Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32000) is highly expressed in striatonigral neurons in which its phosphorylation is regulated by several neurotransmitters including dopamine and glutamate. DARPP-32 becomes a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr-34 by cAMP- or cGMP-dependent protein kinases. DARPP-32 is also phosphorylated on Ser-137 by protein kinase CK1 (CK1), in vitro and in vivo. This phosphorylation has an important regulatory role since it inhibits the dephosphorylation of Thr-34 by calcineurin in vitro and in striatonigral neurons. Here, we show that DARPP-32 phosphorylated by CK1 is a substrate in vitro for protein phosphatases 2A and 2C, but not protein phosphatase 1 or calcineurin. However, in substantia nigra slices, dephosphorylation of Ser-137 was markedly sensitive to decreased temperature, and not detectably affected by the presence of okadaic acid under conditions in which dephosphorylation of Thr-34 by protein phosphatase 2A was inhibited. These results suggest that, in neurons, phospho-Ser-137-DARPP-32 is dephosphorylated by protein phosphatase 2C, but not 2A. Thus, DARPP-32 appears to be a component of a regulatory cascade of phosphatases in which dephosphorylation of Ser-136 by protein phosphatase 2C facilitates dephosphorylation of Thr-34 by calcineurin, removing the cyclic nucleotide-induced inhibition of protein phosphatase 1.

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Protein Phosphatase 1, Protein Phosphatase 2A, and Calcineurin Play a Role in Estrogen-Mediated Neuroprotection

Endocrinology ◽

10.1210/en.2008-0610 ◽

2008 ◽

Vol 149 (10) ◽

pp. 5235-5243 ◽

Cited By ~ 21

Author(s):

Kun Don Yi ◽

James W. Simpkins

Keyword(s):

Protein Phosphatase ◽

Protein Phosphatases ◽

Cellular Function ◽

Artery Occlusion ◽

Protective Effects ◽

The Face ◽

Phosphatase 2A ◽

Cerebral Artery Occlusion

It is becoming increasingly clear that protein phosphatases are important modulators of cellular function and that disruption of these proteins are involved in neurodegenerative disease processes. Serine/threonine protein phosphatases (PP) such as protein phosphatase PP1, PP2A, and calcineurin are involved in hyperphosphorylation of τ- as well as β-amyloid-induced cell death. We have previously shown serine/threonine protein phosphatases to be involved in estrogen-mediated neuroprotection. The purpose of this study was to delineate the role of PP1, PP2A, and calcineurin in the mechanism of estrogen mediated neuroprotection against oxidative stress and excitotoxicity. Treatment with protein phosphatases inhibitor II, endothall, or cyclosporin A, which are specific inhibitors of PP1, PP2A, and calcineurin, respectively, did not have an effect on cell viability. However, in combination, these inhibitors adversely affected cell survival, which suggests the importance of serine/threonine protein phosphatases in maintenance of cellular function. Inhibitors of PP1, PP2A, and calcineurin attenuated the protective effects of estrogen against glutamate-induced -neurotoxicity but did not completely abrogate the estrogen-mediated protection. The attenuation of estrogen-induced neuroprotection was achieved through decrease in the activity of theses serine/threonine phosphatases without the concomitant decrease in protein expression. In an animal model, transient middle cerebral artery occlusion caused a 50% decrease in levels of PP1, PP2A, and PP2B ipsilateral to the lesion in a manner that was prevented by estradiol pretreatment. Therefore, we conclude that in the face of cytotoxic challenges in vitro and in vivo, estrogens maintain the function of PP1, PP2A, and calcineurin.

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A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00669-2 ◽

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.

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The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction.

Molecular Biology of the Cell ◽

10.1091/mbc.e21-06-0307-t ◽

2021 ◽

Author(s):

Margaux R. Audett ◽

Erin L. Johnson ◽

Jessica M. McGory ◽

Dylan M. Barcelos ◽

Evelin Oroszne Szalai ◽

...

Keyword(s):

Protein Binding ◽

Protein Phosphatase ◽

Spindle Assembly Checkpoint ◽

Protein Phosphatase 1 ◽

Intrinsically Disordered ◽

Regulatory Circuit ◽

Cell Assays ◽

Kinetics Of

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of D. melanogaster KNL1 (Spc105) has never been shown to bind MTs nor to recruit PP1. Furthermore, the phospho-regulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell based-assays. A phospho-regulatory circuit, which utilizes Aurora B kinase (ABK), promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag, and deletion/chimera mutants are used to define the interplay of MT- and PP1-binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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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.

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The protein phosphatases involved in cellular regulation. Influence of polyamines on the activities of protein phosphatase-1 and protein phosphatase-2A

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1985.tb08927.x ◽

1985 ◽

Vol 149 (2) ◽

pp. 305-314 ◽

Cited By ~ 57

Author(s):

H. Y. Lim TUNG ◽

Steven PELECH ◽

Michael J. FISHER ◽

Christopher I. POGSON ◽

Philip COHEN

Keyword(s):

Protein Phosphatase ◽

Protein Phosphatases ◽

Protein Phosphatase 2A ◽

Protein Phosphatase 1 ◽

Cellular Regulation ◽

Phosphatase 2A

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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.

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Identification of the Putative Protein Phosphatase Gene PTC1 as a Virulence-Related Gene Using a Silkworm Model of Candida albicans Infection

Eukaryotic Cell ◽

10.1128/ec.00129-08 ◽

2008 ◽

Vol 7 (10) ◽

pp. 1640-1648 ◽

Cited By ~ 42

Author(s):

Nozomu Hanaoka ◽

Yukie Takano ◽

Kazutoshi Shibuya ◽

Hajime Fugo ◽

Yoshimasa Uehara ◽

...

Keyword(s):

Candida Albicans ◽

Mouse Model ◽

Protein Phosphatase ◽

Protein Phosphatases ◽

Hyphal Growth ◽

Marker Genes ◽

Phenotypic Traits ◽

Putative Protein

ABSTRACT Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Δ, yvh1Δ, sit4Δ, and ptc1Δ) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Δ, yvh1Δ, and sit4Δ mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Δ revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.

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Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase 2A family

Journal of Cell Science ◽

10.1242/jcs.106.1.219 ◽

1993 ◽

Vol 106 (1) ◽

pp. 219-226 ◽

Cited By ~ 2

Author(s):

E. Kam ◽

K.A. Resing ◽

S.K. Lim ◽

B.A. Dale

Keyword(s):

Enzymatic Activity ◽

Intermediate Filaments ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Gel Filtration ◽

Phosphatase Inhibitors ◽

Rate Analysis ◽

Phosphatase 2A

The aggregation of cellular intermediate filaments is an important step in the terminal differentiation of keratinocytes. It has been shown that epidermal filaggrin can cause intermediate filaments to aggregate in vitro and may also have the same function in vivo. Filaggrin is derived via dephosphorylation and proteolysis from a highly phosphorylated precursor, profilaggrin, which is found in the granular layer of the epidermis. Using casein kinase II phosphorylated filaggrin as substrate, a profilaggrin phosphatase has been partially purified from rat epidermal homogenate by three chromatographic steps (DE52, hydroxylapatite and S200 gel filtration). Profilaggrin phosphatase activity eluted from the last column has a Km of 0.12 mM and a Vmax of 8 nmol/mg/min with respect to phosphofilaggrin. Results obtained by initial rate analysis showed that the enzymatic activity is not affected by phospho-tyrosyl phosphatase inhibitors and the active fractions preferentially dephosphorylate the alpha subunit of phosphorylase kinase which has been phosphorylated by cAMP-dependent kinase. These results suggest that epidermal profilaggrin phosphatase is not a phospho-tyrosyl phosphatase or a type 1 phospho-seryl/phospho-threonyl phosphatase. Dephosphorylation is not affected by EDTA, calcium or magnesium, but is very sensitive to okadaic acid inhibition (IC50 = 80 pM), suggesting that the enzymatic activity is related to that of the protein phosphatase 2A (PP2A).(ABSTRACT TRUNCATED AT 250 WORDS)

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Activation by phosphorylation of phosphofructokinase from the annelid Lumbricus terrestris and comparison of phosphorylated sites in invertebrate phosphofructokinases

Biochemical Journal ◽

10.1042/bj3170377 ◽

1996 ◽

Vol 317 (2) ◽

pp. 377-383 ◽

Cited By ~ 7

Author(s):

Ralph SCHALOSKE ◽

Michael BIETHINGER ◽

Linda A. FOTHERGILL-GILMORE ◽

Hans Werner HOFER

Keyword(s):

Protein Phosphatase ◽

Helix Pomatia ◽

Lumbricus Terrestris ◽

Catalytic Subunit ◽

Dependent Protein Kinase ◽

N Terminus ◽

Phosphatase 2A ◽

Dependent Protein ◽

Earthworm Lumbricus

Purified phosphofructokinase from the earthworm Lumbricus terrestris was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase from the same organism to an extent of approx. 0.5 mol/mol of subunit. Activation of the enzyme occurred in parallel to the incorporation of covalently bound phosphate and was reversed by the action of the catalytic subunit of protein phosphatase 2A. Phosphorylation decreased the co-operativity of fructose 6-phosphate saturation in the presence of inhibitory concentrations of ATP, and increased the apparent Vmax obtained with saturating concentrations of the activators 5´-AMP and fructose 2,6-bisphosphate. The phosphorylated sites of phosphofructokinase from L. terrestris and from two molluscs (Helix pomatia and Mytilus edulis) were sequenced and shown to exhibit distinct similarity to sequences located near to the N-terminus of nematode phosphofructokinases [Klein, Olson, Favreau, Wintertowed, Hatzenbuhler, Shea, Nulf and Geary (1991) Mol. Biochem. Parasitol. 48, 17–26].

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