires signalling though the mTOR pathway, with the current paradigm suggesting that upregulation of translation initiation via phosphorylation of 4EBP1 is crucial. This model predicts that the mTOR inhibitor rapamycin, which does not efficiently inhibit 4EBP1 function, would be ineffective in limiting development and progression of intestinal adenomas. Methods The inducible in vivo mouse models Lgr5Cre ER and VillinCre ER were used to selectively flox genes from intestinal stem cells and crypts respectively. mTOR complex 1 signalling was inhibited in Apc fl/fl mice either by rapamycin treatment or co-floxing the mTORC1 essential component Raptor. Translational status was assessed by sucrose gradient ultracentrifugation of intestinal epithelial extract from these mice and 35 S methionine incorporation and harringtonine chase assays on organoid cultures. The role of downstream mTORC1 effectors was established by assessing the intestinal regeneration following IR irradiation of 4EBP1/2 DKO , S6K1/2 DKO , rpS6 mut and eEF2k -/- mice. Survival studies for Apc fl/fl mice treated with rapamycin were performed both prior to, and on development of, symptoms Results mTORC1 activity is absolutely required for the proliferation of Apc deficient, but not wild type, intestinal crypts. Surprisingly, although protein synthesis is increased in Apc fl/fl crypts, it is translation elongation and not initiation that is the rate limiting step. Mechanistically, the inhibition of eukaryotic elongation factor (eEF2) kinase, to increase eEF2 activity downstream of mTORC1 and S6K is required for Wnt-mediated proliferation after IR irradiation. Treatment of established Apc fl/fl adenomas with rapamycin (which inhibits the mTORC1-S6K-eEF2k-eEF2 axis) arrests tumour growth and prolongs life. Furthermore, rapamycin treatment of mice immediately following homozygous Apc loss prevents the onset of symptoms. Conclusion These data show that intestinal adenoma formation and growth requires an mTOR mediated increase in translation elongation. Treatment of patients at high risk of developing CRC, such as those with Familial Adenomatous Polyposis, with Rapalogs may therefore be of therapeutic value. Disclosure of Interest None Declared.

Although the molecular mechanism of aging is unknown, a progressive increase with age in the concentration of damaged macromolecules, especially proteins, is likely to play a central role in senescent decline. In this paper, we discuss evidence that the progressive decrease in protein synthesis and turnover can be the primary cause of the increase in the concentration of damaged proteins with age. Conversely, protein damage itself is likely to be the cause of the decrease in protein turnover. This could establish a positive feedback loop where the increase in protein damage decreases the protein turnover rate, leading to a further increase in the concentration of damaged proteins. The establishment of such a feedback loop should result in an exponential increase in the amount of protein damage-a protein damage catastrophe-that could be the basis of the general deterioration observed in senescent organisms.