IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities

In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711→Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1S711E displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1S711E retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1S711E completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1S711E is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.

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GFP Fusions for Fluorescence Detection of Ca2+ and Ca2+-Calmodulin in Living Cells

Microscopy and Microanalysis ◽

10.1017/s1431927600025162 ◽

1998 ◽

Vol 4 (S2) ◽

pp. 1008-1009

Author(s):

Anthony Persechini

Keyword(s):

Mammalian Cells ◽

Dissociation Constants ◽

Hek 293 ◽

Fluorescent Indicators ◽

Calmodulin Binding ◽

Binding Domains ◽

293 Cells ◽

20 Nm ◽

Binding Sequence ◽

Expression In Mammalian Cells

We have previously described fluorescent indicators for Ca2+ (FIP-CAs) and (Ca2+)4-calmodulin (FIP-CBs) whose responses are based on a ligand-dependent decrease in fluorescence energy transfer (FRET) between GFP variants. The indicators for (Ca2+)4-calmodulin contain calmodulin-binding domains, those for Ca2+ also contain an integral calmodulin (CaM) domain. We have developed new versions of these indicators constructed with enhanced blue- and red-shifted GFPs suitable for stable and transient expression in mammalian cells, and have begun to use them to investigate the relationships between the free intracellular concentrations of Ca2+ ([Ca2+]i) and (Ca2+)4-CaM ([(Ca2+)4-CaM]i). When the blue-shifted fluorophore is excited at 380 nm these constructs exhibit an emission peak at 505 nm due to FRET to the red-shifted fluorophore.We have made FIP-CBs with dissociation constants for (Ca2+)4-CaM of 0.5 nM, 20 nM, 300 nM and > 20 μM by introducing R →Q substitutions in the CaM-binding sequence, and have stablyexpressed them in HEK-293 cells (Fig. 1).

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Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event

Biochemical Journal ◽

10.1042/bj20031259 ◽

2004 ◽

Vol 377 (1) ◽

pp. 249-255 ◽

Cited By ~ 189

Author(s):

Adam COLE ◽

Sheelagh FRAME ◽

Philip COHEN

Keyword(s):

Tyrosine Phosphorylation ◽

Mammalian Cells ◽

Protein Tyrosine Kinase ◽

Kinase Inhibitor ◽

Glycogen Synthase ◽

Glycogen Synthase Kinase ◽

Hek 293 ◽

293 Cells ◽

General Protein ◽

The Stability

Phosphorylation of the endogenous GSK3α (glycogen synthase kinase-3α) at Tyr279 and GSK3β at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3β transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not. GSK3β expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3β was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3β lacking phosphate at Tyr216. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.

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KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.4.c859 ◽

2001 ◽

Vol 280 (4) ◽

pp. C859-C866 ◽

Cited By ~ 73

Author(s):

Rikke Søgaard ◽

Trine Ljungstrøm ◽

Kamilla Angelo Pedersen ◽

Søren-Peter Olesen ◽

Bo Skaaning Jensen

Keyword(s):

Mammalian Cells ◽

Inward Rectification ◽

Excitable Cells ◽

Independent Manner ◽

Hek 293 ◽

M Current ◽

Gating Charge ◽

293 Cells ◽

Boltzmann Function ◽

Instantaneous Current

Human cloned KCNQ4 channels were stably expressed in HEK-293 cells and characterized with respect to function and pharmacology. Patch-clamp measurements showed that the KCNQ4 channels conducted slowly activating currents at potentials more positive than −60 mV. From the Boltzmann function fitted to the activation curve, a half-activation potential of −32 mV and an equivalent gating charge of 1.4 elementary charges was determined. The instantaneous current-voltage relationship revealed strong inward rectification. The KCNQ4 channels were blocked in a voltage-independent manner by the memory-enhancing M current blockers XE-991 and linopirdine with IC50 values of 5.5 and 14 μM, respectively. The antiarrhythmic KCNQ1 channel blocker bepridil inhibited KCNQ4 with an IC50 value of 9.4 μM, whereas clofilium was without significant effect at 100 μM. The KCNQ4-expressing cells exhibited average resting membrane potentials of −56 mV in contrast to −12 mV recorded in the nontransfected cells. In conclusion, the activation and pharmacology of KCNQ4 channels resemble those of M currents, and it is likely that the function of the KCNQ4 channel is to regulate the subthreshold electrical activity of excitable cells.

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Human geneSLC41A1encodes for the Na+/Mg2+exchanger

AJP Cell Physiology ◽

10.1152/ajpcell.00289.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. C318-C326 ◽

Cited By ~ 75

Author(s):

Martin Kolisek ◽

Axel Nestler ◽

Jürgen Vormann ◽

Monika Schweigel-Röntgen

Keyword(s):

Mammalian Cells ◽

Molecular Level ◽

Transport Activity ◽

Human Embryonic Kidney ◽

Efflux System ◽

Dependent Protein Kinase ◽

Complete Replacement ◽

Hek 293 ◽

293 Cells ◽

Dependent Protein

Magnesium (Mg2+), the second most abundant divalent intracellular cation, is involved in the vast majority of intracellular processes, including the synthesis of nucleic acids, proteins, and energy metabolism. The concentration of intracellular free Mg2+([Mg2+]i) in mammalian cells is therefore tightly regulated to its optimum, mainly by an exchange of intracellular Mg2+for extracellular Na+. Despite the importance of this process for cellular Mg2+homeostasis, the gene(s) encoding for the functional Na+/Mg2+exchanger is (are) still unknown. Here, using the fluorescent probe mag-fura 2 to measure [Mg2+]ichanges, we examine Mg2+extrusion from hSLC41A1-overexpressing human embryonic kidney (HEK)-293 cells. A three- to fourfold elevation of [Mg2+]iwas accompanied by a five- to ninefold increase of Mg2+efflux. The latter was strictly dependent on extracellular Na+and reduced by 91% after complete replacement of Na+with N-methyl-d-glucamine. Imipramine and quinidine, known unspecific Na+/Mg2+exchanger inhibitors, led to a strong 88% to 100% inhibition of hSLC41A1-related Mg2+extrusion. In addition, our data show regulation of the transport activity via phosphorylation by cAMP-dependent protein kinase A. As these are the typical characteristics of a Na+/Mg2+exchanger, we conclude that the human SLC41A1 gene encodes for the Na+/Mg2+exchanger, the predominant Mg2+efflux system. Based on this finding, the analysis of Na+/Mg2+exchanger regulation and its involvement in the pathogenesis of diseases such as Parkinson's disease and hypertension at the molecular level should now be possible.

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Cytoprotection by glycine against ATP-depletion-induced injury is mediated by glycine receptor in renal cells1

Biochemical Journal ◽

10.1042/bj20050141 ◽

2005 ◽

Vol 390 (2) ◽

pp. 447-453 ◽

Cited By ~ 25

Author(s):

Chao Pan ◽

Xiaoming Bai ◽

Leming Fan ◽

Yong Ji ◽

Xiaoyu Li ◽

...

Keyword(s):

Mammalian Cells ◽

Molecular Mechanisms ◽

Cell Injury ◽

Glycine Receptor ◽

Membrane Integrity ◽

Cellular Membrane ◽

Atp Depletion ◽

Hek 293 ◽

293 Cells ◽

Marker Compounds

It is known that glycine protects mammalian cells against ischaemic cell injury by preventing cellular membrane leakage. However, the molecular mechanisms have not yet been clearly elucidated. The purpose of the present study was to clarify whether GlyR (glycine receptor) acts as a key mediator in cytoprotection of glycine. cDNA encoding human GlyRα1 (α1-subunit of glycine receptor) was transfected into HEK-293 cells. The membrane integrity of the cells with or without GlyRα1 was examined by the uptake of marker compounds, the release of LDH (lactate dehydrogenase) and the exclusion of Trypan Blue. Glycine prevented the permeability of 70 kDa dextrans and 140 kDa LDH in the cells in which GlyR was expressed under conditions of ATP depletion. The inhibition of endogenous GlyR expression by RNA interference attenuated the cytoprotection by glycine. Furthermore, the mutation of Tyr202 to phenylalanine in GlyRα1 blocked the glycine-mediated cytoprotection, while the mutation of Tyr202 to leucine abolished the cytoprotection by strychnine. Our results suggested that the cytoprotection of glycine against ATP-depletion-induced injury might be mediated by GlyR.

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Mutation of protein kinase C phosphorylation site S1076 on α-subunits affects BKCa channel activity in HEK-293 cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90518.2008 ◽

2009 ◽

Vol 297 (4) ◽

pp. L758-L766 ◽

Cited By ~ 10

Author(s):

Shu Zhu ◽

Darren D. Browning ◽

Richard E. White ◽

David Fulton ◽

Scott A. Barman

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Phosphorylation Site ◽

Channel Activity ◽

Excitatory Effect ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Smooth Muscle Function ◽

Α Subunits

Large conductance, calcium- and voltage-activated potassium (BKCa) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BKCa channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BKCa channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BKCa channel α-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BKCa channels. Results showed that mutating S1076 altered the effect of PKC activation on BKCa channels in HEK-293 cells. Specifically, the phospho-deficient mutation BKCa-α(S1076A)/β1 attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BKCa channels, whereas the phospho-mimetic mutation BKCa-α(S1076E)/β1 increased the excitatory effect of PMA on BKCa channels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BKCa channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BKCa channel α-subunits influences BKCa channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

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Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1

Biochemical Journal ◽

10.1042/bj20081340 ◽

2009 ◽

Vol 418 (1) ◽

pp. 93-101 ◽

Cited By ~ 29

Author(s):

Bei You ◽

Guijun Yan ◽

Zhiling Zhang ◽

Lin Yan ◽

Jing Li ◽

...

Keyword(s):

Troponin I ◽

Molecular Conformation ◽

Phosphorylation Site ◽

Dominant Negative ◽

Binding Assays ◽

Interacting Protein ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

293 Cells

Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, cTn [cardiac Tn (troponin)] I was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells (human embryonic kidney cells) and native cardiomyocytes, in which cTnI interacted with full-length Mst1, but not with its N-terminal kinase fragment. in vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1. In contrast, cTnT was phosphorylated by Mst1 only when it was incorporated into the Tn complex. MS analysis indicated that Mst1 phosphorylates cTnI at Thr31, Thr51, Thr129 and Thr143. Substitution of Thr31 with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr51, Thr129 or Thr143 with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr31 is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr31. Protein epitope analysis and binding assays showed that Mst1-mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. The results of the present study suggest that Mst1 is a novel mediator of cTnI phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.

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Direct Observation of Sophorolipid Micelle Docking in Model Membranes and Cells by Single Particle Studies Reveals Optimal Fusion Conditions

Biomolecules ◽

10.3390/biom10091291 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1291 ◽

Cited By ~ 1

Author(s):

Pradeep Kumar Singh ◽

Søren S.-R. Bohr ◽

Nikos S. Hatzakis

Keyword(s):

Cancer Cells ◽

Hela Cells ◽

Single Particle ◽

Mammalian Cells ◽

Model Membranes ◽

Live Cells ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Sophorolipids (SLs) are naturally produced glycolipids that acts as drug delivery for a spectrum of biomedical applications, including as an antibacterial antifungal and anticancer agent, where they induce apoptosis selectively in cancerous cells. Despite their utility, the mechanisms underlying their membrane interactions, and consequently cell entry, remains unknown. Here, we combined a single liposome assay to observe directly and quantify the kinetics of interaction of SL micelles with model membrane systems, and single particle studies on live cells to record their interaction with cell membranes and their cytotoxicity. Our single particle readouts revealed several repetitive docking events on individual liposomes and quantified how pH and membrane charges, which are known to vary in cancer cells, affect the docking of SL micelles on model membranes. Docking of sophorolipids micelles was found to be optimal at pH 6.5 and for membranes with −5% negatively charge lipids. Single particle studies on mammalian cells reveled a two-fold increased interaction on Hela cells as compared to HEK-293 cells. This is in line with our cell viability readouts recording an approximate two-fold increased cytotoxicity by SLs interactions for Hela cells as compared to HEK-293 cells. The combined in vitro and cell assays thus support the increased cytotoxicity of SLs on cancer cells to originate from optimal charge and pH interactions between membranes and SL assemblies. We anticipate studies combining quantitative single particle studies on model membranes and live cell may reveal hitherto unknown molecular insights on the interactions of sophorolipid and additional nanocarriers mechanism.

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Depletion of the polyamines spermidine and spermine by overexpression of spermidine/spermine N1-acetyltransferase 1 (SAT1) leads to mitochondria-mediated apoptosis in mammalian cells

Biochemical Journal ◽

10.1042/bj20150168 ◽

2015 ◽

Vol 468 (3) ◽

pp. 435-447 ◽

Cited By ~ 17

Author(s):

Swati Mandal ◽

Ajeet Mandal ◽

Myung Hee Park

Keyword(s):

Mammalian Cells ◽

Growth Arrest ◽

Mitochondrial Pathway ◽

Human Embryonic Kidney ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Catabolic Enzyme

Adenoviral overexpression of a polyamine catabolic enzyme spermidine (SSAT1)/spermine N1-acetyltransferase 1 (SAT1) in human embryonic kidney (HEK) 293 cells leads to a rapid depletion of spermidine and spermine, growth arrest and apoptosis through the intrinsic mitochondrial pathway.

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Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888

AJP Cell Physiology ◽

10.1152/ajpcell.00194.2013 ◽

2014 ◽

Vol 306 (3) ◽

pp. C298-C306 ◽

Cited By ~ 9

Author(s):

Steven H. Young ◽

Osvaldo Rey ◽

James Sinnett-Smith ◽

Enrique Rozengurt

Keyword(s):

Negative Feedback ◽

Small Interfering Rna ◽

Phosphorylation Site ◽

Hek 293 ◽

Pkc Inhibitor ◽

Identical Response ◽

293 Cells ◽

Intracellular Ca ◽

Interfering Rna

To clarify the mechanism(s) underlying intracellular Ca2+ concentration ([Ca2+]i) oscillations induced by an elevation in extracellular Ca2+ concentration ([Ca2+]e) via the extracellular Ca2+-sensing receptor (CaR), we analyzed the pattern of [Ca2+]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca2+]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca2+]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca2+]i oscillations, cells were exposed to increasing concentrations (0.5–5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca2+. Ro-31-8220 at 3–5 μM completely eliminated the [Ca2+]e-evoked [Ca2+]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca2+]e-evoked [Ca2+]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca2+]e-evoked [Ca2+]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr888 was converted to alanine (CaRT888A) showed [Ca2+]i oscillations after CaR activation. Our results show that [Ca2+]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca2+ or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr888.

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