Role of PP2A in the regulation of p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain

Lee, Taeseung; Kim, Sang Joon; Sumpio, Bauer E.

doi:10.1002/jcp.10211

Cited by 46 publications

(40 citation statements)

References 43 publications

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“…5F, black). These data are consistent with negative p38␣ regulation by PP2A in the cytoplasm (89) and DUSPs in the nucleus (5). Together, our inhibitor results confirm that the assay platform measures all major classes of protein phosphatases in the SE and NE subcellular fractions.…”

Section: Molecular and Cellular Proteomics 16 Supplement 4 S251supporting

confidence: 86%

Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes

Shah¹,

Smolko²,

Kinicki³

et al. 2017

Molecular & Cellular Proteomics

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, and the nucleus. The extracts contain all major classes of protein phosphatases and catalyze dephosphorylation of plate-bound phosphosubstrates in a microtiter format, with cellular activity quantified at the end point by phosphospecific ELISA. The platform is optimized for six phosphosubstrates (ERK2, JNK1, p38␣, MK2, CREB, and STAT1) and measures specific activities from extracts of fewer than 50,000 cells. The assay was exploited to examine viral and antiviral signaling in AC16 cardiomyocytes, which we show can be engineered to serve as susceptible and permissive hosts for CVB3. Phosphatase responses were profiled in these cells by completing a full-factorial experiment for CVB3 infection and type I/II interferon signaling. Over 850 functional measurements revealed several independent, subcellular changes in specific phosphatase activities. During CVB3 infection, we found that type I interferon signaling increases subcellular JNK1 phosphatase activity, inhibiting nuclear JNK1 activity that otherwise promotes viral protein synthesis in the infected host cell. Our assay provides a high-throughput way to capture perturbations in important negative regulators of intracellular signaltransduction networks. Molecular & Cellular Proteomics 16:

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Section: Molecular and Cellular Proteomics 16 Supplement 4 S251supporting

confidence: 86%

Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes

Shah¹,

Smolko²,

Kinicki³

et al. 2017

Molecular & Cellular Proteomics

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“…0.1 mg synthetic EBP50 phosphopeptide (RAHQKR(pS)(pS)KRAPQM) was incubated with 0.1 unit of purified PP1 in PP1 reaction buffer (50 mM Tris-HCl, pH 7.0, 0.1 mM EDTA, 5 mM DTT, 0.01% TX-100 and 1 mM MnCl 2 ) 42 or with 0.1 unit of purified PP2A in PP2A reaction buffer (20 mM HEPES pH7.0, 1 mM DTT, 1 mM MgCl 2 and 100 mg/ml BSA) 43 at 30 1C for 1 h in the absence or presence of calyculin A (100 nM). Subsequently, the phosphorylation status of EBP50 peptide was assessed by immuno-dot blot assay using pEBP50(Ser347-8) antibody.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Chen

Jou

2012

Cell Death Differ

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We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and b-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function. Scaffold proteins have many modular domains that mediate protein-protein interactions involved in orchestrating different signaling cascades. As a result, scaffold proteins are thought to act as stable structural proteins that facilitate many cell signaling pathways. However, this traditional role of scaffold proteins has been challenged, 1 and the dynamic role of scaffold as both an activator and suppressor of cellular signaling remains poorly characterized. Furthermore, the factors that modulate such a bifunctional role of scaffold proteins remain largely unknown.Ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) is a scaffold protein with two tandem PDZ domains and a C-terminal ERM-binding domain. 2,3 EBP50 binds proteins that contain a PDZ-binding motif including ion exchangers, 4,5 ion channels, 6 G protein-coupled receptors, 7-9 growth factor tyrosine kinase receptors 10,11 and PTEN. 10 EBP50 participates in the modulation of ion transport, organization of apical microvilli, cancer development, and the trafficking and stabilization of membrane proteins. 9,12-14 Understanding the mechanisms that regulate the function of EBP50 is, therefore, a critical problem.Ras family proteins are locked in a GDP-bound, inactivated state by binding GDI in the GTP-rich cellular environment. 15,16 Dissociation of small GTPases from GDI is required, but is not sufficient for their activation. GEFs drive the exchange of GTP for GDP, leading to the activation of small GTPases. 17 EBP50 can activate Rho-family small GTPases by recruiting ezrin, which is thought to cause the dissociation of GDI from the small GTPase, and b-PIX, a Rac1 GEF that contains a PDZ-binding motif. 18,19 EBP50 is a serine/threonine-phosphorylated protein. 3 The phosphorylation state of EBP50 regulates the acc...

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“…The kinases that have been found to interact with PP2A include calcium-calmodulin-dependent protein kinase IV (29), p70 S6 kinase (30,31), p21-activated kinases (30), p38 kinase (32), casein kinase II (33), IB kinases (34,35), mitogen-activated protein kinase (MAPK) (36,37), and Akt (15, 38 -41). Although it remains unclear whether B regulatory subunits are involved in the interactions between these kinases and PP2A, several reports using overexpression and RNA interference approaches have demonstrated the roles of the B55 regulatory subunits (42)(43)(44) and the B56 subunits (45) in the MAPK signaling pathway in multiple systems.…”

mentioning

confidence: 99%

Regulation of Phosphorylation of Thr-308 of Akt, Cell Proliferation, and Survival by the B55α Regulatory Subunit Targeting of the Protein Phosphatase 2A Holoenzyme to Akt

Kuo

Huang

Yang

et al. 2008

Journal of Biological Chemistry

338

276

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Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55␣ subunit in NIH3T3 cells. The direct interaction between B55␣ subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55␣ subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55␣ overexpression in these cells. Silencing of B55␣ expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55␣C holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55␣ overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55␣ expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55␣-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.

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Role of PP2A in the regulation of p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain

Cited by 46 publications

References 43 publications

Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes

Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Regulation of Phosphorylation of Thr-308 of Akt, Cell Proliferation, and Survival by the B55α Regulatory Subunit Targeting of the Protein Phosphatase 2A Holoenzyme to Akt

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