scholarly journals The MID1 E3 Ligase Catalyzes the Polyubiquitination of Alpha4 (α4), a Regulatory Subunit of Protein Phosphatase 2A (PP2A)

2013 ◽  
Vol 288 (29) ◽  
pp. 21341-21350 ◽  
Author(s):  
Haijuan Du ◽  
Yongzhao Huang ◽  
Manar Zaghlula ◽  
Erica Walters ◽  
Timothy C. Cox ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
E3 Ligase ◽  
Fold Increase ◽  
Dominant Negative ◽  
Activity Level ◽  
Mass Spectrometry Data ◽  
Regulatory Subunit ◽  
Phosphatase 2A

Alpha4 (α4) is a key regulator of protein phosphatase 2A (PP2A) and mTOR in steps essential for cell-cycle progression. α4 forms a complex with PP2A and MID1, a microtubule-associated ubiquitin E3 ligase that facilitates MID1-dependent regulation of PP2A and the dephosphorylation of MID1 by PP2A. Ectopic overexpression of α4 is associated with hepatocellular carcinomas, breast cancer, and invasive adenocarcinomas. Here, we provide data suggesting that α4 is regulated by ubiquitin-dependent degradation mediated by MID1. In cells stably expressing a dominant-negative form of MID1, significantly elevated levels of α4 were observed. Treatment of cells with the specific proteasome inhibitor, lactacystin, resulted in a 3-fold increase in α4 in control cells and a similar level in mutant cells. Using in vitro assays, individual MID1 E3 domains facilitated monoubiquitination of α4, whereas full-length MID1 as well as RING-Bbox1 and RING-Bbox1-Bbox2 constructs catalyzed its polyubiquitination. In a novel non-biased functional screen, we identified a leucine to glutamine substitution at position 146 within Bbox1 that abolished MID1-α4 interaction and the subsequent polyubiquitination of α4, indicating that direct binding to Bbox1 was necessary for the polyubiquitination of α4. The mutant had little impact on the RING E3 ligase functionality of MID1. Mass spectrometry data confirmed Western blot analysis that ubiquitination of α4 occurs only within the last 105 amino acids. These novel findings identify a new role for MID1 and a mechanism of regulation of α4 that is likely to impact the stability and activity level of PP2Ac.

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.

scholarly journals Protein Phosphatase 2A Subunit PR70 Interacts with pRb and Mediates Its Dephosphorylation

2007 ◽  
Vol 28 (2) ◽  
pp. 873-882 ◽  
Author(s):  
Alessandra Magenta ◽  
Pasquale Fasanaro ◽  
Sveva Romani ◽  
Valeria Di Stefano ◽  
Maurizio C. Capogrossi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Phosphatase ◽  
Molecular Mechanisms ◽  
Protein Phosphatase 2A ◽  
Phosphate Removal ◽  
Regulatory Subunit ◽  
Phosphatase 2A ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma tumor suppressor protein (pRb) regulates cell proliferation and differentiation via phosphorylation-sensitive interactions with specific targets. While the role of cyclin/cyclin-dependent kinase complexes in the modulation of pRb phosphorylation has been extensively studied, relatively little is known about the molecular mechanisms regulating phosphate removal by phosphatases. Protein phosphatase 2A (PP2A) is constituted by a core dimer bearing catalytic activity and one variable B regulatory subunit conferring target specificity and subcellular localization. We previously demonstrated that PP2A core dimer binds pRb and dephosphorylates pRb upon oxidative stress. In the present study, we identified a specific PP2A-B subunit, PR70, that was associated with pRb both in vitro and in vivo. PR70 overexpression caused pRb dephosphorylation; conversely, PR70 knockdown prevented both pRb dephosphorylation and DNA synthesis inhibition induced by oxidative stress. Moreover, we found that intracellular Ca2+ mobilization was necessary and sufficient to trigger pRb dephosphorylation and PP2A phosphatase activity of PR70 was Ca2+ induced. These data underline the importance of PR70-Ca2+ interaction in the signal transduction mechanisms triggered by redox imbalance and leading to pRb dephosphorylation.

scholarly journals The B56α Regulatory Subunit of Protein Phosphatase 2A Is a Target for Regulation by Double-Stranded RNA-Dependent Protein Kinase PKR

2000 ◽  
Vol 20 (14) ◽  
pp. 5285-5299 ◽  
Author(s):  
Zan Xu ◽  
Bryan R. G. Williams
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Regulatory Subunit ◽  
Luciferase Expression ◽  
Regulation Of Transcription ◽  
Pp2a Activity ◽  
Phosphatase 2A

ABSTRACT PKR is a cellular serine/threonine kinase that phosphorylates eukaryotic translation initiation factor 2α (eIF2α) to regulate protein synthesis. PKR also plays a role in the regulation of transcription, programmed cell death and the cell cycle, processes which likely involve other substrates. In a yeast two-hybrid screen, we isolated human protein phosphatase 2A (PP2A) regulatory subunit B56α as a PKR-interacting protein. The interaction between B56α and PKR was confirmed by in vitro binding assays as well as by in vivo coimmunoprecipitation, and this interaction is dependent on the catalytic activity of PKR. Moreover, recombinant B56α was efficiently phosphorylated by PKR in vitro and an isoelectric point shift in B56α was detected in extracts from cells induced with the PKR activator pIC. An in vitro dephosphorylation assay showed that when B56α was phosphorylated by PKR, the activity of PP2A trimeric holoenzyme was increased. A functional interaction between B56α and PKR was observed in cotransfection assays, where a B56α-mediated increase in luciferase expression was inhibited by cotransfection with wild-type PKR. This is likely due to a decreased level of eIF4E phosphorylation caused by an increase in PP2A activity following PKR phosphorylation of B56α. Taken together, our data indicate that PKR can modulate PP2A activity by phosphorylating B56α to regulate cellular activities.

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

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.

scholarly journals Regulatory Subunit B′γ of Protein Phosphatase 2A Prevents Unnecessary Defense Reactions under Low Light in Arabidopsis

2011 ◽  
Vol 156 (3) ◽  
pp. 1464-1480 ◽  
Author(s):  
Andrea Trotta ◽  
Michael Wrzaczek ◽  
Judith Scharte ◽  
Mikko Tikkanen ◽  
Grzegorz Konert ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Regulatory Subunit ◽  
Low Light ◽  
Defense Reactions ◽  
Phosphatase 2A ◽  
Subunit B

scholarly journals Protein Phosphatase 2A Regulatory Subunit B56α Associates with c-Myc and Negatively Regulates c-Myc Accumulation

2006 ◽  
Vol 26 (7) ◽  
pp. 2832-2844 ◽  
Author(s):  
Hugh K. Arnold ◽  
Rosalie C. Sears
Keyword(s):  
Tumor Suppressor ◽  
Protein Phosphatase ◽  
Cell Function ◽  
Cell Transformation ◽  
Protein Phosphatase 2A ◽  
Regulatory Subunit ◽  
Suppressor Activity ◽  
Tumor Suppressor Activity ◽  
Structural Subunit ◽  
Phosphatase 2A

ABSTRACT Protein phosphatase 2A (PP2A) plays a prominent role in controlling accumulation of the proto-oncoprotein c-Myc. PP2A mediates its effects on c-Myc by dephosphorylating a conserved residue that normally stabilizes c-Myc, and in this way, PP2A enhances c-Myc ubiquitin-mediated degradation. Stringent regulation of c-Myc levels is essential for normal cell function, as c-Myc overexpression can lead to cell transformation. Conversely, PP2A has tumor suppressor activity. Uncovering relevant PP2A holoenzymes for a particular target has been limited by the fact that cellular PP2A represents a large heterogeneous population of trimeric holoenzymes, composed of a conserved catalytic subunit and a structural subunit along with a variable regulatory subunit which directs the holoenzyme to a specific target. We now report the identification of a specific PP2A regulatory subunit, B56α, that selectively associates with the N terminus of c-Myc. B56α directs intact PP2A holoenzymes to c-Myc, resulting in a dramatic reduction in c-Myc levels. Inhibition of PP2A-B56α holoenzymes, using small hairpin RNA to knock down B56α, results in c-Myc overexpression, elevated levels of c-Myc serine 62 phosphorylation, and increased c-Myc function. These results uncover a new protein involved in regulating c-Myc expression and reveal a critical interconnection between a potent oncoprotein, c-Myc, and a well-documented tumor suppressor, PP2A.

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