scholarly journals Protein phosphatase 2A plays a role in hydrogen peroxide-induced disruption of tight junctions in Caco-2 cell monolayers

2009 ◽  
Vol 421 (1) ◽  
pp. 59-70 ◽  
Author(s):  
Parimal Sheth ◽  
Geetha Samak ◽  
J. Andrew Shull ◽  
Ankur Seth ◽  
Radhakrishna Rao
Keyword(s):  
Hydrogen Peroxide ◽  
Tight Junction ◽  
Tight Junctions ◽  
Protein Phosphatase ◽  
Electrical Resistance ◽  
Protein Phosphatase 2A ◽  
Src Kinase ◽  
Cell Monolayers ◽  
Pp2a Activity ◽  
Phosphatase 2A

Evidence indicates that PP2A (protein phosphatase 2A) interacts with epithelial tight junctions and negatively regulates the integrity of the tight junction. In the present study, the role of PP2A in the hydrogen peroxide-induced disruption of the tight junction was examined in Caco-2 cell monolayers. Hydrogen peroxide-induced decrease in electrical resistance and increase in inulin permeability was associated with the dephosphorylation of occludin on threonine residues. The hydrogen peroxide-induced decrease in electrical resistance, increase in inulin permeability and redistribution of occludin and ZO (zonula occludens)-1 from the intercellular junctions were significantly attenuated by selective inhibitors of PP2A (okadaic acid and fostriecin) and by knockdown of PP2A-Cα (the catalytic subunit of PP2A). The PP2A-Cα protein and PP2A activity were co-immunoprecipitated with occludin, and this co-immunoprecipitation was rapidly increased by hydrogen peroxide. Hydrogen peroxideinduced increase in co-immunoprecipitation of PP2A-Cα with occludin was prevented by PP2, a Src kinase inhibitor. GST (glutathione transferase)-pull down assays using recombinant GST–Occludin-C (C-terminal tail of occludin) and the purified PP2A showed that PP2A binds to the C-terminal domain of occludin; Src-induced tyrosine phosphorylation of GST–Occludin-C enhanced this binding. The present study shows that hydrogen peroxide increases the association of PP2A with occludin by a Src kinase-dependent mechanism, and that PP2A activity is involved in hydrogen peroxide-induced disruption of tight junctions in Caco-2 cell monolayers.

Na-K-ATPase regulates tight junction permeability through occludin phosphorylation in pancreatic epithelial cells

2007 ◽  
Vol 292 (1) ◽  
pp. G124-G133 ◽  
Author(s):  
Sigrid A. Rajasekaran ◽  
Sonali P. Barwe ◽  
Jegan Gopal ◽  
Sergey Ryazantsev ◽  
Eveline E. Schneeberger ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Protein Phosphatase ◽  
Mammalian Cells ◽  
Protein Phosphatase 2A ◽  
Epithelial Cell Line ◽  
Phosphatase 2A ◽  
Apical Junctions ◽  
Well Differentiated

Tight junctions are crucial for maintaining the polarity and vectorial transport functions of epithelial cells. We and others have shown that Na-K-ATPase plays a key role in the organization and permeability of tight junctions in mammalian cells and analogous septate junctions in Drosophila. However, the mechanism by which Na-K-ATPase modulates tight junctions is not known. In this study, using a well-differentiated human pancreatic epithelial cell line HPAF-II, we demonstrate that Na-K-ATPase is present at the apical junctions and forms a complex with protein phosphatase-2A, a protein known to be present at tight junctions. Inhibition of Na-K-ATPase ion transport function reduced protein phosphatase-2A activity, hyperphosphorylated occludin, induced rearrangement of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na-K-ATPase is required for controlling the tight junction gate function.

Abstract 229: Novel Plasma Membrane Beta Adrenergic Receptor Resensitization By Protein Phosphatase 2a

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Neelakantan T Vasudevan ◽  
Anita Shukla ◽  
Sathyamangla V Naga Prasad
Keyword(s):  
Plasma Membrane ◽  
Protein Phosphatase ◽  
Adrenergic Receptor ◽  
Protein Phosphatase 2A ◽  
Metabolic Labeling ◽  
Specific Expression ◽  
Pp2a Activity ◽  
Pi3k Activity ◽  
Phosphatase 2A

Resensitization of β-adrenergic receptor (βAR) occurs by dephosphorylation of the internalized βAR by protein phosphatase 2A (PP2A) before being recycled back to plasma membrane (PM). Contrary to this classical paradigm, cardiac specific expression of inactive PI3Kγ (PI3Kγ inact ) leads to receptor resensitization at the plasma membrane as measured by adenylyl cyclase activity in mice chronically treated with the agonist. We hypothesized that PI3K activity inhibits PM receptor resensitization. Using cells stably expressing FLAG-β 1 AR alone (single) or along with PI3Kγ inact (double), we show that inhibition of PI3K activity results in novel PM receptor resensitization. Agonist activation of single stables showed significant receptor desensitization as measured by reduced cAMP generation (35.6 ± 4.6 pmol/mg protein). In contrast, double-or wortmannin (Wort, PI3K inhibitor) pre-treated single- stables showed marked generation of cAMP (87.4 ± 3.2 or 83.1 ± 5.7) showing β 1 AR resensitization. Agonist stimulation of metabolically labeled β 1 ARs pre-treated with inhibitors of internalization, sucrose and β-cyclodextrin resulted in accumulation of phosphorylated receptors at the PM, which was abolished with PI3Kγ inact suggesting dephosphorylation of β 1 ARs. Inhibition of PP2A by okadaic acid or Fostriecin resulted in complete loss of β 1 AR resensitization despite the presence of PI3Kγ inact showing that PI3K regulates PP2A activity at the β 1 AR complex. Pre-treatment of single stable cells with Wort resulted in significant increase in β 1 AR-associated phosphatase activity following dobutamine (Dob) treatment (Veh, 4131 ± 14; Dob, 3180 ± 111; Dob + Wort, 17123 ± 680 pmoles/mg protein). Consistently, similar results were obtained in vivo using single transgenic (FLAG-β 1 AR, Veh, 87 ± 12; Dob, 61.7 ± 8.3) and double transgenic (FLAG-β 1 AR and PI3Kγ inact , Veh, 93 ± 9; Dob, 118 ± 6) mice. In vivo metabolic labeling, co-immunoprecipitation and in vitro kinase assays showed that inhibitor of PP2A (I2PP2A) protein as a target of PI3K in regulating PP2A activity at the β 1 AR complex. Indeed, siRNA knock down of I2PP2A results in preservation of β 1 AR function by PM receptor resensitization demonstrating a novel role for PI3K in receptor resensitization. This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).

scholarly journals Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism

2008 ◽  
Vol 294 (4) ◽  
pp. G1060-G1069 ◽  
Author(s):  
A. Seth ◽  
Fang Yan ◽  
D. Brent Polk ◽  
R. K. Rao
Keyword(s):  
Hydrogen Peroxide ◽  
Tight Junction ◽  
Map Kinase ◽  
Tight Junctions ◽  
Barrier Function ◽  
Kinase Inhibitor ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Cell Monolayers ◽  
Dependent Mechanism

Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and β-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCβI and PKCε. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism.

scholarly journals B56-Associated Protein Phosphatase 2A Is Required For Survival and Protects from Apoptosis in Drosophila melanogaster

2002 ◽  
Vol 22 (11) ◽  
pp. 3674-3684 ◽  
Author(s):  
Xinghai Li ◽  
Anne Scuderi ◽  
Anthea Letsou ◽  
David M. Virshup
Keyword(s):  
Cell Death ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Specific Protein ◽  
S2 Cells ◽  
Regulatory Subunits ◽  
Pp2a Activity ◽  
Phosphatase 2A

ABSTRACT Protein phosphorylation and specific protein kinases can initiate signal transduction pathways leading to programmed cell death. The specific protein phosphatases regulating apoptosis have been more elusive. Using double-stranded RNA-mediated interference (RNAi), the role of protein phosphatase 2A (PP2A) in cellular signaling was investigated. Knockdown of A or C subunits individually or of combined B subunits led to concurrent loss of nontargeted PP2A subunits, suggesting that PP2A is an obligate heterotrimer in vivo. Global knockdown of PP2A activity or specific loss of redundant B56 regulatory subunits caused cell death with the morphological and biochemical changes characteristic of apoptosis in cultured S2 cells. B56:PP2A-regulated apoptosis required caspases and the upstream regulators dark, reaper, head involution defective, and dp53. In Drosophila embryos, knockdown of B56-regulated PP2A activity resulted in apoptosis and failure of gastrulation, an effect that was blocked by concurrent RNAi of the caspase Drice. B56-regulated PP2A activity appears to be required upstream of dp53 to maintain a critical proapoptotic substrate in a dephosphorylated, inactive state, thereby preventing apoptosis in Drosophila S2 cells.

scholarly journals Protein phosphatase 2A interacts with the Src kinase substrate p130CAS

Oncogene ◽  
2001 ◽  
Vol 20 (42) ◽  
pp. 6057-6065 ◽  
Author(s):  
Noriko Yokoyama ◽  
W Todd Miller
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Src Kinase ◽  
Kinase Substrate ◽  
Phosphatase 2A

scholarly journals beta-blocker reverses inhibition of beta-2 adrenergic receptor resensitization by hypoxia

2020 ◽  
Author(s):  
Yu Sun ◽  
Manveen K. Gupta ◽  
Kate Stenson ◽  
Maradumane L. Mohan ◽  
Nicholas Wanner ◽  
...  
Keyword(s):  
Heart Failure ◽  
Protein Phosphatase ◽  
Adrenergic Receptor ◽  
Protein Phosphatase 2A ◽  
Receptor Internalization ◽  
Pp2a Activity ◽  
Phosphatase 2A ◽  
Β Blocker ◽  
Underlying Mechanisms

AbstractIschemia/hypoxia is major underlying cause for heart failure and stroke. Although beta-adrenergic receptor (βAR) is phosphorylated in response to hypoxia, less is known about the underlying mechanisms. Hypoxia results in robust GRK2-mediated β2AR phosphorylation but does not cause receptor internalization. However, hypoxia leads to significant endosomal-β2AR phosphorylation accompanied by inhibition of β2AR-associated protein phosphatase 2A (PP2A) activity impairing resensitization. Phosphoinositide 3-kinase γ (PI3Kγ) impedes resensitization by phosphorylating endogenous inhibitor of protein phosphatase 2A, I2PP2A that inhibits PP2A activity. Hypoxia increased PI3Kγ activity leading to significant phosphorylation of I2PP2A resulting in inhibition of PP2A and consequently resensitization. Surprisingly, β-blocker abrogated hypoxia-mediated β2AR phosphorylation instead of phosphorylation in normoxia. Subjecting mice to hypoxia leads to significant cardiac dysfunction and β2AR phosphorylation showing conservation of non-canonical hypoxia-mediated pathway in vivo. These findings provide mechanistic insights on hypoxia-mediated βAR dysfunction which is rescued by β-blocker and will have significant implications in heart failure and stroke.

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