Stimulatory phosphorylation of cAMP-specific PDE4D5 by contractile agonists is mediated by PKC-dependent inactivation of protein phosphatase 2A

2008 ◽  
Vol 294 (1) ◽  
pp. G327-G335 ◽  
Author(s):  
Karnam S. Murthy ◽  
Wimolpak Sriwai
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Dependent Inactivation ◽  
Rhythmic Cycles ◽  
Phosphatase 2A ◽  
Contraction And Relaxation ◽  
Camp Levels

Smooth muscle of the gut undergoes rhythmic cycles of contraction and relaxation. Various constituents in the pathways that mediate muscle contraction could act to cross-regulate cAMP or cGMP levels and terminate subsequent relaxation. We have previously shown that cAMP levels are regulated by PKA-mediated phosphorylation of cAMP-specific phosphodiesterase 3A (PDE3A) and PDE4D5; the latter is the only PDE4D isoform expressed in smooth muscle. In the present study we have elucidated a mechanism whereby cholecystokinin (CCK) and, presumably, other contractile agonists capable of activating PKC can cross-regulate cAMP levels. Forskolin stimulated PDE4D5 phosphorylation and PDE4D5 activity. CCK significantly increased forskolin-stimulated PDE4D5 phosphorylation and activity and attenuated forskolin-stimulated cAMP levels. The effect of CCK on forskolin-induced PDE4D5 phosphorylation and activity and on cAMP levels was blocked by the inhibitors of PLC or PKC and in cultured muscle cells by the expression of Gαq minigene. The effects of CCK on PDE4D5 phosphorylation, PDE4D5 activity, and cAMP levels were mimicked by low (1 nM) concentrations of okadaic acid, but not by a low (10 nM) concentration of tautomycin, suggesting involvement of PP2A. Purified catalytic subunit of PP2A but not PP1 dephosphorylated PDE4D5 in vitro. Coimmunoprecipitation studies demonstrated association of PDE4D5 with PP2A and the association was decreased by the activation of PKC. In conclusion, cAMP levels are cross-regulated by contractile agonists via a mechanism that involves PLC-β-dependent, PKC-mediated inhibition of PP2A activity that leads to increase in PDE4D5 phosphorylation and activity and inhibition of cAMP levels.

scholarly journals Acute regulation of renal Na+/H+ exchanger NHE3 by dopamine: role of protein phosphatase 2A

2010 ◽  
Vol 298 (5) ◽  
pp. F1205-F1213 ◽  
Author(s):  
I. Alexandru Bobulescu ◽  
Henry Quiñones ◽  
Serge M. Gisler ◽  
Francesca Di Sole ◽  
Ming-Chang Hu ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Simian Virus 40 ◽  
Receptor Activation ◽  
T Antigen ◽  
Phosphatase 2A

Nephrogenic dopamine is a potent natriuretic paracrine/autocrine hormone that is central for mammalian sodium homeostasis. In the renal proximal tubule, dopamine induces natriuresis partly via inhibition of the sodium/proton exchanger NHE3. The signal transduction pathways and mechanisms by which dopamine inhibits NHE3 are complex and incompletely understood. This manuscript describes the role of the serine/threonine protein phosphatase 2A (PP2A) in the regulation of NHE3 by dopamine. The PP2A regulatory subunit B56δ (coded by the Ppp2r5d gene) directly associates with more than one region of the carboxy-terminal hydrophilic putative cytoplasmic domain of NHE3 (NHE3-cyto), as demonstrated by yeast-two-hybrid, coimmunoprecipitation, blot overlay, and in vitro pull-down assays. Phosphorylated NHE3-cyto is a substrate for purified PP2A in an in vitro dephosphorylation reaction. In cultured renal cells, inhibition of PP2A by either okadaic acid or by overexpression of the simian virus 40 (SV40) small T antigen blocks the ability of dopamine to inhibit NHE3 activity and to reduce surface NHE3 protein. Dopamine-induced NHE3 redistribution is also blocked by okadaic acid ex vivo in rat kidney cortical slices. These studies demonstrate that PP2A is an integral and critical participant in the signal transduction pathway between dopamine receptor activation and NHE3 inhibition.

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 A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jie Zheng ◽  
Na Tian ◽  
Fei Liu ◽  
Yidian Zhang ◽  
Jingfen Su ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Protein Phosphatase ◽  
High Efficiency ◽  
Microtubule Assembly ◽  
Hyperphosphorylated Tau ◽  
Phosphatase 2A ◽  
Dependent Learning ◽  
The Brain

AbstractIntraneuronal accumulation of hyperphosphorylated tau is a hallmark pathology shown in over twenty neurodegenerative disorders, collectively termed as tauopathies, including the most common Alzheimer’s disease (AD). Therefore, selectively removing or reducing hyperphosphorylated tau is promising for therapies of AD and other tauopathies. Here, we designed and synthesized a novel DEPhosphorylation TArgeting Chimera (DEPTAC) to specifically facilitate the binding of tau to Bα-subunit-containing protein phosphatase 2A (PP2A-Bα), the most active tau phosphatase in the brain. The DEPTAC exhibited high efficiency in dephosphorylating tau at multiple AD-associated sites and preventing tau accumulation both in vitro and in vivo. Further studies revealed that DEPTAC significantly improved microtubule assembly, neurite plasticity, and hippocampus-dependent learning and memory in transgenic mice with inducible overexpression of truncated and neurotoxic human tau N368. Our data provide a strategy for selective removal of the hyperphosphorylated tau, which sheds new light for the targeted therapy of AD and related-tauopathies.

Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements

1993 ◽  
Vol 13 (4) ◽  
pp. 2104-2112
Author(s):  
A S Alberts ◽  
T Deng ◽  
A Lin ◽  
J L Meinkoth ◽  
A Schönthal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Protein Phosphatase ◽  
Signal Transduction Pathways ◽  
Marker Genes ◽  
Direct Mechanism ◽  
Phosphatase 2A ◽  
Beta Galactosidase ◽  
Regulatory Sites

The involvement of serine/threonine protein phosphatases in signaling pathways which modulate the activity of the transcription factor AP-1 was examined. Purified protein phosphatase types 1 (PP1) and 2A (PP2A) were microinjected into cell lines containing stably transfected lacZ marker genes under the control of an enhancer recognized by AP-1. Microinjection of PP2A potentiated serum-stimulated beta-galactosidase expression from the AP-1-regulated promoter. Similarly, transient expression of the PP2A catalytic subunit with c-Jun resulted in a synergistic transactivation of an AP-1-regulated reporter gene. PP2A, but not PP1, potentiated serum-induced c-Jun expression, which has been previously shown to be autoregulated by AP-1 itself. Consistent with these results, PP2A dephosphorylated c-Jun on negative regulatory sites in vitro, suggesting one possible direct mechanism for the effects of PP2A on AP-1 activity. Microinjection of PP2A had no effect on cyclic AMP (cAMP)-induced expression of a reporter gene containing a cAMP-regulated promoter, while PP1 injection abolished cAMP-induced gene expression. Taken together, these results suggest a specific role for PP2A in signal transduction pathways that regulate AP-1 activity and c-Jun expression.

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo

2004 ◽  
Vol 286 (6) ◽  
pp. H2089-H2095 ◽  
Author(s):  
Brian B. Roman ◽  
Paul H. Goldspink ◽  
Elyse Spaite ◽  
Dalia Urboniene ◽  
Ron McKinney ◽  
...  
Keyword(s):  
Troponin I ◽  
Cardiac Contraction ◽  
Phosphorylation Sites ◽  
Developed Pressure ◽  
Contraction And Relaxation ◽  
Intracellular Targets ◽  
Camp Levels

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-ε, may slow the rates of cardiac contraction and relaxation (+dP/d t and −dP/d t). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser43 and Ser45 and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater −dP/d t than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/d t increased by 33% and −dP/d t increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.

scholarly journals Concentration-dependent alpha1-Adrenoceptor Antagonism and Inhibition of Neurogenic Smooth Muscle Contraction by Mirabegron in the Human Prostate

2021 ◽  
Vol 12 ◽  
Author(s):  
Ru Huang ◽  
Yuhan Liu ◽  
Anna Ciotkowska ◽  
Alexander Tamalunas ◽  
Raphaela Waidelich ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Stromal Cells ◽  
Smooth Muscle Contraction ◽  
Human Prostate ◽  
Smooth Muscle Relaxation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Organ Bath ◽  
Response Curves ◽  
Camp Levels

Introduction: Mirabegron is available for treatment of storage symptoms in overactive bladder, which may be improved by β3-adrenoceptor-induced bladder smooth muscle relaxation. In addition to storage symptoms, lower urinary tract symptoms in men include obstructive symptoms attributed to benign prostatic hyperplasia, caused by increased prostate smooth muscle tone and prostate enlargement. In contrast to the bladder and storage symptoms, effects of mirabegron on prostate smooth muscle contraction and obstructive symptoms are poorly understood. Evidence from non-human smooth muscle suggested antagonism of α1-adrenoceptors as an important off-target effect of mirabegron. As α1-adrenergic contraction is crucial in pathophysiology and medical treatment of obstructive symptoms, we here examined effects of mirabegron on contractions of human prostate tissues and on proliferation of prostate stromal cells.Methods: Contractions were induced in an organ bath. Effects of mirabegron on proliferation, viability, and cAMP levels in cultured stromal cells were examined by EdU assays, CCK-8 assays and enzyme-linked immunosorbent assay.Results: Mirabegron in concentrations of 5 and 10 μM, but not 1 µM inhibited electric field stimulation-induced contractions of human prostate tissues. Mirabegron in concentrations of 5 and 10 µM shifted concentration response curves for noradrenaline-, methoxamine- and phenylephrine-induced contractions to the right, including recovery of contractions at high concentrations of α1-adrenergic agonists, increased EC50 values, but unchanged Emax values. Rightshifts of noradrenaline concentration response curves and inhibition of EFS-induced contractions were resistant to L-748,337, l-NAME, and BPIPP. 1 µM mirabegron was without effect on α1-adrenergic contractions. Endothelin-1- and U46619-induced contractions were not affected or only inhibited to neglectable extent. Effects of mirabegron (0.5–10 µM) on proliferation and viability of stromal cells were neglectable or small, reaching maximum decreases of 8% in proliferation assays and 17% in viability assays. Mirabegron did not induce detectable increases of cAMP levels in cultured stromal cells.Conclusion: Mirabegron inhibits neurogenic and α1-adrenergic human prostate smooth muscle contractions. This inhibition may be based on antagonism of α1-adrenoceptors by mirabegron, and does not include activation of β3-adrenoceptors and requires concentrations ranging 50-100fold higher than plasma concentrations reported from normal dosing. Non-adrenergic contractions and proliferation of prostate stromal cells are not inhibited by mirabegron.

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

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.

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