Feedback Inhibition Of Responses Research Articles

Regulation of ERK Kinase by MEK1 Kinase Inhibition in the Brain

Metabotropic (slow) and ionotropic (fast) neurotransmission are integrated by intracellular signal transduction mechanisms involving protein phosphorylation/dephosphorylation to achieve experience-dependent alterations in brain circuitry. ERK is an important effector of both slow and fast forms of neurotransmission and has been implicated in normal brain function and CNS diseases. Here we characterize phosphorylation of the ERK-activating protein kinase MEK1 by Cdk5, ERK, and Cdk1 in vitro in intact mouse brain tissue and in the context of an animal behavioral paradigm of stress. Cdk5 only phosphorylates Thr-292, whereas ERK and Cdk1 phosphorylate both Thr-292 and Thr-286 MEK1. These sites interact in a kinase-specific manner and inhibit the ability of MEK1 to activate ERK. Thr-292 and Thr-286 MEK1 are phosphorylated in most mouse brain regions to stoichiometries of ~5% or less. Phosphorylation of Thr-292 MEK1 is regulated by cAMP-dependent signaling in mouse striatum in a manner consistent with negative feedback inhibition in response to ERK activation. Protein phosphatase 1 and 2A contribute to the maintenance of the basal phosphorylation state of both Thr-292 and Thr-286 MEK1 and that of ERK. Activation of the NMDA class of ionotropic glutamate receptors reduces inhibitory MEK1 phosphorylation, whereas forced swim, a paradigm of acute stress, attenuates Thr-292 MEK1 phosphorylation. Together, the data indicate that these inhibitory MEK1 sites phosphorylated by Cdk5 and ERK1 serve as mechanistic points of convergence for the regulation of ERK signaling by both slow and fast neurotransmission.

Single Cell Mass Cytometry of Dysregulated Signaling Networks in Myeloproliferative Neoplasms and Secondary Acute Myeloid Leukemia

AbstractAbstract ▪703▪This icon denotes a clinically relevant abstractThe classic myeloproliferative neoplasms (MPNs) polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis (PMF) are frequently associated with the JAK2 V617F mutation or other genetic alterations in members of the JAK-STAT axis. These mutations have been shown to cause hyperactivated JAK-STAT signaling in cell lines and mouse models. How accurately these models recapitulate human MPN pathogenesis remains uncertain, as in vivo signaling in MPNs is likely modulated by other genetic changes and regulatory dynamics. In addition, the phenotypic changes that accompany transformation of chronic MPNs to secondary acute myeloid leukemia (sAML) have not been well characterized.While targeted inhibitors of JAK2 have shown activity in MPNs, the incomplete responses observed clinically have called into question the utility of JAK2 as a therapeutic target, suggesting that dysregulation of other signaling pathways may be important in MPN pathogenesis. Therefore, a more complete assessment of JAK-STAT and related signaling pathways in MPNs is needed.Mass cytometry is a novel technology that merges aspects of flow cytometry with mass spectrometry – cells are labeled with antibodies conjugated to elemental isotope reporters and then analyzed on the CyTOF mass cytometer. Each mass channel is distinct, such that no compensation is required, thus circumventing the spectral limitations of fluorescence-based flow cytometry and enabling the simultaneous measurement of 30+ parameters at the single cell level. We have utilized this approach to examine multiple signaling effectors in cell populations throughout hematopoietic differentiation.Our initial experiment included samples from three MPN patients (PV, PMF, post-PV sAML), and one normal donor. Cells were exposed to nine different perturbation conditions ex vivo, including cytokines and the JAK1/2 inhibitor ruxolitinib. Cells were stained with a panel of 17 surface markers and 13 dynamic intracellular signaling effectors and analyzed on the CyTOF. Single cell data was uploaded into SPADE (spanning-tree analysis of density-normalized events), which distills multidimensional data down to interconnected cell subsets and creates 2D tree plots based on shared surface marker expression. These plots identified recognizable cell subsets, including hematopoietic stem/progenitors (HSPCs) and myeloid and lymphoid lineage subsets. Heat maps were constructed to depict the relative induction of each intracellular marker in response to each condition.In the HSPC compartment, several expected responses were observed, particularly in PV. Erythropoietin-mediated activation of STAT3 and thrombopoietin (Tpo)-mediated activation of STAT3/5 were enhanced in PV committed progenitors. On a broader level, PV HSPCs exhibited heightened signaling sensitivities involving several cytokines and downstream effectors. Notably, CREB and S6 phosphorylation were strongly induced by Tpo, G-CSF, and IL-3. Ruxolitinib pre-treatment markedly inhibited signaling mediated by Tpo in PV CD34+ cells, indicating that the HSPC compartment can be effectively targeted by ex vivo JAK1/2 inhibition.In contrast to PV, PMF HSPCs exhibited lesser sensitivity to cytokine stimulation. In several instances, such as IL-3 induction of pSTAT5, the responses were in fact suppressed compared to normal. CD34+ HSPC from the sAML patient generally exhibited subnormal signaling responses. However, widespread hyperactivation following exposure to the phosphatase inhibitor pervanadate (PVO4) was observed in sAML CD34+ cells, suggesting that these signaling pathways were activated in vivo, but that feedback inhibition in response to persistent activity led to downregulation of their ex vivo inducibility.Based on these preliminary findings, we hypothesize that the fundamental chronic phase MPN state is one of heightened cytokine signaling sensitivity, while the advanced phase (especially sAML) state is one of tonic or constitutive downstream signaling activity with persistent feedback inhibition on cytokine signaling pathways. To test this hypothesis, experiments with a larger cohort of MPN samples are currently underway. These studies will provide a comprehensive framework of altered signaling in MPNs and provide deeper insights into the role of targeted therapy for MPNs. Disclosures:No relevant conflicts of interest to declare.

Simulating calcium influx and free calcium concentrations in yeast

Yeast can proliferate in environments containing very high Ca 2+ primarily due to the activity of vacuolar Ca 2+ transporters Pmc1 and Vcx1. Yeast mutants lacking these transporters fail to grow in high Ca 2+ environments, but growth can be restored by small increases in environmental Mg 2+. Low extracellular Mg 2+ appeared to competitively inhibit novel Ca 2+ influx pathways and to diminish the concentration of free Ca 2+ in the cytoplasm, as judged from the luminescence of the photoprotein aequorin. These Mg 2+-sensitive Ca 2+ influx pathways persisted in yvc1 cch1 double mutants. Based on mathematical models of the aequorin luminescence traces, we propose the existence in yeast of at least two Ca 2+ transporters that undergo rapid feedback inhibition in response to elevated cytosolic free Ca 2+ concentration. Finally, we show that Vcx1 helps return cytosolic Ca 2+ toward resting levels after shock with high extracellular Ca 2+ much more effectively than Pmc1 and that calcineurin, a protein phosphatase regulator of Vcx1 and Pmc1, had no detectable effects on these factors within the first few minutes of its activation. Therefore, computational modeling of Ca 2+ transport and signaling in yeast can provide important insights into the dynamics of this complex system.

Principal transcriptional programs regulating plant amino acid metabolism in response to abiotic stresses.

Using a bioinformatics analysis of public Arabidopsis (Arabidopsis thaliana) microarray data, we propose here a novel regulatory program, combining transcriptional and posttranslational controls, which participate in modulating fluxes of amino acid metabolism in response to abiotic stresses. The program includes the following two components: (1) the terminal enzyme of the module, responsible for the first catabolic step of the amino acid, whose level is stimulated or repressed in response to stress cues, just-in-time when the cues arrive, principally via transcriptional regulation of its gene; and (2) the initiator enzyme of the module, whose activity is principally modulated via posttranslational allosteric feedback inhibition in response to changes in the level of the amino acid, just-in-case when it occurs in response to alteration in its catabolism or sequestration into different intracellular compartments. Our proposed regulatory program is based on bioinformatics dissection of the response of all biosynthetic and catabolic genes of seven different pathways, involved in the metabolism of 11 amino acids, to eight different abiotic stresses, as judged from modulations of their mRNA levels. Our results imply that the transcription of the catabolic genes is principally more sensitive than that of the biosynthetic genes to fluctuations in stress-associated signals. Notably, the only exception to this program is the metabolic pathway of Pro, an amino acid that distinctively accumulates to significantly high levels under abiotic stresses. Examples of the biological significance of our proposed regulatory program are discussed.

Altered Neuronal Mitochondrial Coenzyme A Synthesis in Neurodegeneration with Brain Iron Accumulation Caused by Abnormal Processing, Stability, and Catalytic Activity of Mutant Pantothenate Kinase 2

Mutations in the pantothenate kinase 2 (PANK2) gene have been identified in patients with neurodegeneration with brain iron accumulation (NBIA; formerly Hallervorden-Spatz disease). However, the mechanisms by which these mutations cause neurodegeneration are unclear, especially given the existence of multiple pantothenate kinase genes in humans and multiple PanK2 transcripts with potentially different subcellular localizations. We demonstrate that PanK2 protein is localized to mitochondria of neurons in human brain, distinguishing it from other pantothenate kinases that do not possess mitochondrial-targeting sequences. PanK2 protein translated from the most 5' start site is sequentially cleaved at two sites by the mitochondrial processing peptidase, generating a long-lived 48 kDa mature protein identical to that found in human brain extracts. The mature protein catalyzes the initial step in coenzyme A (CoA) synthesis but displays feedback inhibition in response to species of acyl CoA rather than CoA itself. Some, but not all disease-associated point mutations result in significantly reduced catalytic activity. The most common mutation, G521R, results in marked instability of the intermediate PanK2 isoform and reduced production of the mature isoform. These results suggest that NBIA is caused by altered neuronal mitochondrial lipid metabolism caused by mutations disrupting PanK2 protein levels and catalytic activity.

Mechanisms of regulation of the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

The periplasmic glucans of Gram-negative bacteria, including the membrane-derived oligosaccharides (MDO) of Escherichia coli and the cyclic glucans of the Rhizobiaceae, have important but poorly understood functions in osmotic adaptation and, in the case of the Rhizobiaceae, in the complex cell-signaling of these bacteria with specific plant hosts. Experiments on the mechanisms of osmotic regulation of the biosynthesis of MDO in E. coli reported here support a model in which osmotic regulation occurs principally at the level of modulation of enzyme activity rather than at the level of gene expression. 1) Activity of the membrane-bound glucosyltransferase thought to catalyze the first and rate-making step in the biosynthesis of MDO is not altered by the osmolarity of the medium in which cells are grown. 2) Upon dilution of cells growing at high osmolarity into a medium of low osmolarity, the increased synthesis of MDO begins at maximum rate without detectable lag. 3) The activity of the membrane glucosyltransferase in vitro is strongly inhibited by high levels of salts, consistent with the view that synthesis in vivo is regulated chiefly by this mechanism, rather than by regulation of the synthesis of biosynthetic enzymes. We also find that the biosynthesis of MDO is regulated not only osmotically but also by strong feedback inhibition in response to the levels of MDO in the periplasm.