Inhibition of PP2A, but not PP5, mediates p53 activation by low levels of okadaic acid in rat liver epithelial cells

The microbial toxin okadaic acid (OA) specifically inhibits PPP-type ser/thr protein phosphatases. OA is an established tumor promoter with numerous cellular effects that include p53-mediated cell cycle arrest. In T51B rat liver epithelial cells, a model useful for tumor promotion studies, p53 activation is induced by tumor-promoting (low nanomolar) concentrations of OA. Two phosphatases sensitive to these concentrations of OA, PP2A and protein phosphatase 5 (PP5), have been implicated as negative regulators of p53. In this study we examined the respective roles of these phosphatases in p53 activation in non-neoplastic T51B cells. Increases in p53 activity were deduced from levels of p21 (cip1) and/or the rat orthologue of mdm2, two p53-regulated gene products whose induction was blocked by siRNA-mediated knockdown of p53. As observed with 10 nM OA, both phospho-ser15-p53 levels and p53 activity were increased by 10 microM fostriecin or SV40 small t-antigen. Both of these treatments selectively inhibit PP2A but not PP5. siRNA-mediated knockdown of PP2A, but not PP5, also increased p53 activity. Finally, adenoviral-mediated over-expression of an OA-resistant form of PP5 did not prevent increased phospho-ser15-p53, p53 protein, or p53 activity caused by 10 nM OA. Together these results indicate that PP5 blockade is not responsible for OA-induced p53 activation and G1 arrest in T51B cells. In contrast, specific blockade of PP2A mimics p53-related responses to OA in T51B cells, suggesting that PP2A is the target for this response to OA.     

The phosphatase inhibitor, okadaic acid, strongly protects primary rat cortical neurons from lethal oxygen-glucose deprivation

The protein kinase-mediated actions of peptide growth factors such as IGF-1 and bFGF protect cultured neurons from being killed by the oxygen and glucose deprivations (OGD) that prevail in the ‘stroked brain’. Here, we show that neuroprotection by IGF-1 is mediated by PI-3K/Akt, whereas that of bFGF is mediated by MAPK. IGF-1 and bFGF together did not further increase protection suggesting a downstream convergence of their pathways. Since protein kinases mediated the protection, a phosphatase inhibitor such as okadaic acid (OA) might be as protective as the growth factors against OGD. Here, we show that OA is actually a much more effective protector. It increased the phosphorylation of both PI-3K/Akt and MAPK, and stimulated new protein synthesis. OA also acted independently of the CREB activation and FKHRL1 and GSK-3 inactivation which have been implicated in IGF-1 actions.     

Sucrose transport is inhibited by okadaic acid during regeneration of sugar-starved Vicia faba root meristem cells

The sucrose-induced resumption of cell cycle in the Vicia faba root meristem cells, blocked in two principal control points PCP1/2 by carbohydrate starvation, occurs after 12 h of metabolic regeneration comprising increased activity of sucrose synthase (SuSy) and hexokinase (HK) as well as starch grain and cell wall matrix polysaccharide biosynthesis. Okadaic acid (OA), the specific protein phosphatase 1/2A inhibitor, supplied at the beginning of the recovery period (0–3 h) completely blocks these processes, making cell cycle resumption impossible. On the other hand, when added at the end (9–12 h), OA has a weak inhibitory effect. The aim of these studies was: (1) to establish how sucrose is transported into the cells and whether the above-mentioned effects are correlated with the intensity of its uptake at the beginning and at the end of the metabolic regeneration; and (2) to determine whether OA, blocking sucrose metabolism, also interferes with the process of sucrose uptake and distribution. The level of [³H]sucrose uptake was measured by liquid scintillation counting while sugar distribution was analyzed using microautoradiography and electron microscopy. The results showed that sucrose entered the meristematic cells along symplastic or apoplastic pathways and, to a lesser extent, through endocytosis. The cytoplasmic compartments (endoplasmic reticulum, vacuoles, plastids) and the nucleus were labeled. The intensity of [³H]sucrose uptake was nearly 2-fold lower during the initial than during the final period of metabolic regeneration. OA inhibited the apoplastic pathway of radioactive molecule uptake and its distribution between cell compartments, implicating PP1/2A involvement in the regulation of this transport.     

The protein phosphatase inhibitor, okadaic acid, potentiates the stimulatory effect of phorbol ester on phosphatidylcholine synthesis, but not on phospholipid hydrolysis, in fibroblasts.

The potent protein phosphatase inhibitor, okadaic acid, was used to determine the possible role of protein phosphorylation reaction(s) in phorbol ester-induced synthesis and hydrolysis of phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts. Okadaic acid (2 microM) was found to enhance the stimulatory effects of lower concentrations (2.5-25 nM) of phorbol 12-myristate 13-acetate (PMA) on PtdCho synthesis, but not on PtdCho hydrolysis, after treatments for 30-60 min. These data support a view that in fibroblasts PMA stimulates only PtdCho synthesis, and not PtdCho hydrolysis, by a protein phosphorylation-dependent mechanism.     

Mitotic arrest and enhanced nuclear protein phosphorylation in human leukemia K562 cells by okadaic acid, a potent protein phosphatase inhibitor and tumor promoter.

We investigated the effects of the non-phorbol tumor promoter okadaic acid on human leukemia K562 cells. It was found that okadaic acid potently and reversibly inhibited cell growth, with a nearly complete inhibition of thymidine uptake seen at about 10 nM. The cytotoxicity of okadaic acid was characterized by a marked mitotic arrest of the cells exhibiting scattered chromosomes and abnormal anaphase-like structures, a phenomenon distinct from the typical metaphase arrest caused by colchicine. Okadaic acid (10-1,000 nM) greatly stimulated phosphorylation of a number of nuclear proteins in K562 cells. Phosphorylation of many of the same proteins was also stimulated by 12-O-tetradecanoylphorbol-13-O-acetate, a protein kinase C activator. The present findings, consistent with recent reports that okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A) shown to be essential for normal mitosis, provided evidence for the first time that okadaic acid inhibition of PP1/PP2A resulted in enhanced nuclear protein phosphorylation and subsequent mitotic arrest.     

Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide. Potential regulation of eIF2 alpha phosphorylation in differentiated PC12 cells

Oxidative inactivation of protein tyrosine phosphatases and calcineurin is a well established mechanism; however, little information with regard to the effect of oxidants on PP1 and PP2A activity is available. Herein, we show that PP1 activity is inhibited by H(2)O(2) treatment in differentiated PC12 cells both in vitro and in vivo experiments. Thiol-antioxidant N-acetyl-cysteine (NAC) and reduced glutathione (GSH), when added in vitro to lysates from H(2)O(2)-treated cells, reversed PP1 inhibition. H(2)O(2) treatment increased eIF2 alpha phosphorylated levels (eIF2 alpha P) in a time- and dose-dependent fashion and promoted protein synthesis inhibition. Interestingly, NAC pretreatment protected cells from H(2)O(2)-induced PP1 inactivation and, consequently, it abolished increased H(2)O(2)-induced eIF2 alpha phosphorylation and protein synthesis inhibition. In addition, PP1 inhibitor tautomycin prevented both NAC-induced PP1 reactivation and eIF2 alpha P dephosphorylation in H(2)O(2)-treated cells. Taken together, our findings support a role for PP1 in eIF2 alpha phosphorylation and oxidative stress-triggered translation down regulation.     

Effects of phosphatase inhibitors and a protein phosphatase on norepinephrine secretion by permeabilized bovine chromaffin cells

A protein phosphatase and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized bovine chromaffin cells. Addition of okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, or 1-naphthylphosphate, a more general phosphatase inhibitor, to digitonin-permeabilized chromaffin cells caused about a 100% increase in the amount of norepinephrine secreted in the absence of Ca²⁺ (in 5 mM EGTA) without affecting the amount of norepinephrine secreted in the presence of 10 μM free Ca²⁺. This stimulation of norepinephrine secretion by protein phosphatase inhibitors suggests that in the absence of Ca²⁺ there is a slow rate phosphorylation and that this phosphorylation triggers secretion. Addition of an exogenous type 2A protein phosphatase caused almost a 50% decrease in Ca²⁺-dependent norepinephrine secretion. Thus, the amounts of norepinephrine released both in the absence of Ca²⁺ and in the presence of Ca²⁺ appear to depend upon the level of protein phosphorylation.     

Phoslactomycin targets cysteine-269 of the protein phosphatase 2A catalytic subunit in cells

According to the chemical genetic approach, small molecules that bind directly to proteins are used to analyze protein function, thereby enabling the elucidation of complex mechanisms in mammal cells. Thus, it is very important to identify the molecular targets of compounds that induce a unique phenotype in a target cell. Phoslactomycin A (PLMA) is known to be a potent inhibitor of protein Ser/Thr phosphatase 2A (PP2A); however, the inhibitory mechanism of PP2A by PLMA has not yet been elucidated. Here, we demonstrated that PLMA directly binds to the PP2A catalytic subunit (PP2Ac) in cells by using biotinylated PLMA, and the PLMA-binding site was identified as the Cys-269 residue of PP2Ac. Moreover, we revealed that the Cys-269 contributes to the potent inhibition of PP2Ac activity by PLMA. These results suggest that PLMA is a PP2A-selective inhibitor and is therefore expected to be useful for future investigation of PP2A function in cells.     

Retinoic acid induced repression of AP-1 activity is mediated by protein phosphatase 2A in ovarian carcinoma cells

In previous studies we have shown that all-trans retinoic acid (atRA)-treatment of the atRA-sensitive ovarian carcinoma cell line CA-OV3 repressed AP-1 activity by about 50%, while a similar effect was not observed in the atRA-resistant ovarian carcinoma cell line, SK-OV3. These results suggested that the repression of AP-1 activity may be one of the mechanisms by which atRA inhibits the growth of atRA-sensitive CA-OV3 cells. In the present studies, we investigated further the molecular mechanism by which AP-1 activity is repressed by atRA. We show that the repression of AP-1 activity correlates with an increase in JunB protein expression and a decrease in N-terminal phosphorylation of c-Jun. The decrease in N-terminal phosphorylation of c-Jun does not appear to be modulated by JNK or ERK, since their protein expression patterns and kinase activity do not correlate with the repression of AP-1 activity following treatment with atRA. However, the activity of the protein phosphatase PP2A was found to increase 24 h following atRA treatment in CA-OV3 cells. Moreover, the catalytic subunit of PP2A was found to associate with c-Jun in vivo following atRA treatment. Since the inhibition of AP-1 activity following atRA treatment of CA-OV3 cells was abolished in the presence of specific PP2A inhibitors, it is likely that PP2A plays an important role in the atRA-induced repression of AP-1.     

Interaction of nucleoredoxin with protein phosphatase 2A

A trimeric protein phosphatase 2A (PP2A(T55)) composed of the catalytic (PP2Ac), structural (PR65/A), and regulatory (PR55/B) subunits was isolated from rabbit skeletal muscle by thiophosphorylase affinity chromatography, and contained two additional proteins of 54 and 55 kDa, respectively. The 54 kDa protein was identified as eukaryotic translation termination factor 1 (eRF1) and as a PP2A interacting protein. The 55 kDa protein is now identified as nucleoredoxin (NRX). The formation of a complex between GST-NRX, PP2A(C) and PP2A(D) was demonstrated by pull-down experiments with purified forms of PP2A, and by immunoprecipitation of HA-tagged NRX expressed in HEK293 cells complexed endogenous PP2A subunits. Analysis of PP2A activity in the presence of GST-NRX showed that NRX competed with polycations for both stimulatory and inhibitory effects on different forms of PP2A.     

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca2+ oscillation in guard cells of Arabidopsis thaliana

Methyl jasmonate (MeJA) induces stomatal closure similar to abscisic acid (ABA), and MeJA signaling in guard cells shares some signal components with ABA signaling. As part of this process, MeJA as well as ABA induce the elevation and oscillation of cytosolic free-calcium concentrations ([Ca²⁺]_cyt) in guard cells. While abscisic acid-induced [Ca²⁺]_cyt oscillation has been extensively studied, MeJA-induced [Ca²⁺]_cyt oscillation is less well understood. In this study, we investigated the effects of K252a (a broad-range protein kinase inhibitor) and okadaic acid (OA, a protein phosphatase 1 and 2A inhibitor) on MeJA-induced [Ca²⁺]_cyt oscillation in guard cells of Arabidopsis thaliana ecotype Columbia expressing the Ca²⁺ reporter yellow cameleon 3.6. The protein kinase inhibitor K252a abolished MeJA-induced stomatal closure and reduced MeJA-elicited [Ca²⁺]_cyt oscillation. The protein phosphatase inhibitor OA, on the other hand, did not inhibit these processes. These results suggest that MeJA signaling involves activation of K252a-sensitive protein kinases upstream of [Ca²⁺]_cyt oscillation but not activation of an OA-sensitive protein phosphatase in guard cells of A. thaliana ecotype Columbia.     

Interaction between abscisic acid receptor PYL3 and protein phosphatase type 2C in response to ABA signaling in maize

Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. In recent researches, pyrabactin resistance 1-like protein (PYL) and protein phosphatase type 2C (PP2C) were identified as the direct receptor and the second component of ABA signaling pathway, respectively. However, a lot of PYL and PP2C members were found in Arabidopsis and several other plants. Some of them were found not to be involved in ABA signaling. Because of the complex diversity of the genome, few documents have been available on the molecular details of the ABA signal perception system in maize. In the present study, we conducted bioinformatics analysis to find out the candidates (ZmPYL3 and ZmPP2C16) of the PYL and PP2C members most probably involved in ABA signaling in maize, cloned their encoding genes (ZmPYL3 and ZmPP2C16), verified the interaction between these two proteins in response to exogenous ABA induction by yeast two-hybrid assay and bimolecular fluorescence complementation, and investigated the expression patterns of these two genes under the induction of exogenous ABA by real-time fluorescence quantitative PCR. The results indicated that the ZmPYL3 and ZmPP2C16 proteins interacted in vitro and in vivo in response to the induction of exogenous ABA. The downregulated expression of the ZmPYL3 gene and the upregulated expression of the ZmPP2C16 gene are responsive to the induction of exogenous ABA. The ZmPYL3 and ZmPP2C16 proteins are the most probable members of the receptors and the second components of ABA signaling pathway, respectively.     

Phase II enzyme induction by a carotenoid,lutein, in a PC12D neuronal cell line

The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.     

Induced Ca2+ uptake and callose synthesis in suspension-cultured cells of Catharanthus roseus are decreased by the protein phosphatase inhibitor okadaic acid

Subtoxic concentrations of the saponin digitonin. the polyene antibiotic amphotericin B and the bacterial phytotoxin syringomycin induce increased uptake of ⁴⁵Ca²⁺ into suspension-cultured plant cells and a rapid Ca²⁺-dependent defense response, callose synthesis. Both reactions were inhibited by preincubation of the cells with okadaic acid, a specific inhibitor of type 1 and type 2A protein phosphatases. These results suggest that Ca²⁺ uptake induced by the above agents does not occur due to unspecific perturbation of plasma membrane permeability but involves transport proteins which are controlled by protein phosphorylation/dephosphorylation. Phosphoproteins appear also to be involved in the regulation of callose synthesis, although it remains open whether this control is effected at the level of Ca²⁺ transport or at the 1,3-ß-glucan synthase involved in deposition of the polymer.     

Copper-mediated cross-linking of S100A4, but not of S100A2, results in proinflammatory effects in melanoma cells

The aim of this study was to investigate the response to and the physiological consequences of copper-mediated cross-linking of S100A2 and S100A4, two members of the S100 family of EF-hand calcium-binding proteins. As demonstrated by electrophoresis and mass spectrometry techniques S100A2 and S100A4 show formation of cross-links due to copper-mediated oxidation of cysteine residues. For S100A4, but not for S100A2, this results in both increased activation of NFκB and secretion of TNF-α in human A375 and, to a higher extent, in RAGE-transfected melanoma cells. The data suggest that a prooxidative tumor microenvironment enhances proinflammatory and prometastatic action of S100A4.     

Energy adaptive response during parthanatos is enhanced by PD98059 and involves mitochondrial function but not autophagy induction

Parthanatos is a programmed necrotic demise characteristic of ATP (adenosine triphosphate) consumption due to NAD⁺ (nicotinamide adenine dinucleotide) depletion by poly(ADP-ribose) polymerase 1 (PARP1)-dependent poly(ADP-ribosyl)ation on target proteins. However, how the bioenergetics is adaptively regulated during parthanatos, especially under the condition of macroautophagy deficiency, remains poorly characterized. Here, we demonstrated that the parthanatic inducer N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) triggered ATP depletion followed by recovery in mouse embryonic fibroblasts (MEFs). Notably, Atg5^−/− MEFs showed great susceptibility to MNNG with disabled ATP-producing capacity. Moreover, the differential energy-adaptive responses in wild-type (WT) and Atg5^−/− MEFs were unequivocally worsened by inhibition of AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and mitochondrial activity. Importantly, Atg5^−/− MEFs disclosed diminished SIRT1 and mitochondrial activity essential to the energy restoration during parthanatos. Strikingly, however, parthanatos cannot be exasperated by bafilomycin A1 and MNNG neither provokes microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation and p62 elimination, suggesting that parthanatos does not induce autophagic flux. Intriguingly, we reported unexpectedly that PD98059, even at low concentration insufficient to inhibit MEK, can promote mitochondrial activity and facilitate energy-restoring process during parthanatos, without modulating DNA damage responses as evidenced by PARP1 activity, p53 expression, and γH2AX (H2A histone family, member X (H2AX), phosphorylated on Serine 139) induction. Therefore, we propose that Atg5 deficiency confers an infirmity to overcome the energy crisis during parthanatos and further underscore the deficits in mitochondrial quality control, but not incapability of autophagy induction, that explain the vulnerability in Atg5-deficient cells. Collectively, our results provide a comprehensive energy perspective for an improved treatment to alleviate parthanatos-related tissue necrosis and disease progression and also provide a future direction for drug development on the basis of PD98059 as an efficacious compound against parthanatos.     

Cellular prostatic acid phosphatase,a PTEN-functional homologue in prostate epithelia,functions as a prostate-specific tumor suppressor

The inactivation of tumor suppressor genes (TSGs) plays a vital role in the progression of human cancers. Nevertheless, those ubiquitous TSGs have been shown with limited roles in various stages of diverse carcinogenesis. Investigation on identifying unique TSG, especially for early stage of carcinogenesis, is imperative. As such, the search for organ-specific TSGs has emerged as a major strategy in cancer research. Prostate cancer (PCa) has the highest incidence in solid tumors in US males. Cellular prostatic acid phosphatase (cPAcP) is a prostate-specific differentiation antigen. Despite intensive studies over the past several decades on PAcP as a PCa biomarker, the role of cPAcP as a PCa-specific tumor suppressor has only recently been emerged and validated. The mechanism underlying the pivotal role of cPAcP as a prostate-specific TSG is, in part, due to its function as a protein tyrosine phosphatase (PTP) as well as a phosphoinositide phosphatase (PIP), an apparent functional homologue to phosphatase and tensin homolog (PTEN) in PCa cells. This review is focused on discussing the function of this authentic prostate-specific tumor suppressor and the mechanism behind the loss of cPAcP expression leading to prostate carcinogenesis. We review other phosphatases' roles as TSGs which regulate oncogenic PI3K signaling in PCa and discuss the functional similarity between cPAcP and PTEN in prostate carcinogenesis.