Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of Phosphorylation

  • activating phosphorylation
  • b phosphorylation
  • catenin phosphorylation
  • chain phosphorylation
  • constitutive phosphorylation
  • creb phosphorylation
  • dependent phosphorylation
  • eNO phosphorylation
  • egfr phosphorylation
  • erk phosphorylation
  • erk1/2 phosphorylation
  • fak phosphorylation
  • glucose phosphorylation
  • h3 phosphorylation
  • hydroxylase phosphorylation
  • increased phosphorylation
  • induced phosphorylation
  • jnk phosphorylation
  • jun phosphorylation
  • kinase phosphorylation
  • mapk phosphorylation
  • mitochondrial oxidative phosphorylation
  • oxidative phosphorylation
  • pkb phosphorylation
  • protein phosphorylation
  • protein tyrosine phosphorylation
  • rapid phosphorylation
  • receptor phosphorylation
  • reduced phosphorylation
  • serine phosphorylation
  • src-dependent phosphorylation
  • stat3 phosphorylation
  • stat3 tyrosine phosphorylation
  • stat5 phosphorylation
  • sustained phosphorylation
  • tau phosphorylation
  • threonine phosphorylation
  • vitro phosphorylation

  • Terms modified by Phosphorylation

  • phosphorylation event
  • phosphorylation level
  • phosphorylation motif
  • phosphorylation pattern
  • phosphorylation polycondensation
  • phosphorylation site
  • phosphorylation state
  • phosphorylation states
  • phosphorylation status
  • phosphorylation system

  • Selected Abstracts


    Claire Arnaud
    SUMMARY 1.,Heat stress (HS) is known to induce delayed preconditioning against myocardial infarction 24 h later, but the exact signalling pathway of this response remains to be elucidated. In previous studies, we have shown evidence for the implication of protein kinase C (PKC) and p38 mitogen-activated protein kinase (MAPK) in the HS-induced reduction in infarct size. Furthermore, in their phosphorylated state, small heat shock proteins (Hsp27) seem to confer cytoskeletal protection. In the present study, we sought to determine the effect of HS on the subcellular distribution of PKC isoforms and on Hsp27 phosphorylation. 2.,Rats were subjected to either HS (42°C for 15 min; HS group) or sham anaesthesia (sham group) before their hearts were excised. Myocardial tissue extracts obtained 20 min or 24 h after HS were processed for western blot analysis. 3.,In the HS group, PKC, translocated from the cytosolic to the particulate fraction (4426 ± 128 vs 6258 ± 316 arbitrary units; P = 0.002). Chelerythrine (5 mg/kg, i.p.), a PKC inhibitor, abolished this translocation. Western blot analysis of Hsp27 24 h after HS showed a marked increase in protein expression and phosphorylation in the particulate fraction. 4.,In the present study, we have shown that HS induces the translocation of PKC, from the cytosolic to the particulate fraction. Along with our previous observation that PKC is a trigger of HS-induced myocardial preconditioning, the results of the present study suggest an important role of the , isoform of PKC in this cardioprotective mechanism. Furthermore, we have also demonstrated that the cytoprotective protein Hsp27 is phosphorylated following HS. Therefore, we can conclude that PKC and MAPK/Hsp27 are involved in the signalling pathway of HS-induced cardioprotection. [source]

    Resistance exercise increases leg muscle protein synthesis and mTOR signalling independent of sex

    ACTA PHYSIOLOGICA, Issue 1 2010
    H. C. Dreyer
    Abstract Aim:, Sex differences are evident in human skeletal muscle as the cross-sectional area of individual muscle fibres is greater in men than in women. We have recently shown that resistance exercise stimulates mammalian target of rapamycin (mTOR) signalling and muscle protein synthesis in humans during early post-exercise recovery. Therefore, the aim of this study was to determine if sex influences the muscle protein synthesis response during recovery from resistance exercise. Methods:, Seventeen subjects, nine male and eight female, were studied in the fasted state before, during and for 2 h following a bout of high-intensity leg resistance exercise. Mixed muscle protein fractional synthetic rate was measured using stable isotope techniques and mTOR signalling was assessed by immunoblotting from repeated vastus lateralis muscle biopsy samples. Results:, Post-exercise muscle protein synthesis increased by 52% in the men and by 47% in the women (P < 0.05) and was not different between groups (P > 0.05). Akt phosphorylation increased in both groups at 1 h post-exercise (P < 0.05) and returned to baseline during 2 h post-exercise with no differences between groups (P > 0.05). Phosphorylation of mTOR and its downstream effector S6K1 increased significantly and similarly between groups during post-exercise recovery (P < 0.05). eEF2 phosphorylation decreased at 1- and 2 h post-exercise (P < 0.05) to a similar extent in both groups. Conclusion:, The contraction-induced increase in early post-exercise mTOR signalling and muscle protein synthesis is independent of sex and appears to not play a role in the sexual dimorphism of leg skeletal muscle in young men and women. [source]

    Post-ischaemic activation of kinases in the pre-conditioning-like cardioprotective effect of the platelet-activating factor

    ACTA PHYSIOLOGICA, Issue 3 2009
    C. Penna
    Abstract Aim:, Platelet-activating factor (PAF) triggers cardiac pre-conditioning against ischemia/reperfusion injury. The actual protection of ischaemic pre-conditioning occurs in the reperfusion phase. Therefore, we studied in this phase the kinases involved in PAF-induced pre-conditioning. Methods:, Langendorff-perfused rat hearts underwent 30 min of ischaemia and 2 h of reperfusion (group 1, control). Before ischaemia, group 2 hearts were perfused for 19 min with PAF (2 × 10,11 m); groups 3,5 hearts were co-infused during the initial 20 min of reperfusion, with the protein kinase C (PKC) inhibitor chelerythrine (5 × 10,6 m) or the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (5 × 10,5 m) and atractyloside (2 × 10,5 m), a mitochondrial permeability transition pore (mPTP) opener respectively. Phosphorylation of PKC,, PKB/A,t, GSK-3, and ERK1/2 at the beginning of reperfusion was also checked. Left ventricular pressure and infarct size were determined. Results:, PAF pre-treatment reduced infarct size (33 ± 4% vs. 64 ± 5% of the area at risk of control hearts) and improved pressure recovery. PAF pre-treatment enhanced the phosphorylation/activation of PKC,, PKB/A,t and the phosphorylation/inactivation of GSK-3, at reperfusion. Effects on ERK1/2 phosphorylation were not consistent. Infarct-sparing effect and post-ischaemic functional improvement induced by PAF pre-treatment were abolished by post-ischaemic infusion of either chelerythrine, LY294002 or atractyloside. Conclusions:, The cardioprotective effect exerted by PAF pre-treatment involves activation of PKC and PI3K in post-ischaemic phases and might be mediated by the prevention of mPTP opening in reperfusion via GSK-3, inactivation. [source]

    Simultaneous flow cytometric detection of basophil activation marker CD63 and intracellular phosphorylated p38 mitogen-activated protein kinase in birch pollen allergy,

    CYTOMETRY, Issue 1 2009
    Nicolaas E. Aerts
    Abstract Background: Phosphorylation of p38 MAPK is a crucial step in IgE-receptor signaling in basophils. The relation of p38 MAPK to the well-validated diagnostic cell surface marker CD63 has not been evaluated in a clinical allergy model. Methods: Expression of CD63 and phosphorylation of p38 MAPK were analyzed flow cytometrically in anti-IgE-gated basophils from 18 birch pollen allergic patients, five grass pollen allergic patients, and five healthy individuals, after 3 and 20 min of stimulation with recombinant major birch pollen allergen (rBet v 1). Additional time points and the influence of p38 MAPK inhibitor SB203580 were studied in birch pollen allergic patients. Results: Phospho-p38 MAPK and CD63 were expressed dose-dependently in birch pollen allergic patient basophils within 1 minute of rBet v 1 stimulation. P38 MAPK phosphorylation was fastest and subsided gradually while CD63 expression remained elevated for at least 20 min. Inhibition of p38 MAPK significantly inhibited CD63 upregulation. With optimal stimulation of the cells (1 ,g/mL), sensitivity and specificity for the discrimination between patients and a group of control individuals (grass pollen allergic patients and healthy controls) were 94% and 100% for CD63 at 3 and 20 min and for phospho-p38 MAPK at 3 min. Conclusion: Antigen-induced p38 MAPK phosphorylation in human basophils essentially contributes to CD63 upregulation. It is a sensitive and specific intracellular marker for allergy diagnosis and offers new insight into the mechanisms of basophil activation. © 2008 Clinical Cytometry Society [source]

    Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+ -ATPase

    ACTA PHYSIOLOGICA, Issue 2 2002
    F. R. IBARRA
    ABSTRACT The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on , -adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+ -adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+ -ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the , -adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+ -ATPase on the Ser23 residue. The level of PKC induced Na+,K+ -ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+ -ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+ -ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+ -ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+ -ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+ -ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+ -ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis. [source]

    Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

    CYTOSKELETON, Issue 1 2009
    Vijay S. Rao
    Abstract Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]

    In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis

    CYTOSKELETON, Issue 2 2008
    Ryota Uehara
    Abstract Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

    Bioenergetics and the epigenome: Interface between the environment and genes in common diseases

    Douglas C. Wallace
    Abstract Extensive efforts have been directed at using genome-wide association studies (GWAS) to identify the genes responsible for common metabolic and degenerative diseases, cancer, and aging, but with limited success. While environmental factors have been evoked to explain this conundrum, the nature of these environmental factors remains unexplained. The availability of and demands for energy constitute one of the most important aspects of the environment. The flow of energy through the cell is primarily mediated by the mitochondrion, which oxidizes reducing equivalents from hydrocarbons via acetyl-CoA, NADH + H+, and FADH2 to generate ATP through oxidative phosphorylation (OXPHOS). The mitochondrial genome encompasses hundreds of nuclear DNA (nDNA)-encoded genes plus 37 mitochondrial DNA (mtDNA)-encoded genes. Although the mtDNA has a high mutation rate, only milder, potentially adaptive mutations are introduced into the population through female oocytes. In contrast, nDNA-encoded bioenergetic genes have a low mutation rate. However, their expression is modulated by histone phosphorylation and acetylation using mitochondrially-generated ATP and acetyl-CoA, which permits increased gene expression, growth, and reproduction when calories are abundant. Phosphorylation, acetylaton, and cellular redox state also regulate most signal transduction pathways and activities of multiple transcription factors. Thus, mtDNA mutations provide heritable and stable adaptation to regional differences while mitochondrially-mediated changes in the epigenome permit reversible modulation of gene expression in response to fluctuations in the energy environment. The most common genomic changes that interface with the environment and cause complex disease must, therefore, be mitochondrial and epigenomic in origin. © 2010 Wiley-Liss, Inc. Dev Disabil Res Rev 2010;16:114,119. [source]

    MSK regulate TCR-induced CREB phosphorylation but not immediate early gene transcription

    Madlen Kaiser
    Abstract Stimulation of the T cell receptor activates the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) cascades. We demonstrate that TCR stimulation also activates the mitogen- and stress-activated kinases (MSK) downstream of ERK1/2 and p38 in both a T cell line and primary peripheral T cells. MSK1/2-knockout mice were found to have normal numbers of T cells in the thymus, and development of these cells appeared unaffected. Using naive T cells and T lymphoblasts from MSK1/2-knockout mice, it was found that MSK was the kinase responsible for phosphorylation of the transcription factor CREB in response to TCR stimulation. Phosphorylation of CREB by MSK has been linked to the transcription of nur77, nor1 and c-fos downstream of MAPK signalling in various cell types. In T cells, the TCR-dependent transcription of these genes was found to require a MAPK-dependent but MSK-independent signalling pathway. Nevertheless, the number of T cells present in the spleens of MSK1/2-knockout mice and the IL-2-induced proliferation of these cells was reduced compared to wild-type mice. This correlated to a reduction in the TCR-induced up-regulation of the IL-2 receptor CD25 and a requirement for MSK in IL-2-induced CREB phosphorylation. [source]

    Phosphorylation of Artemis following irradiation-induced DNA damage

    Catherine Poinsignon
    Abstract Artemis is a DNA repair factor required for V(D)J recombination, repair of DNA damage induced by ionizing radiation (IR) or radiomimetic drugs, and the maintenance of genome integrity. During V(D)J recombination, Artemis participates in the resolution of hairpin-sealed coding ends, a step crucial to the constitution of the gene encoding for the antigen receptor of lymphocytes. The precise role of Artemis in the repair of IR-induced DNA damage remains to be elucidated. Here we show that Artemis is constitutively phosphorylated in cultured cells and undergoes additional phosphorylation events after irradiation. The IR-induced phosphorylation is mainly, although not solely, dependent on Ataxia-telangiectasia-mutated kinase (ATM). The physiological role of these phosphorylation events remains unknown, as in vitro -generated Artemis mutants, which present impaired IR-induced phosphorylation, still display an activity sufficient to complement the V(D)J recombination defect and the increased radiosensibility of Artemis-deficient cells. Thus, Artemis is an effector of DNA repair that can be phosphorylated by ATM, and possibly by DNA-PKcs and ATR depending upon the type of DNA damage. [source]

    Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472

    Shinji Matsumura
    Abstract Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N -methyl- d -aspartate (NMDA) receptor-mediated Ca2+ influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca2+ response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca2+ response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase C, at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain. [source]

    Hypothermia treatment potentiates ERK1/2 activation after traumatic brain injury

    Coleen M. Atkins
    Abstract Traumatic brain injury (TBI) results in significant hippocampal pathology and hippocampal-dependent memory loss, both of which are alleviated by hypothermia treatment. To elucidate the molecular mechanisms regulated by hypothermia after TBI, rats underwent moderate parasagittal fluid-percussion brain injury. Brain temperature was maintained at normothermic or hypothermic temperatures for 30 min prior and up to 4 h after TBI. The ipsilateral hippocampus was assayed with Western blotting. We found that hypothermia potentiated extracellular signal-regulated kinase 1/2 (ERK1/2) activation and its downstream effectors, p90 ribosomal S6 kinase (p90RSK) and the transcription factor cAMP response element-binding protein. Phosphorylation of another p90RSK substrate, Bad, also increased with hypothermia after TBI. ERK1/2 regulates mRNA translation through phosphorylation of mitogen-activated protein kinase-interacting kinase 1 (Mnk1) and the translation factor eukaryotic initiation factor 4E (eIF4E). Hypothermia also potentiated the phosphorylation of both Mnk1 and eIF4E. Augmentation of ERK1/2 activation and its downstream signalling components may be one molecular mechanism that hypothermia treatment elicits to improve functional outcome after TBI. [source]

    Phosphorylation of voltage-gated ion channels in rat olfactory receptor neurons

    Christian H. Wetzel
    Abstract In olfactory receptor neurons (ORNs), ligand,odorant receptor interactions cause G protein-mediated activation of adenylate cyclase and a subsequent increase in concentration of the intracellular messenger cAMP. Odorant-evoked elevation in cAMP is thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca2+ influx, leading to membrane depolarization and the generation of action potentials. Our data show that in freshly dissociated rat ORNs, odorant-induced elevation in cAMP also activates cAMP-dependent protein kinase (PKA), which is then able to phosphorylate various protein targets in the olfactory signal transduction pathway, specifically voltage-gated sodium and calcium channels. The presence of PKI (PKA inhibitor peptide) blocked the modulatory action of cAMP on voltage-gated ion channels. By modulating the input/output properties of the sensory neurons, this mechanism could take part in the complex adaptation process in odorant perception. In addition, we found modulation of voltage-gated sodium and calcium channel currents by 5-hydroxytryptamine and the dopamine D1 receptor agonist SKF 38393. These findings suggest that in situ ORNs might also be a target for efferent modulation. [source]

    On the Way to Glycoprocessing Inhibitors , Synthesis of an Imidazolo-Nectrisine-Phosphono Acid Derivative: A Potential Glycosyltranferase Inhibitor

    Théophile Tschamber
    Abstract Assuming the transition state of glycosyltransferase inhibitors to be similar to those encountered with potent glycosidase inhibitors , i.e. a flattened conformation with a positively charged anomeric centre , we worked out a synthesis of the D - arabino -configured phosphonic acid target molecule 2 derived from an imidazolo-sugar. The key synthetic intermediate is the linear imidazolo L - xylo compound 10 which could be obtained, either from L - threo precursor 6 by a coupling reaction with imidazole derivative 5, or from L -sorbose. A multi-step and site specific iodination of 10 gave the mono-iodo- L - xylo derivative 14 which was cyclised to the D - arabino -configured bicyclic azasugar 15. Phosphorylation of the Grignard derivative of the latter, followed by mono-esterification with citronellol along with some protection-deprotection steps led to target molecule 2. The potential inhibitor 2 is supposed to be protonated at its most basic N atom by a carboxylic acid residue in the arabinosyl-transferase active site. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source]

    Acute signalling responses to intense endurance training commenced with low or normal muscle glycogen

    Wee Kian Yeo
    We have previously demonstrated that well-trained subjects who completed a 3 week training programme in which selected high-intensity interval training (HIT) sessions were commenced with low muscle glycogen content increased the maximal activities of several oxidative enzymes that promote endurance adaptations to a greater extent than subjects who began all training sessions with normal glycogen levels. The aim of the present study was to investigate acute skeletal muscle signalling responses to a single bout of HIT commenced with low or normal muscle glycogen stores in an attempt to elucidate potential mechanism(s) that might underlie our previous observations. Six endurance-trained cyclists/triathletes performed a 100 min ride at ,70% peak O2 uptake (AT) on day 1 and HIT (8 × 5 min work bouts at maximal self-selected effort with 1 min rest) 24 h later (HIGH). Another six subjects, matched for fitness and training history, performed AT on day 1 then 1,2 h later, HIT (LOW). Muscle biopsies were taken before and after HIT. Muscle glycogen concentration was higher in HIGH versus LOW before the HIT (390 ± 28 versus 256 ± 67 ,mol (g dry wt),1). After HIT, glycogen levels were reduced in both groups (P < 0.05) but HIGH was elevated compared with LOW (229 ± 29 versus 124 ± 41 ,mol (g dry wt),1; P < 0.05). Phosphorylation of 5,AMP-activated protein kinase (AMPK) increased after HIT, but the magnitude of increase was greater in LOW (P < 0.05). Despite the augmented AMPK response in LOW after HIT, selected downstream AMPK substrates were similar between groups. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was unchanged for both groups before and after the HIT training sessions. We conclude that despite a greater activation AMPK phosphorylation when HIT was commenced with low compared with normal muscle glycogen availability, the localization and phosphorylation state of selected downstream targets of AMPK were similar in response to the two interventions. [source]

    The Effects of Steroid Hormones on the Transcription of Genes Encoding Enzymes of Oxidative Phosphorylation

    Klaus Scheller
    Regulation of energy metabolism is one of the major functions of steroid hormones. In this process, mitochondria, by way of oxidative phosphorylation, play a central role. Depending on the energy needs of the cell, on the tissue, on the developmental stage and on the intensity of the hormonal stimulus, the response can be an activation of pre-existing respiratory chain components, an increased transcription of nuclear-encoded and/or mitochondrial-encoded respiratory chain enzyme (OXPHOS) genes and of biosynthesis of the respective enzyme subunits or, in extreme cases of high energy needs, an increase in the number of mitochondria and mitochondrial DNA content per cell. Some of the hormonally regulated systems involving effects on nuclear and mitochondrial OXPHOS genes are reviewed in this paper. The possible molecular mechanisms of steroid hormone action on nuclear and mitochondrial gene transcription and possible ways of coordination of transcription in these two separate cell compartments involving direct interaction of steroid receptors with hormone response elements in nuclear OXPHOS genes and in mitochondria and induction/activation of nuclear-encoded regulatory factors affecting mitochondrial gene transcription are presented. [source]

    Regulation of calpain B from Drosophila melanogaster by phosphorylation

    FEBS JOURNAL, Issue 17 2009
    László Kovács
    Calpain B is one of the two catalytically competent calpain (calcium-activated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombinant protein by protein kinase A as well as by the extracellular signal-regulated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca2+ binding region of an EF-hand motif, and Ser240 close to the autocatalytic activation site of calpain B, as being the residues phosphorylated by protein kinase A. In the transducer region of the protease, Thr747 was shown to be the target of the ERK phosphorylation. Based on the results of three different assays, we concluded that the treatment of calpain B with protein kinase A and ERK1 and ERK2 kinases increases the rate of the autoproteolytic activation of the enzyme, together with the rate of the digestion of external peptide or protein substrates. Phosphorylation also elevates the Ca2+ sensitivity of the protease. The kinetic analysis of phosphorylation mimicking Thr747Glu and Ser845Glu calpain B mutants confirmed the above conclusions. Out of the three phosphorylation events tested in vitro, we verified the in vivo phosphorylation of Thr747 in epidermal growth factor-stimulated Drosophila S2 cells. The data obtained suggest that the activation of the ERK pathway by extracellular signals results in the phosphorylation and activation of calpain B in fruit flies. Structured digital abstract ,,MINT-7214239: ERK1 (uniprotkb:P40417) phosphorylates (MI:0217) CalpainB (uniprotkb:Q9VT65) by protein kinase assay (MI:0424) ,,MINT-7214216, MINT-7214228: PKA (uniprotkb:P12370) phosphorylates (MI:0217) CalpainB (uniprotkb:Q9VT65) by protein kinase assay (MI:0424) ,,MINT-7214325: CalpainB (uniprotkb:Q9VT65) cleaves (MI:0194) MAP2C (uniprotkb:P11137) by protease assay (MI:0435) ,,MINT-7214275: ERK2 (uniprotkb:P40417-2) phosphorylates (MI:0217) CalpainB (uniprotkb:Q9VT65) by protein kinase assay (MI:0424) ,,MINT-7214319: CalpainB (uniprotkb:Q9VT65) and CalpainB (uniprotkb:Q9VT65) cleave (MI:0194) by protease assay (MI:0435) [source]

    Phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization

    FEBS JOURNAL, Issue 16 2008
    Tsui-Yi Peng
    Coronavirus nucleocapsid protein is abundant in infected cells and participates in viral RNA replication and transcription. The central domain of the nucleocapsid protein contains several arginine/serine (RS) dipeptides, the biological significance of which has not been well investigated. In the present study, we demonstrate that the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated primarily within the RS-rich region in cells and by SR protein kinase 1 in vitro. The nucleocapsid protein could suppress translation and its RS motif is essential for such an activity. Moreover, phosphorylation of the RS motif could modulate the translation inhibitory activity of the nucleocapsid protein. We further found that RS motif phosphorylation did not significantly affect RNA binding of the nucleocapsid protein but impaired its multimerization ability. We observed that the nucleocapsid protein could translocate to cytoplasmic stress granules in response to cellular stress. Deletion or mutations of the RS motif enhanced stress granule localization of the nucleocapsid protein, whereas overexpression of SR protein kinase 1 inhibited nucleocapsid protein localization to stress granules. The nucleocapsid protein lacking the RS motif formed high-order RNP complexes, which may also account for its enhanced stress granule localization. Taken together, phosphorylation of the severe acute respiratory syndrome-CoV nucleocapsid protein modulates its activity in translation control and also interferes with its oligomerization and aggregation in stress granules. [source]

    Phosphorylation modulates the local conformation and self-aggregation ability of a peptide from the fourth tau microtubule-binding repeat

    FEBS JOURNAL, Issue 19 2007
    Jin-Tang Du
    Phosphorylation of tau protein modulates both its physiological role and its aggregation into paired helical fragments, as observed in Alzheimer's diseased neurons. It is of fundamental importance to study paired helical fragment formation and its modulation by phosphorylation. This study focused on the fourth microtubule-binding repeat of tau, encompassing an abnormal phosphorylation site, Ser356. The aggregation propensities of this repeat peptide and its corresponding phosphorylated form were investigated using turbidity, thioflavin T fluorescence and electron microscopy. There is evidence for a conformational change in the fourth microtubule-binding repeat of tau peptide upon phosphorylation, as well as changes in aggregation activity. Although both tau peptides have the ability to aggregate, this is weaker in the phosphorylated peptide. This study reveals that both tau peptides are capable of self-aggregation and that phosphorylation at Ser356 can modulate this process. [source]

    Protein kinase C , phosphorylates keratin 8 at Ser8 and Ser23 in GH4C1 cells stimulated by thyrotropin-releasing hormone

    FEBS JOURNAL, Issue 13 2007
    Yoshiko Akita
    Protein kinase C , (PKC,) is activated by thyrotropin-releasing hormone (TRH), a regulator of pituitary function in rat pituitary GH4C1 cells. We analyzed the downstream mechanism after PKC, activation. Exposure of GH4C1 cells to TRH or a phorbol ester increased the phosphorylation of three p52 proteins (p52a, p52b and p52c) and decreased the phosphorylation of destrin and cofilin. GF109203X, an inhibitor of protein kinases including PKC, inhibited phosphorylation of the p52 proteins by TRH stimulation. Peptide mapping, amino-acid sequencing, and immunochemical studies indicated that p52a, p52b, and p52c are the differentially phosphorylated isoforms of keratin 8 (K8), an intermediate filament protein. The unphosphorylated K8 (p52n) localized exclusively to the cytoskeleton, whereas the phosphorylated forms (especially p52c), which are increased in TRH-stimulated cells, localized mainly to the cytosol. K8 phosphorylation was enhanced in PKC,-overexpressing clones, and purified recombinant PKC, directly phosphorylated K8 with a profile similar to that observed in TRH-stimulated cells. PKC, and K8 colocalized near the nucleus under basal conditions and were concentrated in the cell periphery and cell,cell contact area after TRH stimulation. MS analyses of phospho-K8 and K8-synthesized peptide (amino acids 1,53) showed that PKC, phosphorylates Ser8 and Ser23 of K8. Phosphorylation of these sites is enhanced in TRH-stimulated cells and PKC,-overexpressing cells, as assessed by immunoblotting using antibodies to phospho-K8. These results suggest that K8 is a physiological substrate for PKC,, and the phosphorylation at Ser8 and Ser23 transduces, at least in part, TRH,PKC, signaling in pituitary cells. [source]

    Gradual phosphorylation regulates PC4 coactivator function

    FEBS JOURNAL, Issue 7 2006
    Hendrik R. A. Jonker
    The unstructured N-terminal domain of the transcriptional cofactor PC4 contains multiple phosphorylation sites that regulate activity. The phosphorylation status differentially influences the various biochemical functions performed by the structured core of PC4. Binding to ssDNA is slightly enhanced by phosphorylation of one serine residue, which is not augmented by further phosphorylation. The presence of at least two phosphoserines decreases DNA-unwinding activity and abrogates binding to the transcriptional activator VP16. Phosphorylation gradually decreases the binding affinity for dsDNA. These phosphorylation-dependent changes in PC4 activities correlate with the sequential functions PC4 fulfils throughout the transcription cycle. MS and NMR revealed that up to eight serines are progressively phosphorylated towards the N-terminus, resulting in gradual environmental changes in the C-terminal direction of the following lysine-rich region. Also within the structured core, primarily around the interaction surfaces, environmental changes are observed. We propose a model for co-ordinated changes in PC4 cofactor functions, mediated by phosphorylation status-dependent gradual masking of the lysine-rich region causing shielding or exposure of interaction surfaces. [source]

    Phosphorylation of NF-,B proteins by cyclic GMP-dependent kinase

    FEBS JOURNAL, Issue 10 2003
    A noncanonical pathway to NF-, B activation
    The transcription factor NF-,B is activated in cellular stress responses. This requires rapid regulation of its function, which is accomplished, in part, by various modes of phosphorylation. Even though diverse DNA binding subunits of NF-,B proteins may transactivate from distinct recognition sequences, the differential regulation of transcription from the large number of NF-,B responsive sites in various gene promoters and enhancers has been incompletely understood. The cyclic GMP-dependent kinase (PKG) is an important mediator of signal transduction that may induce gene expression through cAMP response element binding protein (CREB) and through other, yet undefined, mechanisms. We have previously characterized a signal transduction pathway that leads to activation-induced cell death in T-lymphocytes and involves the activation of PKG. Here we demonstrate that the NF-,B proteins p65, p49 (also called p52), and p50 are specific substrates for this kinase. PKG dose-dependently increases the transactivating activity of p65 from the NF-,B consensus sequence. It also mediates dose-dependently an increase in transcriptional activity by p49 or p50 from a unique CCAAT/enhance binding protein (C/EBP)-associated NF-,B site, but not from the consensus site. Phosphorylation of p65, p50, or p49 does not alter their subcellular distribution. Because the release of cytosolic p65/p50 heterodimers into the nucleus is by itself insufficient to differentiate all the numerous NF-,B promoter sequences, phosphorylation of the DNA-binding subunits reveals a form of differential regulation of NF-,B activity and it implies a novel pathway for PKG-induced gene transcription. These observations may bear on mechanisms of programmed cell death in T-lymphocytes. They may also be relevant to ongoing efforts to induce cancer cell apoptosis through activation of PKG. [source]

    Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry

    FEBS JOURNAL, Issue 8 2001
    Cyrille Garnier
    HSP90 is one of the most abundant proteins in the cytosol of eukaryotic cells. HSP90 forms transient or stable complexes with several key proteins involved in signal transduction including protooncogenic protein kinases and nuclear receptors, it interacts with cellular structural elements such as actin-microfilament, tubulin-microtubule and intermediate filaments, and also exhibits conventional chaperone functions. This protein exists in two isoforms ,-HSP90 and ,-HSP90, and it forms dimers which are crucial species for its biological activity. PAGE, ESI-MS and MALDI-MS were used to study HSP90 purified from pig brain. The two protein isoforms were clearly distinguished by ESI-MS, the , isoform being ,,six times more abundant than the , isoform. ESI-MS in combination with ,,phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain ,-HSP90, with the diphosphorylated form being the most abundant. For the , isoform, the di-phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross-linking showed a high percentage of ,,, homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained. [source]

    Specific Ser-Pro phosphorylation by the RNA-recognition motif containing kinase KIS

    FEBS JOURNAL, Issue 14 2000
    Alexandre Maucuer
    We present here a first appraisal of the phosphorylation site specificity of KIS (for ,kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks. [source]

    Phosphorylation of phosphodiesterase-5 by cyclic nucleotide-dependent protein kinase alters its catalytic and allosteric cGMP-binding activities

    FEBS JOURNAL, Issue 9 2000
    Jackie D. Corbin
    In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem.265, 14971,14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50,70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2,0.5 ,m PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 ,m. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed. [source]

    AATYK1A phosphorylation by Cdk5 regulates the recycling endosome pathway

    GENES TO CELLS, Issue 7 2010
    Tetsuya Takano
    Trafficking of recycling endosomes (REs) is regulated by the small GTPase, Rab11A; however, the regulatory mechanism remains elusive. Apoptosis-associated tyrosine kinase 1A (AATYK1A) is a Ser/Thr kinase expressed highly in brain. We have recently shown that AATYK1A localizes to Rab11A-positive RE and is phosphorylated at Ser34 by cyclin-dependent kinase 5 (Cdk5). Here, we have investigated a role of AATYK1A and its phosphorylation in recycling endosomal trafficking using Chinese hamster ovary-K1 (CHO-K1) cells. AATYK1A localizes predominantly to Rab11A-positive pericentrosomal endocytic recycling compartment (ERC). Phosphorylation at Ser34 of AATYK1A disrupts its accumulation in the pericentrosomal ERC. Consistently, phosphorylation-mimic mutant (AATYK1A-S34D) did not accumulate in the ERC and additionally attenuated ERC formation. ERC formation suppression can be reversed by constitutively active Rab11A-Q70L, suggesting a functional link between AATYK1A phosphorylation and Rab11A activity. Although no direct interaction between AATYK1A and Rab11A could be detected, the exchange of guanine nucleotides bound to Rab11A was significantly reduced in the presence of the phosphorylation-mimic AATYK1A-S34D. Together, our results reveal a regulatory role for AATYK1A in the formation of pericentrosomal ERC. They furthermore indicate that Cdk5 can disrupt ERC formation via Ser34 phosphorylation of AATYK1A. Finally, our data suggest a mechanism by which AATYK1A signaling couples Cdk5 to Rab11A activity. [source]

    In vivo potentiation of human oestrogen receptor , by Cdk7-mediated phosphorylation

    GENES TO CELLS, Issue 10 2004
    Saya Ito
    Phosphorylation of the Ser118 residue in the N-terminal A/B domain of the human oestrogen receptor , (hER,) by mitogen-activated protein kinase (MAPK), stimulated via growth factor signalling pathways, is known to potentiate ER, ligand-induced transactivation function. Besides MAPK, cyclin dependent kinase 7 (Cdk7) in the TFIIH complex has also been found to potentiate hER, transactivation in vitro through Ser118 phosphorylation. To investigate an impact of Cdk7 on hER, transactivation in vivo, we assessed activity of hER, in a wild-type and cdk7 inactive mutant Drosophila that ectopically expressed hER, in the eye disc. Ectopic expression of the wild-type or mutant receptors, together with a green fluorescent protein (GFP) reporter gene, allowed us to demonstrate that hER, expressed in the fly tissues was transcriptionally functional and adequately responded to hER, ligands in the patterns similar to those observed in mammalian cells. Replacement of Ser118 with alanine in hER, (S118A mutant) significantly reduced the ligand-induced hER, transactivation function. Importantly, while in cdk7 inactive mutant Drosophila the wild-type hER, exhibited reduced response to the ligand; levels of transactivation by the hER, S118A mutant were not affected in these inactive cdk7 mutant flies. Furthermore, phosphorylation of hER, at Ser118 has been observed in vitro by both human and Drosophila Cdk7. Our findings demonstrate that Cdk7 is involved in regulation of the ligand-induced transactivation function of hER,in vivo via Ser118 phosphorylation. [source]

    Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK)

    GENES TO CELLS, Issue 2 2002
    Hidemasa Goto
    Background: Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments. Results: We determined if the vimentin filaments can be regulated by p21-activated kinase (PAK), one of targets downstream of Cdc42 or Rac. In vitro analyses revealed that vimentin served as an excellent substrate for PAK. This phosphorylated vimentin lost the potential to form 10 nm filaments. We identified Ser25, Ser38, Ser50, Ser65 and Ser72 in the amino-terminal head domain as the major phosphorylation sites on vimentin for PAK. The ectopic expression of constitutively active PAK in COS-7 cells induced vimentin phosphorylation. Fibre bundles or granulates of vimentin were frequent in these transfected cells. However, the kinase-inactive mutant induced neither vimentin phosphorylation nor filament reorganization. Conclusion: Our observations suggest that PAK may regulate the reorganization of vimentin filaments through direct vimentin phosphorylation. [source]

    Phosphorylation and lipid raft association of fibroblast growth factor receptor-2 in oligodendrocytes,

    GLIA, Issue 9 2009
    M. R. Bryant
    Abstract Fibroblast growth factors (FGFs) and their receptors (FGFRs) initiate diverse cellular responses that contribute to the regulation of oligodendrocyte (OL) function. To understand the mechanisms by which FGFRs elicit these cellular responses, we investigated the phosphorylation of signal transduction proteins and the role of cholesterol-glycosphingolipid-enriched "lipid raft" microdomains in differentiated OLs. Surprisingly, we found that the most abundant tyrosine-phosphorylated protein in OLs was the 120-kd isoform of FGFR2 and that it was phosphorylated even in the absence of FGF2, suggesting a potential ligand-independent function for this receptor. Furthermore, FGFR2, but not FGFR1, was associated with lipid raft microdomains in OLs and myelin (but not in astrocytes). This provides the first evidence for the association of FGFR with TX-100-insoluble lipid raft fractions. FGFR2 phosphorylated the key downstream target, FRS2 in OLs. Raft disruption resulted in loss of phosphorylated FRS2 from lipid rafts, coupled with the loss of Akt but not of Mek or Erk phosphorylation. This suggests that FGFR2-FRS2 signaling in lipid rafts operates via the PI3-Kinase/Akt pathway rather than the Ras/Mek/Erk pathway, emphasizing the importance of microenvironments within the cell membrane. Also present in lipid rafts in OLs and myelin, but not in astrocytes, was a novel 52-kd isoform of FGFR2 that lacked the extracellular ligand-binding region. These results demonstrate that FGFR2 in OLs and myelin possess unique characteristics that are specific both to receptor type and to OLs and provide a novel mechanism to elicit distinct cellular responses that mediate both FGF-dependent and -independent functions. © 2008 Wiley-Liss, Inc. [source]

    2,4-Dioxa-7-aza-, 2,4-Dioxa-8-aza-, and 2,4-Dioxa-9-aza-3-phosphadecalins as Rigid Acetylcholine Mimetics: Syntheses and Characterization

    Stefan Furegati
    Phosphorylation of suitable piperidine precursors yielded a series of novel decalin-type O,N,P-heterocycles. The title compounds, P(3)-axially and P(3)-equatorially X-substituted, cis- and trans- configurated 2,4-dioxa-7-aza-, 2,4-dioxa-8-aza-, and 2,4-dioxa-9-aza-3-phosphabicyclo[4.4.0]decane 3-oxides (X=Cl, F, 4-nitrophenoxy, and 2,4-dinitrophenoxy), are configuratively fixed and conformationally constrained P-analogues of acetylcholine and as such represent acetylcholine (7-aza and 9-aza isomers) or , -homo-acetylcholine mimetics (8-aza isomers). Being irreversible inhibitors of acetylcholinesterase (AChE), the compounds are considered to be suitable probes for the investigation of the stereochemical course of the inhibition reaction by 31P-NMR spectroscopy. Moreover, the design of these mimetics will enable studies of molecular interactions with AChE, in particular, the recognition conformation of acetylcholine. [source]