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KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes.
McKenzie AJ, Hoshino D, Hong NH, Cha DJ, Franklin JL, Coffey RJ, Patton JG, Weaver AM
(2016) Cell Rep 15: 978-987
MeSH Terms: Argonaute Proteins, Cell Line, Tumor, Exosomes, Humans, MicroRNAs, Mitogen-Activated Protein Kinase Kinases, Multivesicular Bodies, Mutant Proteins, Phosphorylation, Phosphoserine, Protein Transport, Proto-Oncogene Proteins p21(ras), Signal Transduction, Subcellular Fractions
Show Abstract · Added April 29, 2016
Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
1 Communities
3 Members
0 Resources
14 MeSH Terms
In vivo neuronal function of the fragile X mental retardation protein is regulated by phosphorylation.
Coffee RL, Williamson AJ, Adkins CM, Gray MC, Page TL, Broadie K
(2012) Hum Mol Genet 21: 900-15
MeSH Terms: Animals, Animals, Genetically Modified, Brain, Cytoskeleton, Disease Models, Animal, Drosophila Proteins, Drosophila melanogaster, Fragile X Mental Retardation Protein, Fragile X Syndrome, Humans, Learning, Microtubule-Associated Proteins, Mutation, Neuromuscular Junction, Neurons, Phosphorylation, Phosphoserine, Protein Biosynthesis, Serine, Transgenes
Show Abstract · Added May 29, 2014
Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene product (FMRP), is the most common heritable cause of intellectual disability and autism spectrum disorders. It has been long hypothesized that the phosphorylation of serine 500 (S500) in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, we employed neuronally targeted expression of three human FMR1 transgenes, including wild-type (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1), in the Drosophila FXS disease model to investigate phosphorylation requirements. At the molecular level, dfmr1 null mutants exhibit elevated brain protein levels due to loss of translational repressor activity. This defect is rescued for an individual target protein and across the population of brain proteins by the phosphomimetic, whereas the dephosphomimetic phenocopies the null condition. At the cellular level, dfmr1 null synapse architecture exhibits increased area, branching and bouton number. The phosphomimetic fully rescues these synaptogenesis defects, whereas the dephosphomimetic provides no rescue. The presence of Futsch-positive (microtubule-associated protein 1B) supernumerary microtubule loops is elevated in dfmr1 null synapses. The human phosphomimetic restores normal Futsch loops, whereas the dephosphomimetic provides no activity. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. We conclude that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior.
1 Communities
2 Members
0 Resources
20 MeSH Terms
The stability and transactivation potential of the mammalian MafA transcription factor are regulated by serine 65 phosphorylation.
Guo S, Burnette R, Zhao L, Vanderford NL, Poitout V, Hagman DK, Henderson E, Ozcan S, Wadzinski BE, Stein R
(2009) J Biol Chem 284: 759-65
MeSH Terms: Amino Acid Sequence, Animals, Cells, Cultured, Conserved Sequence, Electrophoresis, Polyacrylamide Gel, Gene Expression Regulation, Glycogen Synthase Kinase 3, Humans, Maf Transcription Factors, Large, Mice, Molecular Sequence Data, Mutation, Phosphoserine, Rats, Sequence Alignment, Transcriptional Activation, Ubiquitin, Ubiquitination
Show Abstract · Added December 10, 2013
The level of the MafA transcription factor is regulated by a variety of effectors of beta cell function, including glucose, fatty acids, and insulin. Here, we show that phosphorylation at Ser(65) of mammalian MafA influences both protein stability and transactivation potential. Replacement of Ser(65) with Glu to mimic phosphorylation produced a protein that was as unstable as the wild type, whereas Asp or Ala mutation blocked degradation. Analysis of MafA chimeric and deletion constructs suggests that protein phosphorylation at Ser(65) alone represents the initial degradation signal, with ubiquitinylation occurring within the C terminus (amino acids 234-359). Although only wild type MafA and S65E were polyubiquitinylated, both S65D and S65E potently stimulated transactivation compared with S65A. Phosphorylation at Ser(14) also enhanced activation, although it had no impact on protein turnover. The mobility of MafA S65A was profoundly affected upon SDS-PAGE, with the S65E and S65D mutants influenced less due to their ability to serve as substrates for glycogen synthase kinase 3, which acts at neighboring N-terminal residues after Ser(65) phosphorylation. Our observations not only illustrate the sensitivity of the cellular transcriptional and degradation machinery to phosphomimetic mutants at Ser(65), but also demonstrate the singular importance of phosphorylation at this amino acid in regulating MafA activity.
0 Communities
2 Members
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18 MeSH Terms
PDGF receptor activation induces p120-catenin phosphorylation at serine 879 via a PKCalpha-dependent pathway.
Brown MV, Burnett PE, Denning MF, Reynolds AB
(2009) Exp Cell Res 315: 39-49
MeSH Terms: Catenins, Cell Adhesion Molecules, Cell Line, Cell Membrane, Enzyme Activation, Fibroblasts, Humans, Isoenzymes, Phosphoproteins, Phosphorylation, Phosphoserine, Platelet-Derived Growth Factor, Protein Kinase C-alpha, Receptors, G-Protein-Coupled, Receptors, Platelet-Derived Growth Factor
Show Abstract · Added March 5, 2014
p120-catenin (p120) is required for cadherin stability and is thought to have a central role in modulating cell-cell adhesion. Several lines of evidence suggest that S/T phosphorylation may regulate p120 activity, but the upstream kinases involved have not been established, nor has a discreet measurable function been assigned to an individual site. To approach these issues, we have generated p120 phospho-specific monoclonal antibodies to several individual phosphorylation sites and are using them to pinpoint upstream kinases and signaling pathways that control p120 activity. Protein Kinase C (PKC) has been implicated as a signaling intermediate in several cadherin-associated cellular activities. Signaling events that activate PKC induce rapid phosphorylation at p120 Serine 879 (S879), suggesting that p120 activity is regulated, in part, by one or more PKC isoforms. Here, we find that physiologic activation of a G-protein coupled receptor (i.e., endothelin receptor), as well as several Receptor Tyrosine Kinases, induce rapid and robust p120 phosphorylation at S879, suggesting that these pathways crosstalk to cadherin complexes via p120. Using Va2 cells and PDGF stimulation, we show for the first time that PDGFR-mediated phosphorylation at this site is dependent on PKCalpha, a conventional PKC isoform implicated previously in disruption of adherens junctions.
1 Communities
1 Members
0 Resources
15 MeSH Terms
Ryanodine receptor phosphorylation at Serine 2030, 2808 and 2814 in rat cardiomyocytes.
Huke S, Bers DM
(2008) Biochem Biophys Res Commun 376: 80-5
MeSH Terms: Animals, Antibodies, Phospho-Specific, Base Sequence, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Cells, Cultured, Cyclic AMP-Dependent Protein Kinases, Isoproterenol, Myocytes, Cardiac, Phosphoric Monoester Hydrolases, Phosphorylation, Phosphoserine, Rats, Ryanodine Receptor Calcium Release Channel, Serine
Show Abstract · Added May 27, 2014
The cardiac ryanodine receptor (RyR) controls Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling. Three phosphorylation sites have been identified: Serine-(S)2808, S2814 and recently S2030. We measured phosphorylation with at least two different antibodies per site and demonstrate that for S2808 results were highly antibody-dependent and two out of three S2808 antibodies did not accurately report phosphorylation level. The RyR was substantially phosphorylated in quiescent rat cardiomyocytes at S2808 and less so at S2814, but appeared to be unphosphorylated at S2030. Basal phosphorylation at S2808/S2814 was maintained by a Ca2+ dependent kinase other than Ca2+/Calmodulin-dependent kinase (CaMKII). During stimulation with Isoproterenol S2808 was phosphorylated by protein kinase A (PKA) and S2814 was phosphorylated by CaMKII. Phosphatase 1 appears to be the main phosphatase dephosphorylating S2808/S2814, but phosphatase 2a may also dephosphorylate S2814. RyR phosphorylation is complex, but important in understanding RyR functional modulation.
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1 Members
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15 MeSH Terms
Glucocorticoid receptor phosphorylation differentially affects target gene expression.
Chen W, Dang T, Blind RD, Wang Z, Cavasotto CN, Hittelman AB, Rogatsky I, Logan SK, Garabedian MJ
(2008) Mol Endocrinol 22: 1754-66
MeSH Terms: Amino Acid Sequence, Antibodies, Phospho-Specific, Cell Line, Tumor, Curcumin, Egtazic Acid, Gene Expression Regulation, Humans, Kinetics, Ligands, Mediator Complex, Models, Biological, Models, Molecular, Molecular Sequence Data, Mutation, Peptides, Phosphorylation, Phosphoserine, Protein Structure, Secondary, Receptors, Glucocorticoid, Signal Transduction, Trans-Activators, Transcriptional Activation
Show Abstract · Added August 18, 2015
The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.
0 Communities
1 Members
0 Resources
22 MeSH Terms
Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity.
Kumar A, Harris TE, Keller SR, Choi KM, Magnuson MA, Lawrence JC
(2008) Mol Cell Biol 28: 61-70
MeSH Terms: Animals, Biological Transport, Carrier Proteins, GTPase-Activating Proteins, Gene Expression Regulation, Glucose, Glycogen, Glycogen Synthase, Insulin, Male, Mice, Mice, Knockout, Muscles, Phosphorylation, Phosphoserine, Phosphothreonine, Proto-Oncogene Proteins c-akt, Rapamycin-Insensitive Companion of mTOR Protein
Show Abstract · Added August 22, 2011
Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.
3 Communities
1 Members
0 Resources
18 MeSH Terms
Global assessment of regulation of phosphorylation of insulin receptor substrate-1 by insulin in vivo in human muscle.
Yi Z, Langlais P, De Filippis EA, Luo M, Flynn CR, Schroeder S, Weintraub ST, Mapes R, Mandarino LJ
(2007) Diabetes 56: 1508-16
MeSH Terms: Adult, Biopsy, Blood Glucose, Female, Glucose Clamp Technique, Glucose Tolerance Test, Homeostasis, Humans, Insulin, Insulin Receptor Substrate Proteins, Kinetics, Male, Muscle, Skeletal, Phosphoproteins, Phosphorylation, Phosphoserine, Phosphothreonine, Reference Values
Show Abstract · Added December 10, 2013
OBJECTIVE - Research has focused on insulin receptor substrate (IRS)-1 as a locus for insulin resistance. Tyrosine phosphorylation of IRS-1 initiates insulin signaling, whereas serine/threonine phosphorylation alters the ability of IRS-1 to transduce the insulin signal. Of 1,242 amino acids in IRS-1, 242 are serine/threonine. Serine/threonine phosphorylation of IRS-1 is affected by many factors, including insulin. The purpose of this study was to perform global assessment of phosphorylation of serine/threonine residues in IRS-1 in vivo in humans.
RESEARCH DESIGN AND METHODS - In this study, we describe our use of capillary high-performance liquid chromotography electrospray tandem mass spectrometry to identify/quantify site-specific phosphorylation of IRS-1 in human vastus lateralis muscle obtained by needle biopsy basally and after insulin infusion in four healthy volunteers.
RESULTS - Twenty-two serine/threonine phosphorylation sites were identified; 15 were quantified. Three sites had not been previously identified (Thr495, Ser527, and S1005). Insulin increased the phosphorylation of Ser312, Ser616, Ser636, Ser892, Ser1101, and Ser1223 (2.6 +/- 0.4, 2.9 +/- 0.8, 2.1 +/- 0.3, 1.6 +/- 0.1, 1.3 +/- 0.1, and 1.3 +/- 0.1-fold, respectively, compared with basal; P < 0.05); phosphorylation of Ser348, Thr446, Thr495, and Ser1005 decreased (0.4 +/- 0.1, 0.2 +/- 0.1, 0.1 +/- 0.1, and 0.3 +/- 0.2-fold, respectively; P < 0.05).
CONCLUSIONS - These results provide an assessment of IRS-1 phosphorylation in vivo and show that insulin has profound effects on IRS-1 serine/threonine phosphorylation in healthy humans.
0 Communities
1 Members
0 Resources
18 MeSH Terms
Temporal dissociation of frequency-dependent acceleration of relaxation and protein phosphorylation by CaMKII.
Huke S, Bers DM
(2007) J Mol Cell Cardiol 42: 590-9
MeSH Terms: Animals, Calcium-Binding Proteins, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium-Calmodulin-Dependent Protein Kinases, Enzyme Inhibitors, Heart, Muscle Cells, Phosphoric Monoester Hydrolases, Phosphorylation, Phosphoserine, Phosphothreonine, Rats, Ryanodine Receptor Calcium Release Channel, Time Factors
Show Abstract · Added May 27, 2014
Frequency-dependent acceleration of relaxation (FDAR) is an important intrinsic mechanism that allows for diastolic filling of the ventricle at higher heart rates, yet its molecular mechanism is still not understood. Previous studies showed that FDAR is dependent on functional sarcoplasmic reticulum (SR) and can be abolished by phosphatase or by Ca/CaM kinase (CaMKII) inhibition. Additionally, CaMKII activity/autophosphorylation has been shown to be frequency-dependent. Thus, we tested the hypothesis that CaMKII phosphorylation of SR Ca(2+)-handling proteins (Phospholamban (PLB), Ca(2+) release channel (RyR)) mediates FDAR. Here we show that FDAR occurs abruptly in fluo-4 loaded isolated rat ventricular myocytes when frequency is raised from 0.1 to 2 Hz. The effect is essentially complete within four beats (2 s) with the tau of [Ca(2+)](i) decline decreasing by 42+/-3%. While there is a detectable increase in PLB Thr-17 and RyR Ser-2814 phosphorylation, the increase is quantitatively small (PLB<5%, RyR approximately 8%) and the time-course is clearly delayed with regard to FDAR. The low substrate phosphorylation indicates that pacing of myocytes only mildly activates CaMKII and consistent with this CaMKIIdelta autophosphorylation did not increase with pacing alone. However, in the presence of phosphatase 1 inhibition pacing triggered a net-increase in autophosphorylated CaMKII and also greatly enhanced PLB and RyR phosphorylation. We conclude that FDAR does not rely on phosphorylation of PLB or RyR. Even though CaMKII does become activated when myocytes are paced, phosphatases immediately antagonize CaMKII action, limit substrate phosphorylation and also prevent sustained CaMKII autophosphorylation (thereby suppressing global CaMKII effects).
0 Communities
1 Members
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14 MeSH Terms
Serine-phosphorylated STAT1 is a prosurvival factor in Wilms' tumor pathogenesis.
Timofeeva OA, Plisov S, Evseev AA, Peng S, Jose-Kampfner M, Lovvorn HN, Dome JS, Perantoni AO
(2006) Oncogene 25: 7555-64
MeSH Terms: Apoptosis Regulatory Proteins, Casein Kinase II, Cell Line, Cell Line, Tumor, Cell Survival, Child, Green Fluorescent Proteins, HSP27 Heat-Shock Proteins, Heat-Shock Proteins, Humans, Kidney, Kidney Neoplasms, Myeloid Cell Leukemia Sequence 1 Protein, Neoplasm Proteins, Phosphorylation, Phosphoserine, Proto-Oncogene Proteins c-bcl-2, STAT1 Transcription Factor, Transfection, Wilms Tumor
Show Abstract · Added December 26, 2013
Wilms' tumor (WT), one of the most common pediatric solid cancers, arises in the developing kidney as a result of genetic and epigenetic changes that lead to the abnormal proliferation and differentiation of the metanephric blastema. As activation of signal transducers and activators of transcription (STATs) plays an important role in the maintenance/growth and differentiation of the metanephric blastema, and constitutively activated STATs facilitate neoplastic behaviors of a variety of cancers, we hypothesized that dysregulation of STAT signaling may also contribute to WT pathogenesis. Accordingly, we evaluated STAT phosphorylation patterns in tumors and found that STAT1 was constitutively phosphorylated on serine 727 (S727) in 19 of 21 primary WT samples and two WT cell lines. An inactivating mutation of S727 to alanine reduced colony formation of WT cells in soft agar by more than 80% and induced apoptosis under conditions of growth stress. S727-phosphorylated STAT1 provided apoptotic resistance for WT cells via upregulation of expression of the heat-shock protein (HSP)27 and antiapoptotic protein myeloid cell leukemia (MCL)-1. The kinase responsible for STAT1 S727 phosphorylation in WT cells was identified based upon the use of selective inhibitors as protein kinase CK2, not p38, MAP-kinase kinase (MEK)1/2, phosphatidylinositol 3'-kinase, protein kinase C or Ca/calmodulin-dependent protein kinase II (CaMKII). The inhibition of CK2 blocked the anchorage-independent growth of WT cells and induced apoptosis under conditions of growth stress. Our findings suggest that serine-phosphorylated STAT1, as a downstream target of protein kinase CK2, plays a critical role in the pathogenesis of WT and possibly other neoplasms with similar STAT1 phosphorylation patterns.
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1 Members
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20 MeSH Terms