14-3-3ζ regulates nuclear trafficking of protein phosphatase 1α (PP1α) in HEK-293 cells

2014 ◽  
Vol 558 ◽  
pp. 28-35 ◽  
Author(s):  
Myrka Jérôme ◽  
Hemant K. Paudel
Keyword(s):  
Protein Phosphatase ◽  
Nuclear Trafficking ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

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.

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 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

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
Author(s):  
Kevin J. Gingrich ◽  
Son Tran ◽  
Igor M. Nikonorov ◽  
Thomas J. Blanck
Keyword(s):  
Charge Transfer ◽  
Calcium Channels ◽  
Dependent Manner ◽  
Current Time ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

2007 ◽  
Vol 9 (4) ◽  
pp. 475-485 ◽  
Author(s):  
R. M. Johann ◽  
Ch. Baiotto ◽  
Ph. Renaud
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Calcium Induces Expression of Cytoplasmic Gelsolin in SH-SY5Y and HEK-293 Cells

2010 ◽  
Vol 35 (7) ◽  
pp. 1075-1082 ◽  
Author(s):  
Lina Ji ◽  
Abha Chauhan ◽  
Ved Chauhan
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Cytoplasmic Gelsolin

Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells

2007 ◽  
Vol 454 (3) ◽  
pp. 441-450 ◽  
Author(s):  
Christian Barmeyer ◽  
Jeff Huaqing Ye ◽  
Shafik Sidani ◽  
John Geibel ◽  
Henry J. Binder ◽  
...  
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Abstract 5316: hERG 1b as a Potential Target for Inherited and Acquired Long QT Syndrome

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Gail A Robertson ◽  
Harinath Sale ◽  
David Tester ◽  
Thomas J O’Hara ◽  
Pallavi Phartiyal ◽  
...  
Keyword(s):  
Action Potential ◽  
Long Qt Syndrome ◽  
Time Course ◽  
Drug Sensitivity ◽  
Long Qt ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Acquired Long Qt Syndrome ◽  
293 Cells ◽  
Qt Syndrome

Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long QT syndrome. Biochemical studies show that native I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homo-oligomers of the original hERG 1a isolate. The hERG 1a and 1b subunits are identical except at the amino (NH2) terminus, which in hERG 1b is much shorter and has a unique primary sequence. We compared the biophysical properties of currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We found that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a two-fold increase in the apparent rates of activation and recovery from inactivation, which reduces rectification and facilitates current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the two channel types. Depending on the action potential model used, loss of 1b predicts an increase in action potential duration of 27 ms (7%) or 41 ms (17%), respectively. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding four-fold positive shift in the IC 50 . Differences in current kinetics and drug sensitivity were modeled by “NH2 mode” gating with conformational states bound by the amino terminus in hERG 1a homomers but not 1a/1b heteromers. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients and absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells nearly eliminated 1b protein. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr , prolong QT interval and enhance drug sensitivity, thus representing a potential mechanism underlying inherited or acquired LQTS.

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