Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding

2008 ◽  
Vol 410 (1) ◽  
pp. 195-204 ◽  
Author(s):  
Ingeborg Winge ◽  
Jeffrey A. Mckinney ◽  
Ming Ying ◽  
Clive S. D'Santos ◽  
Rune Kleppe ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tryptophan Hydroxylase ◽  
Enzyme Stability ◽  
Enzyme Activation ◽  
Site Directed Mutagenesis ◽  
Peripheral Tissues ◽  
Hek 293 ◽  
Rate Limiting Step ◽  
293 Cells ◽  
Dependent Protein

TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca2+/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser19, but PKA also phosphorylated Ser104, as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser19, Ser99, Ser104 and Ser306. On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser19. This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins γ, ϵ and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.

scholarly journals Molecular mechanism of TMEM16A regulation: role of CaMKII and PP1/PP2A

2019 ◽  
Vol 317 (6) ◽  
pp. C1093-C1106 ◽  
Author(s):  
Ramon J. Ayon ◽  
Matthew B. Hawn ◽  
Joydeep Aoun ◽  
Michael Wiwchar ◽  
Abigail S. Forrest ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Inhibitory Peptide ◽  
Dependent Protein Kinase ◽  
Initial Current ◽  
Pipette Solution ◽  
Hek 293 ◽  
293 Cells ◽  
Dependent Protein

This study explored the mechanism by which Ca2+-activated Cl− channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl− currents ( IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.

ClC-2 Cl− channels in human lung epithelia: activation by arachidonic acid, amidation, and acid-activated omeprazole

2001 ◽  
Vol 281 (1) ◽  
pp. C46-C54 ◽  
Author(s):  
John Cuppoletti ◽  
Kirti P. Tewari ◽  
Ann M. Sherry ◽  
Elena Y. Kupert ◽  
Danuta H. Malinowska
Keyword(s):  
Cystic Fibrosis ◽  
Arachidonic Acid ◽  
Protein Kinase ◽  
Human Lung ◽  
Kinase Inhibitor ◽  
Hek 293 ◽  
Potential Target ◽  
293 Cells ◽  
Cl Channels ◽  
Dependent Protein

ClC-2 Cl− channels represent a potential target for therapy in cystic fibrosis. Key questions regarding the feasibility of using ClC-2 as a therapeutic target are addressed in the present studies, including whether the channels are present in human lung epithelia and whether activators of the channel can be identified. Two new mechanisms of activation of human recombinant ClC-2 Cl− channels expressed in HEK-293 cells were identified: amidation with glycine methyl ester catalyzed by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and treatment with acid-activated omeprazole. ClC-2 mRNA was detected by RT-PCR. Channel function was assessed by measuring Cl− currents by patch clamp in the presence of a cAMP-dependent protein kinase (PKA) inhibitor, myristoylated protein kinase inhibitor, to prevent PKA-activated Cl− currents. Calu-3, A549, and BEAS-2B cell lines derived from different human lung epithelia contained ClC-2 mRNA, and Cl− currents were increased by amidation, acid-activated omeprazole, and arachidonic acid. Similar results were obtained with buccal cells from healthy individuals and cystic fibrosis patients. The ClC-2 Cl− channel is thus a potential target for therapy in cystic fibrosis.

Layers of organization of cAMP microdomains in a simple cell

2006 ◽  
Vol 34 (4) ◽  
pp. 480-483 ◽  
Author(s):  
A.C.L. Martin ◽  
D.M.F. Cooper
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Simple Cell ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Phosphodiesterase 4 ◽  
293 Cells ◽  
Phosphatase 2A ◽  
Dependent Protein

Based on a variety of single-cell measurements, the notion that cAMP microdomains exist in cells is being increasingly embraced. The cellular and molecular underpinnings of this organization are also steadily being revealed. A dependence of Ca2+-sensitive ACs (adenylate cyclases) in HEK-293 cells (human embryonic kidney cells) on capacitative Ca2+ entry is enforced by their presence in lipid rafts and protein–protein interactions. In these cells, many of the participants in the cAMP cascade, including AC, phosphodiesterase 4, cAMP-dependent protein kinase [PKA (protein kinase A)] and protein phosphatase 2A, are now seen to be involved in higher order assemblies. Moreover, the presence of Na+/H+ exchanger 1 in these domains creates a microclimate, protected against global swings in cellular pH. The Ca2+-stimulatable AC8, which is targeted to these regions, can sequester calmodulin for its own regulatory purposes. These devices are a sampling of the multiple layers of organization that are in place – even in a simple cell – to ensure faithful and economical communication of the cAMP message.

scholarly journals Effect of thyroliberin on the concentration of adenosine 3′:5′-phosphate and on the activity of adenosine 3′:5′-phosphate-dependent protein kinase in prolactin-producing cells in culture

1977 ◽  
Vol 162 (2) ◽  
pp. 379-386 ◽  
Author(s):  
K M Gautvik ◽  
E Walaas ◽  
O Walaas
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Time Course ◽  
Pituitary Tumour ◽  
Enzyme Activation ◽  
Protein Kinase Activity ◽  
Half Maximum ◽  
Prolactin Release ◽  
Dependent Protein Kinase ◽  
Dependent Protein

1. The effects of thyroliberin were studied in cultured rat pituitary-tumour cells that synthesize and secrete prolactin (the GH4C1 cell strain). 2. Prolactin and cyclic AMP were measured by radioimmunological methods, and a cyclic AMP-dependent protein kinase was characterized by using histone as substrate. 3. Prolactin release was studied after 5-60min of treatment, and synthesis after 48h of treatment with thyroliberin. One-half maximum stimulation of release and synthesis were observed at 0.25 and at 4nM respectively. 4. Cyclic AMP was temporarily increased in cell suspensions after treatment with thyroliberin, and one-half maximum stimulation was observed at 25nM. 5. Dibutyryl cyclic AMP increased prolactin release and synthesis, one-half maximum effects being obtained at 20 micronM. 6. A cyclic AMP-dependent protein kinase, which was one-half maximally stimulated at 30 nM-cyclic AMP, was demonstrated. 7. An increase in the activity ratio (-cyclic AMP/+cyclic AMP) of the cyclic AMP-dependent protein kinase was observed after treatment with thyroliberin. Total protein kinase activity in the presence of cyclic AMP was unaltered. The time-course of enzyme activation was similar to that of cyclic AMP formation and corresponded to the time when prolactin release was first observed. 8. It is concluded that thyroliberin induces cyclic AMP formation, resulting in the activation of a cyclic AMP-dependent protein kinase.

scholarly journals Expression of tachykinin receptors (tacr1a and tacr1b) in zebrafish: influence of cocaine and opioid receptors

2012 ◽  
Vol 50 (2) ◽  
pp. 115-129 ◽  
Author(s):  
Roger López-Bellido ◽  
Katherine Barreto-Valer ◽  
Raquel E Rodríguez
Keyword(s):  
Embryonic Development ◽  
Opioid Receptors ◽  
Phylogenetic Analyses ◽  
Critical Role ◽  
Cocaine Exposure ◽  
Peripheral Tissues ◽  
Tachykinin Receptors ◽  
Hek 293 ◽  
293 Cells ◽  
Gene Study

Opioid and tachykinin receptors (TACRs) are closely related in addiction and pain processes. In zebrafish, opioid receptors have been cloned and characterized both biochemically and pharmacologically. However, thetacr1gene has not yet been described in zebrafish. The aim of this research was to identify thetacr1gene, study the effects of cocaine ontacr1, and analyze the interaction betweentacr1and opioid receptors. We have identified a duplicate oftacr1gene in zebrafish, designated astacr1aandtacr1b. Phylogenetic analyses revealed an alignment of these receptors in the Tacr1 fish cluster, with a clear distinction from other TACR1s of amphibians, birds, and mammals. Our qPCR results showed thattacr1aandtacr1bmRNAs are expressed during embryonic development. Whole-mountin situhybridization showedtacr1expression in the CNS and in the peripheral tissues. Cocaine (1.5 μM) induced an upregulation oftacr1aandtacr1bat 24 and 48 h post-fertilization (hpf; except fortacr1aat 48 hpf, which was downregulated). By contrast, HEK-293 cells transfected withtacr1aandtacr1band exposed to cocaine showed a downregulation oftacr1s. The knockdown of ZfDOR2 and ZfMOR, opioid receptors, induced a down- and upregulation oftacr1aandtacr1brespectively. In conclusion,tacr1aandtacr1bin zebrafish are widely expressed throughout the CNS and peripherally, suggesting a critical role of thesetacr1sduring embryogenesis.tacr1aandtacr1bmRNA expression is altered by cocaine exposure and by the knockdown of opioid receptors. Thus, zebrafish can provide clues for a better understanding of the relationship between tachykinin and opioid receptors in pain and addiction during embryonic development.

scholarly journals The methylated-DNA binding protein MBD2 enhances NGFI-A (egr-1)-mediated transcriptional activation of the glucocorticoid receptor

2014 ◽  
Vol 369 (1652) ◽  
pp. 20130513 ◽  
Author(s):  
Ian C. G. Weaver ◽  
Ian C. Hellstrom ◽  
Shelley E. Brown ◽  
Stephen D. Andrews ◽  
Sergiy Dymov ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Signalling Pathways ◽  
Hek 293 ◽  
293 Cells ◽  
Exon 1 ◽  
Inducible Protein

Variations in maternal care in the rat influence the epigenetic state and transcriptional activity of glucocorticoid receptor (GR) gene in the hippocampus. The mechanisms underlying this maternal effect remained to be defined, including the nature of the relevant maternally regulated intracellular signalling pathways. We show here that increased maternal licking/grooming (LG), which stably enhances hippocampal GR expression, paradoxically increases hippocampal expression of the methyl-CpG binding domain protein-2 (MBD2) and MBD2 binding to the exon 1 7 GR promoter. Knockdown experiments of MBD2 in hippocampal primary cell culture show that MBD2 is required for activation of exon 1 7 GR promoter. Ectopic co-expression of nerve growth factor-inducible protein A (NGFI-A) with MBD2 in HEK 293 cells with site-directed mutagenesis of the NGFI-A response element within the methylated exon 1 7 GR promoter supports the hypothesis that MBD2 collaborates with NGFI-A in binding and activation of this promoter. These data suggest a possible mechanism linking signalling pathways, which are activated by behavioural stimuli and activation of target genes.

Splice variants of a ClC-2 chloride channel with differing functional characteristics

2000 ◽  
Vol 279 (4) ◽  
pp. C1198-C1210 ◽  
Author(s):  
L. Pablo Cid ◽  
María-Isabel Niemeyer ◽  
Alfredo Ramírez ◽  
Francisco V. Sepúlveda
Keyword(s):  
Splice Variants ◽  
Donor Site ◽  
Proximal Colon ◽  
Site Directed Mutagenesis ◽  
Intestinal Epithelial ◽  
Chloride Currents ◽  
Hek 293 ◽  
Internal Donor ◽  
293 Cells ◽  
Functional Consequences

We identified two ClC-2 clones in a guinea pig intestinal epithelial cDNA library, one of which carries a 30-bp deletion in the NH2 terminus. PCR using primers encompassing the deletion gave two products that furthermore were amplified with specific primers confirming their authenticity. The corresponding genomic DNA sequence gave a structure of three exons and two introns. An internal donor site occurring within one of the exons accounts for the deletion, consistent with alternative splicing. Expression of the variants gpClC-2 and gpClC-2Δ77–86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2Δ77–86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2Δ77–86 is not responsible for the observed change. The deletion-carrying variant is found in most tissues examined, and it appears more abundant in proximal colon, kidney, and testis. The presence of a splice variant of ClC-2 modified in its NH2-terminal domain could have functional consequences in tissues where their relative expression levels are different.

scholarly journals Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

2007 ◽  
Vol 407 (2) ◽  
pp. 231-241 ◽  
Author(s):  
Kathryn M. Geraghty ◽  
Shuai Chen ◽  
Jean E. Harthill ◽  
Adel F. Ibrahim ◽  
Rachel Toth ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Glucose Transporter ◽  
Kinase Inhibitors ◽  
Hek 293 ◽  
293 Cells ◽  
Ribosomal S6 Kinase ◽  
Phosphoinositide 3 Kinase ◽  
Amp Activated Protein Kinase

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA–AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.

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