scholarly journals Down-regulation of alphav/beta3 integrin via misrouting to lysosomes by overexpression of a beta3Lamp1 fusion protein

2003 ◽  
Vol 370 (2) ◽  
pp. 703-711 ◽  
Author(s):  
Magali CONESA ◽  
Annik PRAT ◽  
John S. MORT ◽  
Jacques MARVALDI ◽  
Jean-Claude LISSITZKY ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Cell Types ◽  
Degradation Pathway ◽  
Dominant Negative ◽  
General Strategy ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Cellular Aggregation ◽  
Beta3 Integrin

We present a general strategy for the dominant negative reduction in the levels of type-1 membrane-bound heterodimeric proteins within the secretory pathway through fusion of the soluble ectodomain of one of the partners to the transmembrane-cytosolic tail of the lysosomal protein Lamp1. Thus, in human embryonic kidney (HEK)-293 cells, overexpression of an integrin β3Lamp1 chimera resulted in a drastic reduction of its endogenous partner, the integrin αv subunit. The mechanism involves the formation in the endoplasmic reticulum of a αv/β3Lamp1 complex that is subsequently sorted towards a lysosomal/endosomal degradation pathway. The specificity of this approach is afforded by the invariance in the levels of the endogenous integrins α5 and β1 as compared with control cells. Conversely overexpression of integrin β3 in HEK-293 cells led to an increased level of αvβ3 at the cell surface. Functionally β3Lamp1 and β3 overexpressors exhibit decreased and increased adhesion to vitronectin, respectively, as well as diminished cellular aggregation. The application of this technology should enable the analysis of the functional importance of homodimers or heterodimers in the cell types of choice and the identification of novel partner proteins by proteomic approaches.

Extracellular ATP dissociates nonmuscle myosin from P2X7complex: this dissociation regulates P2X7pore formation

2009 ◽  
Vol 297 (2) ◽  
pp. C430-C439 ◽  
Author(s):  
Ben J. Gu ◽  
Catherine Rathsam ◽  
Leanne Stokes ◽  
Andrew B. McGeachie ◽  
James S. Wiley
Keyword(s):  
Specific Inhibitor ◽  
Life Time ◽  
Cell Types ◽  
Interferon Γ ◽  
Extracellular Atp ◽  
Nonmuscle Myosin ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Myosin Va

The P2X7receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X7channel and massive K+efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X7were isolated by using anti-P2X7monoclonal antibody-coated Dynabeads from both interferon-γ plus LPS-stimulated monocytic THP-1 cells and P2X7-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X7in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X7receptor by ATP caused dissociation of P2X7from nonmuscle myosin in both cell types. The interaction of P2X7and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X7pore function without any increase in surface expression or ion channel function of P2X7receptors. S- l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X7-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X7and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X7channel to a pore.

scholarly journals Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cϵ

2007 ◽  
Vol 405 (3) ◽  
pp. 591-596 ◽  
Author(s):  
Jun-ichi Saito ◽  
Shinnosuke Toriumi ◽  
Kenjiro Awano ◽  
Hidenori Ichijo ◽  
Keiichi Sasaki ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Feedback Regulation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Regulation Mechanism ◽  
H2o2 Treatment ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
293 Cells

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFα (tumour necrosis factor α). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cϵ (protein phosphatase 2Cϵ), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cϵ inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cϵ mutant enhanced AP-1 activity. Exogenous PP2Cϵ associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cϵ and ASK1 was also observed in mouse brain extracts. PP2Cϵ directly dephosphorylated ASK1 at Thr845in vitro. In contrast with PP5, PP2Cϵ transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cϵ maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cϵ and PP5 play different roles in H2O2-induced regulation of ASK1 activity.

Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase

2013 ◽  
Vol 394 (5) ◽  
pp. 685-693 ◽  
Author(s):  
Petra Cordes ◽  
Wei Sun ◽  
Rainer Wolber ◽  
Ludger Kolbe ◽  
Gerhard Klebe ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Heterologous Expression ◽  
Western Blotting ◽  
Kluyveromyces Lactis ◽  
Cell Types ◽  
Hek 293 ◽  
Activity Staining ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Human Tyrosinase

Abstract Mammalian tyrosinases are key enzymes of melanin formation. Their native forms undergo complex maturation and sorting processes before being integrated into the melanosomal membrane, which greatly complicates their heterologous expression in other cell types. In the present work, we constructed several differently truncated, soluble variants of human tyrosinase and studied their properties after expression in HEK 293 cells. In addition, we prepared two affinity-tagged forms of the enzyme for expression in the yeast Kluyveromyces lactis and HEK cells, respectively. A Strep-tagged variant was secreted by K. lactis in excellent yields but found to be inactive, whereas a His-tagged variant secreted by HEK 293 cells in an active state could be purified from cell supernatants to near homogeneity. The resulting preparation consisted of an inactive, probably unglycosylated species of about 57 kDa and several glycosylated forms with masses between 63 and 75 kDa, as confirmed by activity staining, Western blotting and mass spectrometry.

PKC-δ sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells

2005 ◽  
Vol 289 (3) ◽  
pp. C543-C556 ◽  
Author(s):  
Sean G. Brown ◽  
Alison Thomas ◽  
Lodewijk V. Dekker ◽  
Andrew Tinker ◽  
Joanne L. Leaney
Keyword(s):  
Fluorescent Protein ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Membrane Phospholipid ◽  
Channel Activity ◽  
Receptor Stimulation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Channel Inhibition ◽  
293 Cells

G protein-gated inward rectifier (Kir3) channels are inhibited by activation of Gq/11-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP2). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M3 receptor activation. In perforated patch mode, staurosporine prevented the Gq/11-mediated, M3 receptor, inhibition of channel activity. Recovery from M3-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP2, were still irreversibly inhibited by M3 receptor stimulation. When adenosine A1 receptors were co-expressed, inclusion of PIP2 rescued the A1-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-δ mediates channel inhibition because recombinant PKC-δ inhibited channel activity, M3-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-δ constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-δ to the plasma membrane on M3 receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-δ after M3 receptor activation in HEK-293 cells.

scholarly journals Calcitonin stimulates expression of the rat 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter in HEK-293 cells expressing calcitonin receptor: identification of signaling pathways

2004 ◽  
Vol 32 (1) ◽  
pp. 87-98 ◽  
Author(s):  
XH Gao ◽  
PP Dwivedi ◽  
JL Omdahl ◽  
HA Morris ◽  
BK May
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Basal Expression ◽  
293 Cells ◽  
Gc Box

Regulation of the gene for renal 25-hydroxyvitamin D-24-hydroxylase (CYP24) is important for controlling the level of circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We report here for the first time that the peptide hormone calcitonin significantly stimulates expression of a rat CYP24 promoter-luciferase construct in both transiently and stably transfected kidney HEK-293 cells. A GC box at -114/-101 and a CCAAT box at -62/-51 have been identified that underlie both basal expression of the CYP24 promoter and the calcitonin inductive response. Data from overexpression studies suggested that Sp1 and NF-Y are the proteins that function through the GC and CCAAT boxes respectively. ERK1/2 signaling pathways were not involved in the calcitonin-mediated response, since stimulation of the promoter was unaffected by the pharmacological ERK1/2 inhibitor PD98059 and by a dominant negative mutant of ERK1/2 (ERK1K71R). In contrast, calcitonin induction but not basal expression was dependent on protein kinase A and protein kinase C (PKC) activities with the inhibitors H89 and calphostin C lowering induction by 50-60%. The atypical PKC, PKCzeta contributes to calcitonin induction, but not to basal expression of the CYP24 promoter, since overexpression of a dominant negative clone PKCzetaK281 M lowered induction by 50%. Cotransfection of a dominant negative form of Ras resulted in calcitonin-mediated induction being reduced also by about 50%. A Ras-PKCzeta signaling pathway for calcitonin action is proposed, which acts through the GC box. The findings have been extrapolated to the in vivo situation where we suggest that induction of renal CYP24 by calcitonin could be important under hypercalcemic conditions thus contributing to the lowering of circulating 1,25(OH)2D3 levels.

scholarly journals Protein phosphatase 1 regulates the stability of the circadian protein PER2

2006 ◽  
Vol 399 (1) ◽  
pp. 169-175 ◽  
Author(s):  
Monica Gallego ◽  
Heeseog Kang ◽  
David M. Virshup
Keyword(s):  
Protein Phosphatase ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Protein Phosphatase 1 ◽  
Casein Kinase I ◽  
Hek 293 ◽  
Over Expression ◽  
Hek 293 Cells ◽  
Egg Extract ◽  
293 Cells

The circadian clock is regulated by a transcription/translation negative feedback loop. A key negative regulator of circadian rhythm in mammals is the PER2 (mammalian PERIOD 2) protein. Its daily degradation at the end of the night accompanies de-repression of transcription. CKIϵ (casein kinase I ϵ) has been identified as the kinase that phosphorylates PER2, targeting it for ubiquitin-mediated proteasomal degradation. We now report that PER2 degradation is also negatively regulated by PP1 (protein phosphatase 1)-mediated dephosphorylation. In Xenopus egg extract, PP1 inhibition by Inhibitor-2 accelerated mPER2 degradation. Co-immunoprecipitation experiments showed that PER2 bound to PP1c in transfected HEK-293 cells. PP1 immunoprecipitated from HEK-293 cells, mouse liver and mouse brain, dephosphorylated CKIϵ-phosphorylated PER2, showing that PER2 is a substrate for mammalian endogenous PP1. Moreover, over-expression of the dominant negative form of PP1c, the D95N mutant, accelerated ubiquitin and proteasome-mediated degradation of PER2, and shortened the PER2 half-life in HEK-293 cells. Over-expression of the PP1 inhibitors, protein phosphatase 1 holoenzyme inhibitor-1 and Inhibitor-2, confirmed these results. Thus PP1 regulates PER2 stability and is therefore a candidate to regulate mammalian circadian rhythms.

scholarly journals A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells

2003 ◽  
Vol 369 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Sanna PARTANEN ◽  
Stephan STORCH ◽  
Hans-Gerhard LÖFFLER ◽  
Andrej HASILIK ◽  
Jaana TYYNELÄ ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Active Site ◽  
Cathepsin D ◽  
Secretory Pathway ◽  
Neuronal Ceroid Lipofuscinosis ◽  
Aspartic Acid Residue ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Lysosomal Protease ◽  
293 Cells

The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786—2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme.

scholarly journals Wild-type and mutant ferroportins do not form oligomers in transfected cells

2006 ◽  
Vol 396 (2) ◽  
pp. 265-275 ◽  
Author(s):  
Ana Sofia Gonçalves ◽  
Françoise Muzeau ◽  
Rand Blaybel ◽  
Gilles Hetet ◽  
Fathi Driss ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Kidney ◽  
Cell Types ◽  
Dominant Negative ◽  
Wild Type ◽  
Dominant Form ◽  
Hek 293 ◽  
C Terminus ◽  
293 Cells ◽  
Iron Export

Ferroportin [FPN; Slc40a1 (solute carrier family 40, member 1)] is a transmembrane iron export protein expressed in macrophages and duodenal enterocytes. Heterozygous mutations in the FPN gene result in an autosomal dominant form of iron overload disorder, type-4 haemochromatosis. FPN mutants either have a normal iron export activity but have lost their ability to bind hepcidin, or are defective in their iron export function. The mutant protein has been suggested to act as a dominant negative over the wt (wild-type) protein by multimer formation. Using transiently transfected human epithelial cell lines expressing mouse FPN modified by the addition of a haemagglutinin or c-Myc epitope at the C-terminus, we show that the wtFPN is found at the plasma membrane and in Rab5-containing endosomes, as are the D157G and Q182H mutants. However, the delV162 mutant is mostly intracellular in HK2 cells (human kidney-2 cells) and partially addressed at the cell surface in HEK-293 cells (human embryonic kidney 293 cells). In both cell types, it is partially associated with the endoplasmic reticulum and with Rab5-positive vesicles. However, this mutant is complex-glycosylated like the wt protein. D157G and G323V mutants have a defective iron export capacity as judged by their inability to deplete the intracellular ferritin content, whereas Q182H and delV162 have normal iron export function and probably have lost their capacity to bind hepcidin. In co-transfection experiments, the delV162 mutant does not co-localize with the wtFPN, does not prevent its normal targeting to the plasma membrane and cannot be immunoprecipitated in the same complex, arguing against the formation of FPN hetero-oligomers.

pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells: implications for pH regulation in the secretory pathway

2003 ◽  
Vol 285 (1) ◽  
pp. C205-C214 ◽  
Author(s):  
Terry E. Machen ◽  
Mary Jae Leigh ◽  
Carmen Taylor ◽  
Tohru Kimura ◽  
Shinji Asano ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Ph Regulation ◽  
Buffer System ◽  
Β Cells ◽  
Hek 293 ◽  
Recycling Endosomes ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Golgi Network

The influences of the gastric H+/K+ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the α- and β-subunits of human H+/K+-ATPase (H+/K+-α,β cells). The pH values of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-α,β cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H+/K+-ATPase was present in the plasma membrane, TGN, and RE. The pHTGN was similar in both H+/K+-α,β cells (pHTGN 6.36) and vector-transfected (“mock”) cells (pHTGN 6.34); pHRE was also similar in H+/K+-α,β (pHRE 6.40) and mock cells (pHRE 6.37). SCH28080 (inhibits H+/K+-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H+ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pHTGN of 7.0–7.5, close to pHcytosol. Similar results were observed in RE. Thus H+/K+-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8,000) active in the TGN to balance a large, passive H+ leak (with PH ∼10–3 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H+/K+-ATPases (estimated to be ∼4,000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane.

scholarly journals Leukemia inhibitory factor regulates trafficking of T-type Ca2+ channels

2011 ◽  
Vol 300 (3) ◽  
pp. C576-C587 ◽  
Author(s):  
Deblina Dey ◽  
Andrew Shepherd ◽  
Judith Pachuau ◽  
Miguel Martin-Caraballo
Keyword(s):  
Membrane Trafficking ◽  
Leukemia Inhibitory Factor ◽  
Fluorescent Protein ◽  
Expression System ◽  
Functional Expression ◽  
Dominant Negative ◽  
Inhibitory Factor ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca2+ channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca2+ channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca2+ channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca2+ channel α1H-subunit resulted in the generation of transient Ca2+ currents. Overnight treatment of α1H-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca2+ channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca2+ channels. Trafficking of α1H-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α1H-GFP subunits. Trafficking of T-type Ca2+ channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca2+ channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca2+ channels.

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