Comparison of Classical P81 and SVI-P Cation-Exchange Papers in Radiometric Protein Kinase Assays.

Radiometric kinase assays have been widely used due to their high sensitivity and dynamic range. The assay measures the transfer of 32Pi from [γ-32P]-ATP to specific substrates, typically synthetic peptides. The 32P-phosphorylated peptide product is captured by binding it to phosphocellulose paper, specifically P81.

Enhanced Expression of Mitochondrial Magmas Protein in Ovarian Carcinomas: Magmas Inhibition Facilitates Antitumour Effects, Signifying a Novel Approach for Ovarian Cancer Treatment.

Mitochondrial-associated granulocyte macrophage colony-stimulating factor (Magmas) is a unique protein located in the inner membrane of mitochondria, with an active role in scavenging reactive oxygen species (ROS) in cellular systems. Ovarian cancer (OC), one of the deadliest gynaecological cancers, is characterised by genomic instability, affected by ROS production in the tumour microenvironment. This manuscript discusses the role of Magmas and efficacy of its novel small molecule inhibitor BT#9 in OC progression, metastasis, and chemoresistance.

AMPK phosphosite profiling by label-free mass spectrometry reveals a multitude of mTORC1-regulated substrates.

The nutrient-sensitive protein kinases AMPK and mTORC1 form a fundamental negative feedback loop that governs cell growth and proliferation. mTORC1 phosphorylates α2-S345 in the AMPK αβγ heterotrimer to suppress its activity and promote cell proliferation under nutrient stress conditions. Whether AMPK contains other functional mTORC1 substrates is unknown.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death.

An AMPKα2-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.

CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins.

The calcium-calmodulin-dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications.

Long-chain fatty acyl-CoA esters regulate metabolism via allosteric control of AMPK β1 isoforms.

Long-chain fatty acids (LCFAs) play important roles in cellular energy metabolism, acting as both an important energy source and signalling molecules. LCFA-CoA esters promote their own oxidation by acting as allosteric inhibitors of acetyl-CoA carboxylase, which reduces the production of malonyl-CoA and relieves inhibition of carnitine palmitoyl-transferase 1, thereby promoting LCFA-CoA transport into the mitochondria for β-oxidation. Here we report a new level of regulation wherein LCFA-CoA esters per se allosterically activate AMP-activated protein kinase (AMPK) β1-containing isoforms to increase fatty acid oxidation through phosphorylation of acetyl-CoA carboxylase.

Functional analysis of an R311C variant of Ca -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 (Ca -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 Ca -CaM.

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.

mTORC1 directly inhibits AMPK to promote cell proliferation under nutrient stress.

Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK), dysregulation of which are implicated in pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex-1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly down-regulates AMPK signalling by phosphorylating the evolutionarily conserved residue Ser367 in the fission yeast AMPK catalytic subunit Ssp2, and AMPK α1Ser347/α2Ser345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop Thr172.

Construction and reconstruction of brain circuits: normal and pathological axon guidance.

Perception of our environment entirely depends on the close interaction between the central and peripheral nervous system. In order to communicate each other, both systems must develop in parallel and in coordination. During development, axonal projections from the CNS as well as the PNS must extend over large distances to reach their appropriate target cells.

Quantitative proteomic analyses of dynamic signalling events in cortical neurons undergoing excitotoxic cell death.

Excitotoxicity, caused by overstimulation or dysregulation of ionotropic glutamate receptors (iGluRs), is a pathological process directing neuronal death in many neurological disorders. The aberrantly stimulated iGluRs direct massive influx of calcium ions into the affected neurons, leading to changes in expression and phosphorylation of specific proteins to modulate their functions and direct their participation in the signalling pathways that induce excitotoxic neuronal death. To define these pathways, we used quantitative proteomic approaches to identify these neuronal proteins (referred to as the changed proteins) and determine how their expression and/or phosphorylation dynamically changed in association with excitotoxic cell death.

AMP-activated protein kinase selectively inhibited by the type II inhibitor SBI-0206965.

Inhibition of the metabolic regulator AMP-activated protein kinase (AMPK) is increasingly being investigated for its therapeutic potential in diseases where AMPK hyperactivity results in poor prognoses, as in established cancers and neurodegeneration. However, AMPK-inhibitory tool compounds are largely limited to compound C, which has a poor selectivity profile. Here we identify the pyrimidine derivative SBI-0206965 as a direct AMPK inhibitor.

Structural Determinants for Small-Molecule Activation of Skeletal Muscle AMPK α2β2γ1 by the Glucose Importagog SC4.

The AMP-activated protein kinase (AMPK) αβγ heterotrimer regulates cellular energy homeostasis with tissue-specific isoform distribution. Small-molecule activation of skeletal muscle α2β2 AMPK complexes may prove a valuable treatment strategy for type 2 diabetes and insulin resistance. Herein, we report the small-molecule SC4 is a potent, direct AMPK activator that preferentially activates α2 complexes and stimulates skeletal muscle glucose uptake.

Mitochondrial fission protein Drp1 inhibition promotes cardiac mesodermal differentiation of human pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear.

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.

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.

Impact of Genetic Variation on Human CaMKK2 Regulation by Ca-Calmodulin and Multisite Phosphorylation.

The Ca-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.

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.

Dual role of Src kinase in governing neuronal survival.

Background: Src-family kinases (SFKs) are involved in neuronal survival and their aberrant regulation contributes to neuronal death. However, how they control neuronal survival and death remains unclear.

Objective: To define the effect of inhibition of Src activity and expression on neuronal survival.

Isolation and molecular identification of Cronobacter spp. from powdered infant formula (PIF) in Bangladesh.

Cronobacter spp. formerly known as Enterobacter sakazakii is an occasional contaminant of powdered infant formula (PIF). This pathogen has been associated with out-breaks of a rare form of infant meningitis, necrotizing enterocolitis (NEC), bacteremia and neonate deaths.