scholarly journals STEM-23. INHIBITING PROTEIN PHOSPHATASE 2A INCREASES THE ANTITUMOR EFFECT OF PROTEIN ARGININE METHYLTRANSFERASE 5 INHIBITION IN MODELS OF GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii201-ii201
Author(s):  
Yeshavanth Kumar Banasavadi Siddegowda ◽  
Hannah Sur ◽  
Yoshihiro Otani ◽  
Guruprasad Rachaiah ◽  
Sriya Namagiri ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Tumor Cells ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Xenograft Model ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Pp2a Activity ◽  
Phosphatase 2A

Abstract BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor treating fields, median survival of Glioblastoma (GBM) patients is less than two years. Protein Arginine Methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence of immature GBM tumor cells. LB100, first-in-class small molecule inhibitor of Protein Phosphatase 2A (PP2A) can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Methods: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. Results: We found that PRMT5-depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5 intact GBMNS. LB100 enhanced the G1 cell cycle arrest induced by PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged mice survival, while LB100 treatment alone had no survival benefit. Conclusion: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

scholarly journals Inhibiting protein phosphatase 2A increases the antitumor effect of protein arginine methyltransferase 5 inhibition in models of glioblastoma

2021 ◽  
Author(s):  
Yoshihiro Otani ◽  
Hannah Sur ◽  
Guruprasad Rachaiah ◽  
Sriya Namagiri ◽  
Ashis Chowdhury ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Tumor Cells ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Xenograft Model ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Pp2a Activity ◽  
Phosphatase 2A

Abstract Background Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor treating fields, the median survival of Glioblastoma (GBM) patients is less than 15 months. Protein Arginine Methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of Protein Phosphatase 2A (PP2A) can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Methods Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. Results We found that PRMT5-depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5 intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. Conclusion Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

scholarly journals STEM-16. DUAL INHIBITION OF PROTEIN ARGININE METHYLTRANSFERASE 5 AND PROTEIN PHOSPHATASE 2A ENHANCES THE ANTI-TUMOR EFFICACY IN PRIMARY GLIOBLASTOMA NEUROSPHERES

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi237-vi237
Author(s):  
Hannah Sur ◽  
Arunakumar Gangaplara ◽  
Xiang Wang ◽  
Abhik Ray-Chaudhury ◽  
Zhengping Zhuang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Combination Therapy ◽  
Tumor Cells ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Pp2a Activity ◽  
Phosphatase 2A ◽  
Protein Arginine Methyltransferase 5

Abstract INTRODUCTION Glioblastoma (GBM) is the most common malignant primary brain tumor and has a heterogeneous tumor cell population. The median survival of GBM patients is less than two years with current multi-model therapy of maximal surgical resection followed by chemotherapy, radiation and tumor treating fields. Protein Arginine Methyltransferase 5 (PRMT5) regulates cellular functions through symmetric di-methylation of arginine residues of histone and non-histone proteins. Our recent findings show that PRMT5 is overexpressed in GBM; its inhibition causes apoptosis and senescence of mature and immature GBM tumor cells respectively. Protein Phosphatase 2A (PP2A), a serine-threonine phosphatase, is associated with senescent tumor cells that contribute to therapy resistance; LB100 is a first-in-class small molecule inhibitor of PP2A that can sensitize tumor cells that are chemo- and radiation-therapy resistant. In this study, using patient-derived primary GBM neurospheres (GBMNS), we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. METHODS Patient-derived primary GBM neurospheres transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays such as proliferation assay, cell cycle analysis, cytotoxicity assay, PP2A activity and western blot. RESULTS LB100 treatment significantly reduced the viability of PRMT5-depleted GBM cells as compared to PRMT5 intact cells. LB100 caused G1 cell cycle arrest through inactivation of Rb protein; this effect is further enhanced in combination with PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL; Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100 indicating that necroptosis caused the enhanced cytotoxicity in combination therapy. We also found that PRMT5-depletion increased the PP2A activity in GBM neurospheres. CONCLUSION Combining the PRMT5 inhibition and PP2A inhibition produced greater antitumor effects than with PRMT5 inhibition alone.

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 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 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.

scholarly journals The Adenovirus E4orf4 Protein Induces G2/M Arrest and Cell Death by Blocking Protein Phosphatase 2A Activity Regulated by the B55 Subunit

2009 ◽  
Vol 83 (17) ◽  
pp. 8340-8352 ◽  
Author(s):  
Suiyang Li ◽  
Claudine Brignole ◽  
Richard Marcellus ◽  
Sara Thirlwell ◽  
Olivier Binda ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Growth Arrest ◽  
Human Tumor ◽  
Cell Killing ◽  
Pp2a Activity ◽  
Phosphatase 2A

ABSTRACT Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70S6K, were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I1 PP2A enhanced E4orf4-induced cell killing and G2/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.

scholarly journals The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo.

1994 ◽  
Vol 269 (23) ◽  
pp. 16311-16317 ◽  
Author(s):  
B. Favre ◽  
S. Zolnierowicz ◽  
P. Turowski ◽  
B.A. Hemmings
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Phosphatase 2A

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

2000 ◽  
Vol 20 (21) ◽  
pp. 8143-8156 ◽  
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.

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