Impact of genetic variation on human CaMKK2 regulation by Ca2+ -calmodulin and multisite phosphorylation

The Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) is a key regulator of neuronal function and whole-body energy metabolism. Elevated CaMKK2 activity is strongly associated with prostate and hepatic cancers, whereas reduced CaMKK2 activity has been linked to schizophrenia and bipolar disease in humans. Here we report the functional effects of nine rare-variant point mutations that were detected in large-scale human genetic studies and cancer tissues, all of which occur close to two regulatory phosphorylation sites and the catalytic site on human CaMKK2. Four mutations (G87R, R139W, R142W and E268K) cause a marked decrease in Ca2+-independent autonomous activity, however S137L and P138S mutants displayed increased autonomous and Ca2+-CaM stimulated activities. Furthermore, the G87R mutant is defective in Thr85-autophosphorylation dependent autonomous activity, whereas the A329T mutation rendered CaMKK2 virtually insensitive to Ca2+-CaM stimulation. The G87R and R139W mutants behave as dominant-negative inhibitors of CaMKK2 signaling in cells as they block phosphorylation of the downstream substrate AMP-activated protein kinase (AMPK) in response to ionomycin. Our study provides insight into functionally disruptive, rare-variant mutations in human CaMKK2, which have the potential to influence risk and burden of disease associated with aberrant CaMKK2 activity in human populations carrying these variants

The autophagy initiator ULK1 sensitizes AMPK to allosteric drugs

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the β1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a β1-Ser108 kinase in cells. Cellular β1-Ser108 phosphorylation by ULK1 was dependent on AMPK β-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation

An AMPKa2-specific phospho-switch controls lysosomal targeting for activation

AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are metabolic kinases that co-ordinate nutrient supply with cell growth. AMPK negatively regulates mTORC1, and mTORC1 reciprocally phosphorylates S345/7 in both AMPK α-isoforms. We report that genetic or torin1-induced loss of α2-S345 phosphorylation relieves suppression of AMPK signaling; however, the regulatory effect does not translate to α1-S347 in HEK293T or MEF cells. Dephosphorylation of α2-S345, but not α1-S347, transiently targets AMPK to lysosomes, a cellular site for activation by LKB1. By mass spectrometry, we find that α2-S345 is basally phosphorylated at 2.5-fold higher stoichiometry than α1-S347 in HEK293T cells and, unlike α1, phosphorylation is partially retained after prolonged mTORC1 inhibition. Loss of α2-S345 phosphorylation in endogenous AMPK fails to sustain growth of MEFs under amino acid starvation conditions. These findings uncover an α2-specific mechanism by which AMPK can be activated at lysosomes in the absence of changes in cellular energy

Blocking AMPK β1 myristoylation enhances AMPK activity and protects mice from high-fat diet-induced obesity and hepatic steatosis

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis and a therapeutic target for metabolic diseases. Co/post-translational N-myristoylation of glycine-2 (Gly2) of the AMPK β subunit has been suggested to regulate the distribution of the kinase between the cytosol and membranes through a “myristoyl switch” mechanism. However, the relevance of AMPK myristoylation for metabolic signaling in cells and in vivo is unclear. Here, we generated knockin mice with a Gly2-to-alanine point mutation of AMPKβ1 (β1-G2A). We demonstrate that non-myristoylated AMPKβ1 has reduced stability but is associated with increased kinase activity and phosphorylation of the Thr172 activation site in the AMPK α subunit. Using proximity ligation assays, we show that loss of β1 myristoylation impedes colocalization of the phosphatase PPM1A/B with AMPK in cells. Mice carrying the β1-G2A mutation have improved metabolic health with reduced adiposity, hepatic lipid accumulation, and insulin resistance under conditions of high-fat diet-induced obesity

Recent Advances in Functional Fabric‐Based Wearable Supercapacitors

Abstract With the advent of wearable electronics, the urge to devise new and flexible energy storage devices to power up wearable systems has steadily risen over the past few decades. Wearable fabric‐based supercapacitors have emerged as a fantastic solution for powering up these systems. Functionalizing fabric surfaces with electroactive materials has proven to be the ideal way to fabricate high‐performance wearable supercapacitors. In this review, the recent progress in functional fabric‐based wearable supercapacitors is summarized. The article begins with the introduction of different fabric structures and outlining the functional materials implemented in wearable supercapacitors. The emphasis shifts toward summarizing the fabrication of functional fabric‐based electrodes according to different fabric architectures. Then, different novel fabric‐based supercapacitor configurations, the current state of integration, and the durability of such devices are analyzed. The review is concluded by emphasizing the existing drawbacks, envisaging potential applications, and, most importantly, the future perspective of the technology

Exploring the signalling mechanism of excitotoxic neuronal injury by molecular and quantitative proteomic approaches

© 2016 Dr. Md Ashfaqul HoqueExcitotoxicity caused by over-stimulation of the ionotropic glutamate receptors is a key neuronal cell death process underpinning brain damage in acute and chronic neurological disorders such as ischaemic stroke, traumatic brain injury, and neurodegenerative diseases. Exactly how neurons die in excitotoxicity still remains unclear and is an important area of research in the field of neuroscience. In my PhD study, I employed the targeted biochemical and molecular approaches and the unbiased mass spectrometry-based systems biology approaches to address this outstanding question. The protein tyrosine kinase Src, which co-localises with and phosphorylates the ionotropic glutamate receptor, is implicated in regulation of neuronal survival in excitotoxicity. Previous study from our laboratory demonstrated that upon excitotoxic stimulation Src is cleaved by the Ca2+-dependent cysteine protease calpain in the unique domain to form a ~52-kDa truncated Src fragment (Src∆N). Lentiviral expression of this recombinant Src∆N in cultured neurons revealed that Src∆N is neurotoxic and its expression inhibits the Akt signalling pathway. A cell-membrane permeable fusion peptide (Tat-Src) derived from the unique domain of Src was found to prevent Src cleavage by calpain and also protects against excitotoxic neuronal death (Hossain et al., 2013). These findings indicate that calpain cleavage of Src is a key event directing neuronal death in excitotoxicity. As a logical continuation, I employed the targeted biochemical and molecular approaches to decipher the mechanism by which Src governs neuronal survival. Treatment with specific Src kinase inhibitor (A419259) and knockdown of Src using shRNA significantly reduced neuronal viability and caused inactivation of the pro-survival protein kinase Erk1/2, indicating that intact Src is indispensable for neuronal survival and it exerts its pro-survival function in part by activating Erk1/2. Based upon these results we conclude that intact Src performs neurotrophic function under normal physiological conditions and it is converted by calpain cleavage into a potent cell death executor in excitotoxicity. Exactly how Src∆N exerts its neurotoxic action in excitotoxicity is not fully clear. Next, I sought to define the neurotoxic mechanism of Src∆N and understand how Src∆N interplays with other neuronal signalling proteins to direct neuronal death in excitotoxicity. To this end, I employed the unbiased quantitative proteomics-based systems biology approaches to identify the neuronal proteins of which the abundance and/or phosphorylation are significantly perturbed in excitotoxicity. Using the stable-isotope dimethyl labelling-based quantitative proteomic method, I was able to identify at least 80 neuronal proteins with significantly perturbed expression in cultured primary cortical neurons after 15 min or 4 h of glutamate-induced excitotoxicity. Most of these proteins exhibited decreased expression in excitotoxicity. Signalling network analysis using the Ingenuity Pathway Analysis (IPA) software to ascertain the potential functions of the identified neuronal proteins revealed the following canonical pathways as the top perturbed signalling pathways in excitotoxicity: (i) 14-3-3-mediated signalling, (ii) remodelling of epithelial adherens junctions, (iii) cell cycle including G2/M DNA damage checkpoint regulation, (iv) Myc-mediated apoptosis signalling, (v) PI3K/Akt signalling and (vi) Erk/MAPK signalling pathways. Additionally, I performed quantitative phosphoproteomic analysis and identified 59 neuronal proteins showing significant changes in phosphorylation in either one or more phosphorylation sites upon over-stimulation by glutamate for 15 min and 4 h. Most of the identified phosphoproteins showed decreased phosphorylation, implying that inactivation of the upstream kinases or activation of the specific phosphatases targeting those phosphorylation sites as the causes for the decreased phosphorylation. In agreement with the pathway and network analysis, Western blot analysis also confirmed that phosphorylation of Erk1/2 and Akt are perturbed in excitotoxicity. My results collectively indicate that inactivation of a number of pro-survival signalling pathways and activation of a series of pro-death signalling pathways cooperate to cause neuronal demise in excitotoxicity. Specific types of the N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and Kainate receptors are the ionotropic glutamate receptors from which the cytotoxic signals directing excitotoxic neuronal death originate. Among them, the extrasynaptic NMDA receptor is the major neurotoxic receptor directing excitotoxic neuronal death. Upon over-stimulation by glutamate, the extrasynaptic NMDA receptor allows massive influx of Ca2+ into the neuronal cytosol, where the excess Ca2+ over-activates a number of calcium-dependent enzymes. The calcium-dependent proteases calpains are among these over-activated enzymes. The over-activated calpains cleave a number of neuronal proteins such as Src to direct neuronal death. Since Ifenprodil, an antagonist of the extrasynaptic NMDA receptor, calpeptin, a cell-permeable pan-calpain inhibitor and Tat-Src have been shown to protect neurons against excitotoxic cell death, I therefore used them to further define the signalling networks underpinning excitotoxic neuronal death by quantitative global proteomic and phosphoproteomic approaches. Specifically, I identified specific neuronal proteins of which perturbation of expression and/or phosphorylation induced by glutamate treatment are offset by co-treatment with Ifenprodil, calpeptin and Tat-Src. Some of these neuronal proteins are likely downstream effectors mediating the neurotoxic signals of the extrasynaptic NMDA receptors, over-activated calpains and Src∆N. Several identified neuronal proteins were selected for validation of their perturbed expression and/or phosphorylation in excitotoxicity by Western blot and label-free full-scan precursor ions (MS1) quantitation analysis using isotopically labelled synthetic peptide standards. In summary, my findings shed light on the molecular mechanism of excitotoxic neuronal death. The neuronal proteins involved in excitotoxic neuronal death identified in my PhD project are potential targets for the development of neuroprotectants to reduce excitotoxic brain damage in neurological disorders

Mdivi-1 protects human W8B2+Cardiac stem cells from oxidative stress and simulated ischemia-reperfusion injury

Cardiac stem cell (CSC) therapy is a promising approach to treat ischemic heart disease. However, the poor survival of transplanted stem cells in the ischemic myocardium has been a major impediment in achieving an effective cell-based therapy against myocardial infarction. Inhibiting mitochondrial fission has been shown to promote survival of several cell types. However, the role of mitochondrial morphology in survival of human CSC remains unknown. In this study, we investigated whether mitochondrial division inhibitor-1 (Mdivi-1), an inhibitor of mitochondrial fission protein dynamin-related protein-1 (Drp1), can improve survival of a novel population of human W8B2+ CSCs in hydrogen peroxide (H2O2)-induced oxidative stress and simulated ischemia-reperfusion injury models. Mdivi-1 significantly reduced H2O2-induced cell death in a dose-dependent manner. This cytoprotective effect was accompanied by an increased proportion of cells with tubular mitochondria, but independent of mitochondrial membrane potential recovery and reduction of mitochondrial superoxide production. In simulated ischemia-reperfusion injury model, Mdivi-1 given as a pretreatment or throughout ischemia-reperfusion injury significantly reduced cell death. However, the cytoprotective effect of Mdivi-1 was not observed when given at reperfusion. Moreover, the cytoprotective effect of Mdivi-1 in the simulated ischemia-reperfusion injury model was not accompanied by changes in mitochondrial morphology, mitochondrial membrane potential, or mitochondrial reactive oxygen species production. Mdivi-1 also did not affect mitochondrial bioenergetics of intact W8B2+ CSCs. Taken together, these experiments demonstrated that Mdivi-1 treatment of human W8B2+ CSCs enhances their survival and can be employed to improve therapeutic efficacy of CSCs for ischemic heart disease

Functional analysis of an R311C variant of Ca2+-calmodulin-dependent protein kinase kinase-2 (CaMKK2) found as a de novo mutation in a patient with bipolar disorder

Objectives Loss-of-function mutations in the gene encoding the calcium-calmodulin (Ca2+-CaM)-dependent protein kinase kinase-2 (CaMKK2) enzyme are linked to bipolar disorder. Recently, a de novo arginine to cysteine (R311C) mutation in CaMKK2 was identified from a whole exome sequencing study of bipolar patients and their unaffected parents. The aim of the present study was to determine the functional consequences of the R311C mutation on CaMKK2 activity and regulation by Ca2+-CaM. Methods The effects of the R311C mutation on CaMKK2 activity and Ca2+-CaM activation were examined using a radiolabeled adenosine triphosphate (ATP) kinase assay. We performed immunoblot analysis to determine whether the R311C mutation impacts threonine-85 (T85) autophosphorylation, an activating phosphorylation site on CaMKK2 that has also been implicated in bipolar disorder. We also expressed the R311C mutant in CaMKK2 knockout HAP1 cells and used immunoblot analysis and an MTS reduction assay to study its effects on Ca2+-dependent downstream signaling and cell viability, respectively. Results The R311C mutation maps to the conserved HRD motif within the catalytic loop of CaMKK2 and caused a marked reduction in kinase activity and Ca2+-CaM activation. The R311C mutation virtually abolished T85 autophosphorylation in response to Ca2+-CaM and exerted a dominant-negative effect in cells as it impaired the ability of wild-type CaMKK2 to initiate downstream signaling and maintain cell viability. Conclusions The highly disruptive, loss-of-function impact of the de novo R311C mutation in human CaMKK2 provides a compelling functional rationale for being considered a potential rare monogenic cause of bipolar disorder

A beacon of hope in stroke therapy - blockade of pathologically activated cellular events in excitotoxic neuronal death as potential neuroprotective strategies

Excitotoxicity, a pathological process caused by over-stimulation of ionotropic glutamate receptors, is a major cause of neuronal loss in acute and chronic neurological conditions such as ischaemic stroke, Alzheimer's and Huntington's diseases. Effective neuroprotective drugs to reduce excitotoxic neuronal loss in patients suffering from these neurological conditions are urgently needed. One avenue to achieve this goal is to clearly define the intracellular events mediating the neurotoxic signals originating from the over-stimulated glutamate receptors in neurons. In this review, we first focus on the key cellular events directing neuronal death but not involved in normal physiological processes in the neurotoxic signalling pathways. These events, referred to as pathologically activated events, are potential targets for the development of neuroprotectant therapeutics. Inhibitors blocking some of the known pathologically activated cellular events have been proven to be effective in reducing stroke-induced brain damage in animal models. Notable examples are inhibitors suppressing the ion channel activity of neurotoxic glutamate receptors and those disrupting interactions of specific cellular proteins occurring only in neurons undergoing excitotoxic cell death. Among them, Tat-NR2B9c and memantine are clinically effective in reducing brain damage caused by some acute and chronic neurological conditions. Our second focus is evaluation of the suitability of the other inhibitors for use as neuroprotective therapeutics. We also discuss the experimental approaches suitable for bridging our knowledge gap in our current understanding of the excitotoxic signalling mechanism in neurons and discovery of new pathologically activated cellular events as potential targets for neuroprotectio

The eEF2 kinase-induced STAT3 inactivation inhibits lung cancer cell proliferation by phosphorylation of PKM2

Background Eukaryotic elongation factor-2 kinase (eEF2K) is a Ca 2+ /calmodulin (CaM)-dependent protein kinase that inhibits protein synthesis. However, the role of eEF2K in cancer development was reported paradoxically and remains to be elucidated. Methods Herein, A549 cells with eEF2K depletion or overexpression by stably transfected lentivirus plasmids were used in vitro and in vivo study. MTT and colony assays were used to detect cell proliferation and growth. Extracellular glucose and lactate concentration were measured using test kit. Immunoblot and co-immunoprecipitation assays were used to examine the molecular biology changes and molecular interaction in these cells. LC-MS/MS analysis and [γ- 32 P] ATP kinase assay were used to identify combining protein and phosphorylation site. Nude mice was utilized to study the correlation of eEF2K and tumor growth in vivo. Results We demonstrated that eEF2K inhibited lung cancer cells proliferation and affected the inhibitory effects of EGFR inhibitor gefitinib. Mechanistically, we showed that eEF2K formed a complex with PKM2 and STAT3, thereby phosphorylated PKM2 at T129, leading to reduced dimerization of PKM2. Subsequently, PKM2 impeded STAT3 phosphorylation and STAT3-dependent c-Myc expression. eEF2K depletion promoted the nuclear translocation of PKM2 and increased aerobic glycolysis reflected by increased lactate secretion and glucose. Conclusions Our findings define a novel mechanism underlying the regulation of cancer cell proliferation by eEF2K independent of its role in protein synthesis, disclosing the diverse roles of eEF2K in cell biology, which lays foundation for the development of new anticancer therapeutic strategies