Potential involvement of serine/threonine protein phosphatases in apoptosis of HepG2 cells during selenite treatment

Selenium, an essential biological trace element present in both prokaryotic and eukaryotic cells, exerts its regulatory effect in a variety of cellular events, including cell growth, survival, and death. Selenium compunds have been shown in different cell lines to inhibit apoptosis by several mechanisms. Serine/threonine phosphatases (STPs) are potentially important in selenite-induced apoptosis because of their role in regulation of diverse set of cellular processes. In this study, the regulatory role of STPs in selenite-induced apoptosis has been implied by the use of two specific inhibitors: ocadaic acid and calyculin A. Our results show a decrease in cell density in HepG2 cells under selenite treatment. Resulting specific enzyme activities showed a concentration-dependent increase in all three phosphatase activities after 24 h in cells treated with 5 μM selenite and these activities decreased at 48 and 72 h. However, in cells treated with 10μM selenite, PP2A and PP2B decreased at 48 h, whereas PP2C activity did not change at this dose. In cells treated with 25μM, there was not a significant change in PP2C activity. These data suggest that the most specific response to selenite treatment was in PP2A and PP2B activities in a dose-dependent manner. Our results with OA and Cal-A further support the view that PP1 and PP2A might act as negative regulators of growth. With these data, we have first demonstrated the role of serine/threonine protein phosphatases in the signaling pathway of selenite-induced apoptosis and resulting cytotoxicity     

SET improved oocyte maturation by serine/threonine protein phosphatase 2A and inhibited oocyte apoptosis in mouse oocytes

SET is a multifunctional protein involved in a variety of molecular processes such as cell apoptosis and cell-cycle regulation. In ovaries SET is predominantly expressed in theca cells and oocytes. In polycystic ovary syndrome (PCOS) patients the expression of SET was increased than healthy people. The current study was designed to determine whether SET plays a role in oocyte maturation and apoptosis, which may provide clues for the underlying pathological mechanism of follicular development in PCOS patients. Oocytes at germinal vesicle (GV) stage were collected from 6-week-old female ICR mice ovaries. The expression of SET was manipulated by AdCMV-SET and AdH1-SiRNA/SET adenoviruses. SET overexpression improved oocyte maturation whereas SET knockdown inhibited oocyte maturation. Moreover, SET negatively regulated serine/threonine protein phosphatase 2A (PP2A) activity in oocytes. Treatment with PP2A inhibitor okadaic acid (OA) promoted oocyte maturation. Furthermore, PP2A knockdown confirmed the role of PP2A in oocyte maturation, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition on oocyte maturation. The central role of PP2A in SET-mediated regulation of oocyte maturation was confirmed by the finding that SET increased the expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) and PP2A inhibited their expressions. Besides, SET inhibited oocyte apoptosis through decreasing the expression of caspase 3 and caspases 8, while PP2A had no effect on oocyte apoptosis. SET promoted oocyte maturation by inhibiting PP2A activity and inhibited oocyte apoptosis in mouse in-vitro cultured oocytes, which may provide a pathologic pathway leading to impaired oocyte developmental competence in PCOS.     

Disruption of serine/threonine protein phosphatase 5 (PP5:PPP5c) in mice reveals a novel role for PP5 in the regulation of ultraviolet light-induced phosphorylation of serine/threonine protein kinase Chk1 (CHEK1)

PP5 is a ubiquitously expressed Ser/Thr protein phosphatase. High levels of PP5 have been observed in human cancers, and constitutive PP5 overexpression aids tumor progression in mouse models of tumor development. However, PP5 is highly conserved among species, and the roles of PP5 in normal tissues are not clear. Here, to help evaluate the biological actions of PP5, a Cre/loxP-conditional mouse line was generated. In marked contrast to the early embryonic lethality associated with the genetic disruption of other PPP family phosphatases (e.g. PP2A and PP4), intercrosses with mouse lines that ubiquitously express Cre recombinase starting early in development (e.g. MeuCre40 and ACTB-Cre) produced viable and fertile PP5-deficient mice. Phenotypic differences caused by the total disruption of PP5 were minor, suggesting that small molecule inhibitors of PP5 will not have widespread systemic toxicity. Examination of roles for PP5 in fibroblasts generated from PP5-deficient embryos (PP5(-/-) mouse embryonic fibroblasts) confirmed some known roles and identified new actions for PP5. PP5(-/-) mouse embryonic fibroblasts demonstrated increased sensitivity to UV light, hydroxyurea, and camptothecin, which are known activators of ATR (ataxia-telangiectasia and Rad3-related) kinase. Further study revealed a previously unrecognized role for PP5 downstream of ATR activation in a UV light-induced response. The genetic disruption of PP5 is associated with enhanced and prolonged phosphorylation of a single serine (Ser-345) on Chk1, increased phosphorylation of the p53 tumor suppressor protein (p53) at serine 18, and increased p53 protein levels. A comparable role for PP5 in the regulation of Chk1 phosphorylation was also observed in human cells.     

Characterization of DNA sequences encoding a novel isoform of the 55 kDa B regulatory subunit of the type 2A protein serine/threonine phosphatase of Arabidopsis thaliana

We have indentified a novel gene (AtB) encoding a previously uncharacterized isoform of the B regulatory subunit of the type 2A serine/threonine protein phosphatase (PP2A) of Arabidopsis, and show that mRNA derived from the AtB gene accumulates in all Arabidopsis organs. In addition, we examined the expression of the three genes encoding the A regulatory subunit of Arabidopsis PP2A and show these genes are expressed in all organs as well. Taken together, our results suggest a myriad of PP2A subunit combinations, possibly with distinct substrate specificities, may occur within each Arabidopsis cell.     

Trithorax interacts with type 1 serine/threonine protein phosphatase in Drosophila

The catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c) was shown to bind trithorax (TRX) in the yeast two-hybrid system. Interaction between PP1c and TRX was confirmed in vivo by co-immunoprecipitation from Drosophila extracts. An amino-terminal fragment of TRX, containing a putative PP1c-binding motif, was shown to be sufficient for binding to PP1c by in vitro glutathione S-transferase pull-down assays using recombinant protein and fly extracts expressing epitope tagged PP1c. Disruption of the PP1c-binding motif abolished binding, indicating that this motif is necessary for interaction with PP1. On polytene chromosomes, PP1c is found at many discrete bands, which are widely distributed along the chromosomes. Many of the sites that stain strongly for PP1c correspond to sites of TRX, consistent with a physical association of PP1c with chromatin-bound TRX. Homeotic transformations of haltere to wing in flies mutant for trx are dominantly suppressed by PP1c mutants, indicating that PP1c not only binds TRX, but is a physiologically relevant regulator of TRX function in vivo.     

Tautomycetin is a novel and specific inhibitor of serine/threonine protein phosphatase type 1, PP1

Here we isolated tautomycetin, TC, and examined its phosphatase inhibitory activity. Recently we have reported that the left-hand moiety of tautomycin, TM, and the right one containing the spiroketal are essentially required for inhibition of protein phosphatase, PP, and induction of apoptosis, respectively. TC is structurally almost identical to TM except that TC is lacking the spiroketal, which has the potential apoptosis-inducing activity. TC specifically inhibited PP1 activity, IC50 values for purified PP1 and PP2A enzymes being 1.6 and 62 nM, respectively, whereas the IC50 values of TM were 0.21 and 0.94 nM, respectively. These results demonstrate that TC is the most specific PP1 inhibitor out of over 40 species of natural phosphatase inhibitors reported, strongly suggesting that TC is a novel powerful tool to elucidate the physiological roles of PP1 in various biological events.     

Differential susceptibilities of serine/threonine phosphatases to oxidative and nitrosative stress

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are signal-transducing molecules that regulate the activities of a variety of proteins. In the present investigation, we have compared the effects of superoxide (O2-), nitric oxide (NO), and hydrogen peroxide (H2O2) on the activities of three highly homologous serine/threonine phosphatases, protein phosphatase type 1 (PP1), protein phosphatase type 2A (PP2A), and calcineurin (protein phosphatase type 2B). Although superoxide, generated from xanthine/xanthine oxidase or paraquat, and NO, generated from (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide or sodium nitroprusside, potently inhibited the phosphatase activity of calcineurin in neuroblastoma cell lysates, they had relatively little effect on the activities of PP1 or PP2A. In contrast, H2O2 inhibited the activities of all three phosphatases in lysates but was not a potent inhibitor for any of the enzymes. Calcineurin inactivated by O2-, NO, and H2O2 could be partially reactivated by the reducing agent ascorbate or by the thiol-specific reagent dithiothreitol (DTT). Maximal reactivation was achieved by the addition of both reagents, which suggests that ROS and RNS inhibit calcineurin by oxidizing both a catalytic metal(s) and a critical thiol(s). Reactivation of H2O2-treated PP1 also required the combination of both ascorbate and DTT, whereas PP2A required only DTT for reactivation. These results suggest that, despite their highly homologous structures, calcineurin is the only major Ser/Thr phosphatase that is a sensitive target for inhibition by superoxide and nitric oxide and that none of the phosphatases are sensitive to inhibition by hydrogen peroxide.     

Human T-lymphotropic virus type I tax activates I-kappa B kinase by inhibiting I-kappa B kinase-associated serine/threonine protein phosphatase 2A

I-kappa B kinase (IKK) is a serine/threonine kinase that phosphorylates I-kappa B alpha and I-kappa B beta and targets them for polyubiquitination and proteasome-mediated degradation. IKK consists of two highly related catalytic subunits, alpha and beta, and a regulatory gamma subunit, which becomes activated after serine phosphorylation of the activation loops of the catalytic domains. The human T-lymphotropic retrovirus type-I trans-activator, Tax, has been shown to interact directly with IKK gamma and activates IKK via a mechanism not fully understood. Here we demonstrate that IKK binds serine/threonine protein phosphatase 2A (PP2A), and via a tripartite protein-protein interaction, Tax, IKK gamma, and PP2A form a stable ternary complex. In vitro, PP2A down-regulates active IKK prepared from Tax-producing MT4 cells. In the presence of Tax, however, the ability of PP2A to inactivate IKK is diminished. Despite their interaction with IKK gamma, PP2A-interaction-defective Tax mutants failed to activate NF-kappa B. Our data support the notion that IKK gamma-associated PP2A is responsible for the rapid deactivation of IKK, and inhibition of PP2A by Tax in the context of IKK x PP2A x Tax ternary complex leads to constitutive IKK and NF-kappa B activation.     

Process formation of podocytes: morphogenetic activity of microtubules and regulation by protein serine/threonine phosphatase PP2A

Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.     

Probing the function of conserved residues in the serine/threonine phosphatase PP2Calpha

Human PP2Calpha is a metal-dependent phosphoserine/phosphothreonine protein phosphatase and is the representative member of the large PPM family. The X-ray structure of human PP2Calpha has revealed an active site containing a dinuclear metal ion center that is coordinated by several invariant carboxylate residues. However, direct evidence for the catalytic function of these and other active-site residues has not been established. Using site-directed mutagenesis and enzyme kinetic analyses, we probed the roles of conserved active-site amino acids within PP2Calpha. Asp-60 bridges metals M1 and M2, and Asp-239 coordinates metal M2, both of which were replaced individually to asparagine residues. These point mutations resulted in >or=1000-fold decrease in k(cat) and >or=30-fold increase in K(m) value for Mn(2+). Mutation of Asp-282 to asparagine caused a 100-fold decrease in k(cat), but no significant effect on K(m) values for metal and substrate, consistent with Asp-282 activating a metal-bound water nucleophile. Mutants T128A, E37Q, D38N, and H40A displayed little or no alterations on k(cat) and K(m) values for substrate or metal ion (Mn(2+)). Analysis of H62Q and R33A yielded k(cat) values that were 20- and 2-fold lower than wild-type, respectively. The mutant R33A showed a 8-fold higher K(m) for substrate, while the K(m) observed with H62Q was unaffected. A pH-rate profile of the H62Q mutant showed loss of the ionization that must be protonated for activity. Br?nsted analysis of substrate leaving group pK(a) values for H62Q indicated a greater dependency (slope -0.84) on leaving group pK(a) in comparison to wild-type (slope -0.33). These data provide strong evidence that His-62 acts as a general acid during the cleavage of the P-O bond.     

Kinetic analysis of human serine/threonine protein phosphatase 2Calpha.

The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes. Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family. Although it is known that these phosphatases require metal cations for activity, the molecular details of these important reactions have not been established. Therefore, here we report a detailed biochemical study to elucidate the kinetic and chemical mechanism of PP2Calpha. Steady-state kinetic and product inhibition studies revealed that PP2Calpha employs an ordered sequential mechanism, where the metal cations bind before phosphorylated substrate, and phosphate is the last product to be released. The metal-dependent activity of PP2C (as reflected in kcat and kcat/Km), indicated that Fe2+ was 1000-fold better than Mg2+. The pH rate profiles revealed two ionizations critical for catalytic activity. An enzyme ionization with a pKa value of 7 must be unprotonated for catalysis, and an enzyme ionization with a pKa of 9 must be protonated for substrate binding. Br?nsted analysis of substrate leaving group pKa indicated that phosphomonoester hydrolysis is rate-limiting at pH 7. 0, but not at pH 8.5 where a common step independent of the nature of the substrate and alcohol product limits turnover (kcat). Rapid reaction kinetics between phosphomonoester and PP2C yielded exponential "bursts" of product formation, consistent with phosphate release being the slow catalytic step at pH 8.5. Dephosphorylation of synthetic phosphopeptides corresponding to several protein kinases revealed that PP2C displays a strong preference for diphosphorylated peptides in which the phosphorylated residues are in close proximity.     

丝氨酸/苏氨酸蛋白磷酸酯酶在tau蛋白异常磷酸化中的作用

Tau蛋白过度磷酸化在阿尔采末病（Alzheimer’s disease,AD）发病过程中发挥重要作用,抑制蛋白磷酸酯酶活性,可诱导tau的过度磷酸化和聚积。本文拟就近年来蛋白磷酸酯酶在tau蛋白异常磷酸化中的作用作一综述。     

Role of serine/threonine protein phosphatase in Alzheimer's disease

Accumulating evidence indicates that serine/threonine (Ser/Thr) protein phosphatases (PPs), such as PP1, PP2A and PP2B, participate in the neurodegenerative progress in Alzheimer's disease (AD). The general characteristics and pathologic changes of PP1, PP2A and PP2B in AD, and their relations with microtubule-associated proteins, focusing mainly on tau protein, neurofilament (NF), amyloid precursor protein (APP) processing and synaptic plasticity are discussed. Deriving novel insight into the particular topic will attract greater attention to more active investigation and effective therapeutic intervention in the future.     

Plasmodium berghei serine/threonine protein phosphatase PP5 plays a critical role in male gamete fertility

Sexual development in malaria parasites involves multiple signal transduction pathways mediated by reversible protein phosphorylation. Here, we functionally characterised a protein phosphatase, Ser/Thr protein phosphatase 5 (PbPP5), during sexual development of the rodent malaria parasite Plasmodium berghei. The recombinant protein phosphatase domain displayed obvious protein phosphatase activity and was sensitive to PP1/PP2A inhibitors including cantharidic acid (IC₅₀ = 122.2 nM), cantharidin (IC₅₀ = 74.3 nM), endothall (IC₅₀ = 365.5 nM) and okadaic acid (IC₅₀ = 1.3 nM). PbPP5 was expressed in both blood stages and ookinetes with more prominent expression during sexual development. PbPP5 was localised in the cytoplasm of the parasite and highly concentrated beneath the parasite plasma membrane in free merozoites and ookinetes. Targeted deletion of the pbpp5 gene had no influence on asexual blood-stage parasite multiplication or the survival curve of the infected hosts. However, male gamete formation and fertility were severely affected, resulting in almost complete blockade of ookinete conversion and oocyst development in the Δpbpp5 lines. This sexual development defect was rescued by crossing Δpbpp5 with the female defective Δpbs47 parasite line, but not with the male defective Δpbs48/45 line, thus confirming the essential function of the pbpp5 gene in male gamete fertility. Furthermore, the aforementioned PP1/PP2A inhibitors all had inhibitory effects on exflagellation of male gametocytes and ookinete conversion. In particular, endothall, a selective inhibitor of PP2A, completely blocked exflagellation and ookinete conversion at ～548.3 nM. This study elucidated an essential function of PbPP5 during male gamete development and fertility.     

Induction of abnormal nuclear shapes in two distinct modes by overexpression of serine/threonine protein phosphatase 5 in Hela cells

Okadaic acid-sensitve serine/threonine protein phosphatase 5 (PP5) is expressed ubiquitously in various tissues and is considered to participate in many cellular processes. PP5 has a catalytic domain in the C-terminal region and three tetratricopeptide repeat (TPR) motifs in the N-terminal region, which are suspected to function as a protein-protein interaction domain. Physiological roles of PP5 are still largely unknown, although several PP5-binding proteins were reported and a few in vivo functions of PP5 were suggested. In the present study, the effects of expression of the full-length wild-type PP5 fused with EGFP (EGFP-PP5(WT)) and its phosphatase-dead mutant EGFP-PP5(H304A) were investigated. Transient expression of either EGFP-PP5(WT) or EGFP-PP5(H304A) in HeLa cells induced deformed nuclei with a 10-fold frequency compared to that of EGFP. Abnormal-shaped nuclei were also substantially increased by induced moderate expression of PP5 in tet-on HeLa cells. Many HeLa cells expressing EGFP-PP5(WT) possessed multi-nuclei separated from each other by nuclear membrane, while expression of EGFP-PP5(H304A) induced deformed nuclei which were multiple-like in shape, but not separated completely and were surrounded by one nuclear membrane. These results suggest that PP5 plays important roles at the M-phase of the cell cycle, especially in separation of chromosomes and formation of nuclear membrane.     

Development of an assay method for activities of serine/threonine protein phosphatase type 2B (calcineurin) in crude extracts

Despite the physiological importance of serine/threonine protein phosphatase type 2B (PP2B/calcineurin), an accurate assay method of PP2B in crude tissue extracts has not been established. By using recombinant protein phosphatase inhibitor-1 as a substrate and ascorbic acid as an antioxidant, we developed an improved assay method for PP2B activity in crude extracts from mouse tissues and investigated tissue distribution of its activity. Under the assay conditions, the PP2B activities were stable for at least 30 min with more than 100-fold higher sensitivity than those previously reported. The specific activities of PP2B were 22.3, 0.85, 2.9, 0.36, and 1.5 mU/mg protein in mouse brain, heart, spleen, liver, and testis, respectively, and furthermore in each region of the brain they were 26.1, 13.7, 42.8, 40.5, 15.1, and 8.6 mU/mg protein in cerebrum, midbrain plus interbrain, striatum, hippocampus, cerebellum, and brain stem, respectively. This is the first paper to demonstrate a close correlation between tissue distributions and content of PP2B. These results showed that the present assay method is extremely powerful for precise measurement of a wide range of PP2B activities including not only high PP2B activity in the brain but also low PP2B activities in other tissues.     

Protein phosphatase type 2A,PP2A,is involved in degradation of gp130

The interleukin-6 (IL-6) stimulates growth in cells such as multiple myeloma and B-cell plasmacytomas/hybridomas, while it inhibits growth in several myeloid leukemia cells. The IL-6 receptor has subunit called gp130. It was reported that Ser-782 of gp130 is phosphorylated by unidentified kinase(s) in cell extracts, and level of gp130 (S782A) transiently expressed on the cell surface of COS-7 is 6-times higher than that of the wild type. These results motivated us to analyze whether the phosphorylation of gp130 at Ser-782 is involved in its degradation or not. In this study, we demonstrated here that treatment of HepG2 cells with okadaic acid (OA), a potent inhibitor for PP2A, promotes phosphorylation of gp130 at Ser-782 and degradation of gp130. MG115, a proteasome inhibitor, suppressed this degradation. These effects of OA could not be replaced with tautomycetin (TC), an inhibitor for PP1. Purified PP2A dephosphorylated phospho-Ser-782 of gp130 in vitro. IL-6-induced activation of Stat3 was suppressed by preincubation of the cells with OA, suggesting that the IL-6 signaling pathway was blocked by OA through degradation of gp130. Taken together, present results strongly suggest that degradation of gp130 is regulated through a phosphorylation-dephosphorylation mechanism in which PP2A is crucially involved and that gp130 is a potential therapeutic target in cancers. (Mol Cell Biochem 269: 183–187, 2005)