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 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.

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 The over-expression of TRPC6 channels in HEK-293 cells favours the intracellular accumulation of zinc

2011 ◽  
Vol 1808 (12) ◽  
pp. 2807-2818 ◽  
Author(s):  
Julien Gibon ◽  
Peng Tu ◽  
Sylvain Bohic ◽  
Pierre Richaud ◽  
Josiane Arnaud ◽  
...  
Keyword(s):  
Intracellular Accumulation ◽  
Hek 293 ◽  
Over Expression ◽  
Hek 293 Cells ◽  
293 Cells

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.

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.

scholarly journals Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽  
2013 ◽  
Vol 9 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Patience Musiwaro ◽  
Matthew Smith ◽  
Maria Manifava ◽  
Simon A. Walker ◽  
Nicholas T. Ktistakis
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells
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