In vitro neutralization of the inhibitory effect of microcystin-LR to protein phosphatase 2A by antibody against the toxin

Abstract Rapamycin is an immunosuppressant that effectively controls various immune responses; however, its action in the signal transduction of lymphocytes has remained largely unknown. We show here that a phosphoprotein encoded by mouse α4 (mα4) gene transmitting a signal through B-cell antigen receptor (BCR) is associated with the catalytic subunit of protein phosphatase 2A (PP2Ac). The middle region of α4, consisting of 109 amino acids (94-202), associates directly with PP2Ac, irrespective of any other accessory molecule. Rapamycin treatment disrupts the association of PP2Ac/α4 in parallel with the inhibitory effect of lymphoid cell proliferation. The effect of rapamycin was inhibited with an excess amount of FK506 that potentially completes the binding to FKBP. Rapamycin treatment also suppresses the phosphatase activity of cells measured by in vitro phosphatase assay. Introduction of the mα4 cDNA into Jurkat cells or the increased association of PP2Ac/α4 by the culture with low serum concentration confers cells with rapamycin resistance. Moreover, glutathione S-transferase (GST)-α4 augments the PP2A activity upon myelin basic protein (MBP) and histone in the in vitro assay. These results suggest that α4 acts as a positive regulator of PP2A and as a new target of rapamycin in the activation of lymphocytes.

Download Full-text

Ig Receptor Binding Protein 1 (α4) Is Associated With a Rapamycin-Sensitive Signal Transduction in Lymphocytes Through Direct Binding to the Catalytic Subunit of Protein Phosphatase 2A

Blood ◽

10.1182/blood.v92.2.539.414k23_539_546 ◽

1998 ◽

Vol 92 (2) ◽

pp. 539-546

Author(s):

Seiji Inui ◽

Hideki Sanjo ◽

Kazuhiko Maeda ◽

Hideyuki Yamamoto ◽

Eishichi Miyamoto ◽

...

Keyword(s):

Signal Transduction ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Inhibitory Effect ◽

Catalytic Subunit ◽

Jurkat Cells ◽

Vitro Assay ◽

Rapamycin Treatment ◽

Phosphatase 2A

Rapamycin is an immunosuppressant that effectively controls various immune responses; however, its action in the signal transduction of lymphocytes has remained largely unknown. We show here that a phosphoprotein encoded by mouse α4 (mα4) gene transmitting a signal through B-cell antigen receptor (BCR) is associated with the catalytic subunit of protein phosphatase 2A (PP2Ac). The middle region of α4, consisting of 109 amino acids (94-202), associates directly with PP2Ac, irrespective of any other accessory molecule. Rapamycin treatment disrupts the association of PP2Ac/α4 in parallel with the inhibitory effect of lymphoid cell proliferation. The effect of rapamycin was inhibited with an excess amount of FK506 that potentially completes the binding to FKBP. Rapamycin treatment also suppresses the phosphatase activity of cells measured by in vitro phosphatase assay. Introduction of the mα4 cDNA into Jurkat cells or the increased association of PP2Ac/α4 by the culture with low serum concentration confers cells with rapamycin resistance. Moreover, glutathione S-transferase (GST)-α4 augments the PP2A activity upon myelin basic protein (MBP) and histone in the in vitro assay. These results suggest that α4 acts as a positive regulator of PP2A and as a new target of rapamycin in the activation of lymphocytes.

Download Full-text

IMMU-47. PROTEIN PHOSPHATASE 2A INHIBITION IN NK CELL THERAPY FOR TREATMENT OF MEDULLOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.477 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii115-ii115

Author(s):

Rongze Olivia Lu ◽

Winson Ho ◽

Brandon Chiou

Keyword(s):

Nk Cells ◽

Cell Line ◽

Protein Phosphatase ◽

Nk Cell ◽

Protein Phosphatase 2A ◽

T Cell Response ◽

Intrinsic Activity ◽

Cd107a Expression ◽

Phosphatase 2A

Abstract Checkpoint immunotherapy (ICB) thus far has shown limited efficacy against brain tumors, such as medulloblastoma (MB). Its low mutational burden is thought to result in a paucity of neoantigen to trigger an effective T-cell response. Natural killer (NK) cells, can recognize tumor cells independently of neoantigens, making them appealing against MBs. Modulation of NK cells to enhance cytotoxicity against MBs could be a novel treatment strategy. Protein Phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase, has been shown to inhibit IFNg and Granzyme B production by NK cells. We hypothesize that NK92, a transformed human NK cell line, has intrinsic activity against human MB cells and that inhibiting PP2A pharmacologically can enhance cytotoxicity of NK92 cells. We performed NK cytotoxicity assay and granulation assay against human MB cell line D425. We also used a small molecular inhibitor, LB100, to modulate PP2A activity in NK92. NK92 cells were co-cultured with D425, in increasing E:T (Effector:Target) ratio for 4 hours. D425 cells were pre-labeled with CellTrace Violet dye. The percentage of D425 (Violet+) cells in apoptosis (Cas3/7+) or necrosis (AAD+) were compared with different ET ratios to quantify NK mediated cell cytotoxicity. We also measured CD107a expression in NK92 to assess granulation with LB100 treatment. D425 cells were sensitive to NK92 killing. Percentage of D425 cells either apoptotic or necrotic increased with increasing ET ratio, suggesting that there was NK92 mediated cytotoxicity. Percentage of killed D425 cells ranged from 18% at baseline (without NK92) to 80% at ET ratio of 20. Inhibition of PP2A using LB100, enhanced NK92 degranulation. CD107a+ NK92 cells increased from 19% to 28% with 8uM of LB100. NK92 cells are cytotoxic against MB cells in vitro and inhibition of PP2A in NK cells can enhance their activity against MB cells.

Download Full-text

Abstract 212: Inhibition of Protein Phosphatase 2A (PP2A) Leads to Beta-adrenergic Receptor Dysfunction With Cardiac Stress Following Pressure-overload Hypertrophy

Circulation Research ◽

10.1161/res.117.suppl_1.212 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Arunachal Chatterjee ◽

Neelakantan Vasudevan ◽

Maradumane Mohan ◽

Elizabeth Martelli ◽

John George ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Adenylyl Cyclase ◽

Cardiac Dysfunction ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Cyclase Activity ◽

Adenylyl Cyclase Activity ◽

Cardiac Stress ◽

Phosphatase 2A

Beta-Adrenergic receptors (bARs) play a key role in regulating cardiac function. Loss of surface receptors and desensitization (impaired G-protein coupling) of bARs are hallmarks of a failing heart. Desensitization occurs by phosphorylation of bARs. The bARs are resensitized by protein phosphatase 2A (PP2A) mediated dephosphorylation in the endosomes before recycling to the plasma membrane. While mechanisms of desensitization are well understood, little is known about mechanisms regulating resensitization. Our previous work has shown that PI3Kg phosphorylates an endogenous inhibitor of PP2A (I2PP2A) on serine 9 & 93, which then robustly binds to PP2A inhibiting bAR resensitization. Since it is not known whether resensitization is altered in response to cardiac stress or whether altered bAR resensitization contributes to cardiac hypertrophy and failure, we generated transgenic mice with cardiomyocyte specific overexpression of wild type I2PP2A (WT I2PP2A Tg), I2PP2A phospho-mimetic mutants S9, 93D and mutants with constitutively dephosphorylated S9, 93A state. To test whether resensitization is critical in the development of bAR dysfunction during cardiac hypertrophy, WT I2PP2A Tg mice were subjected to transverse aortic constriction (TAC) for 8 weeks. Echocardiographic analysis post-TAC showed that WT I2PP2A Tg mice had accelerated cardiac dysfunction compared to their littermate controls [HW (mg)/BW(g): Sham: WT - 4.83, WT I2PP2A Tg - 4.82, TAC: WT- 6.47, WT I2PP2A Tg - 7.61; %EF: Sham: WT - 83.53, WT I2PP2A Tg - 74.72, TAC: WT - 70.47, WT I2PP2A Tg - 49.62]. To directly test whether resensitization mechanisms are altered, plasma membranes and endosomes were isolated and in vitro Adenylyl Cyclase activity assessed. Our studies show that compared to littermate controls, WT I2PP2A Tg had altered in vitro adenylyl cyclase activity showing that resensitization mechanisms in the endosomes may in part, contribute to cardiac dysfunction. Mechanistic underpinnings of the resensitization pathways using the I2PP2A S9, 93A and S9, 93D will be presented showing that bAR resensitization a process considered passive is altered in conditions of cardiac stress that in part may contribute to bAR dysfunction leading to cardiac hypertrophy and heart failure.

Download Full-text

Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A.

The EMBO Journal ◽

10.1002/j.1460-2075.1989.tb08568.x ◽

1989 ◽

Vol 8 (12) ◽

pp. 3891-3898 ◽

Cited By ~ 66

Author(s):

D.M. Virshup ◽

M.G. Kauffman ◽

T.J. Kelly

Keyword(s):

Dna Replication ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Cellular Protein ◽

Phosphatase 2A ◽

Sv40 Dna

Download Full-text

Loss of a Protein Phosphatase 2A Regulatory Subunit (Cdc55p) Elicits Improper Regulation of Swe1p Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.20.21.8143-8156.2000 ◽

2000 ◽

Vol 20 (21) ◽

pp. 8143-8156 ◽

Cited By ~ 37

Author(s):

Haifeng Yang ◽

Wei Jiang ◽

Matthew Gentry ◽

Richard L. Hallberg

Keyword(s):

Phosphatase Activity ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Cell Types ◽

Regulatory Subunit ◽

Cyclin Dependent Kinase ◽

Wild Type ◽

Phosphatase 2A

ABSTRACT CDC55 encodes a Saccharomyces cerevisiaeprotein phosphatase 2A (PP2A) regulatory subunit.cdc55-null cells growing at low temperature exhibit a failure of cytokinesis and produce abnormally elongated buds, butcdc55-null cells producing the cyclin-dependent kinase Cdc28-Y19F, which is unable to be inhibited by Y19 phosphorylation, show a loss of the abnormal morphology. Furthermore,cdc55-null cells exhibit a hyperphosphorylation of Y19. For these reasons, we have examined in wild-type and cdc55-null cells the levels and activities of the kinase (Swe1p) and phosphatase (Mih1p) that normally regulate the extent of Cdc28 Y19 phosphorylation. We find that Mih1p levels are comparable in the two strains, and an estimate of the in vivo and in vitro phosphatase activity of this enzyme in the two cell types indicates no marked differences. By contrast, while Swe1p levels are similar in unsynchronized and S-phase-arrested wild-type and cdc55-null cells, Swe1 kinase is found at elevated levels in mitosis-arrestedcdc55-null cells. This excess Swe1p incdc55-null cells is the result of ectopic stabilization of this protein during G2 and M, thereby accounting for the accumulation of Swe1p in mitosis-arrested cells. We also present evidence indicating that, in cdc55-null cells, misregulated PP2A phosphatase activity is the cause of both the ectopic stabilization of Swe1p and the production of the morphologically abnormal phenotype.

Download Full-text

An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator

Biochemical Journal ◽

10.1042/bj20031643 ◽

2004 ◽

Vol 380 (1) ◽

pp. 111-119 ◽

Cited By ~ 72

Author(s):

Sari LONGIN ◽

Jan JORDENS ◽

Ellen MARTENS ◽

Ilse STEVENS ◽

Veerle JANSSENS ◽

...

Keyword(s):

Phosphatase Activity ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Phosphatase 2A ◽

Inactive Protein

We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.

Download Full-text

Protein Phosphatase 2A Stabilizes Human Securin, Whose Phosphorylated Forms Are Degraded via the SCF Ubiquitin Ligase

Molecular and Cellular Biology ◽

10.1128/mcb.01904-05 ◽

2006 ◽

Vol 26 (11) ◽

pp. 4017-4027 ◽

Cited By ~ 39

Author(s):

Ana M. Gil-Bernabé ◽

Francisco Romero ◽

M. Cristina Limón-Mortés ◽

María Tortolero

Keyword(s):

Ubiquitin Ligase ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Anaphase Promoting Complex ◽

Anaphase Onset ◽

Chromatid Segregation ◽

Phosphatase 2A ◽

F Box Protein

ABSTRACT Sister chromatid segregation is triggered at the metaphase-to-anaphase transition by the activation of the protease separase. For most of the cell cycle, separase activity is kept in check by its association with the inhibitory chaperone securin. Activation of separase occurs at anaphase onset, when securin is targeted for destruction by the anaphase-promoting complex or cyclosome E3 ubiquitin protein ligase. This results in the release of the cohesins from chromosomes, which in turn allows the segregation of sister chromatids to opposite spindle poles. Here we show that human securin (hSecurin) forms a complex with enzymatically active protein phosphatase 2A (PP2A) and that it is a substrate of the phosphatase, both in vitro and in vivo. Treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cul1/F-box protein complex ubiquitin ligase. We propose that PP2A regulates hSecurin levels by counteracting its phosphorylation, which promotes its degradation. Misregulation of this process may lead to the formation of tumors, in which overproduction of hSecurin is often observed.

Download Full-text