mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected

Protein kinase B (PKB, Akt) is a Ser/Thr kinase involved in the regulation of cell survival, proliferation, and metabolism and is activated by dual phosphorylation on Thr(308) in the activation loop and Ser(473) in the hydrophobic motif. It plays a contributory role to platelet function, although little is known about its regulation. In this study, we investigated the role of the mammalian target of rapamycin complex (mTORC)-2 in Akt regulation using the recently identified small molecule ATP competitive mTOR inhibitors PP242 and Torin1. Both PP242 and Torin1 blocked thrombin and insulin-like growth factor 1-mediated Akt Ser(473) phosphorylation with an IC(50) between 1 and 5 nm, whereas the mTORC1 inhibitor rapamycin had no effect. Interestingly, PP242 and Torin1 had no effect on Akt Thr(308) phosphorylation, Akt1 activity, and phosphorylation of the Akt substrate glycogen synthase kinase 3β, indicating that Ser(473) phosphorylation is not necessary for Thr(308) phosphorylation and maximal Akt1 activity. In contrast, Akt2 activity was significantly reduced, concurrent with inhibition of PRAS40 phosphorylation, in the presence of PP242 and Torin1. Other signaling pathways, including phospholipase C/PKC and the MAPK pathway, were unaffected by PP242 and Torin1. Together, these results demonstrate that mTORC2 is the kinase that phosphorylates Akt Ser(473) in human platelets but that this phosphorylation is dispensable for Thr(308) phosphorylation and Akt1 activity.     

Induction of actin cytoskeleton rearrangement by methyl okadaate--comparison with okadaic acid

Methyl okadaate is a derivative of the lipophilic polyether okadaic acid (OA), a well-known inducer of apoptosis. OA inhibits Ser/Thr protein phosphatases (PPs), among them types 1 and 2A (PP1 and PP2A), whereas methyl okadaate lacks PP1/PP2A inhibitory activity in vitro. As progressive loss of neuronal cytoarchitecture is a major event that precedes neuronal death, in this work we studied comparatively the effects of both toxins on actin cytoskeleton organization in human neuroblastoma cells by filamentous actin (F-actin) labeling with the specific dye Oregon Green 514 Phalloidin. Neither methyl okadaate nor OA modified the amount of F-actin per cell. However, confocal microscopy imaging showed that methyl okadaate induced reorganization of actin cytoskeleton, loss of the typical flattened morphology and adoption of a round shape, and a reduction in the number of neurites, with a consequent loss of cell attachment. These effects were identical to those induced by OA, although methyl okadaate potency was approximately 10-fold lower. In order to investigate the role of membrane potential and cytosolic Ca2+ concentration in morphological changes induced by these toxins, the cells were stained with bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and fura-2. No toxin effect was detected on membrane potential or calcium influx, indicating that these two signals are not responsible for cytoskeletal/morphological change induction. Methyl okadaate induced an increase of Ser/Thr phosphorylation of cellular proteins detected by western blot, showing similar phosphorylation profiles to OA. Our data suggest that methyl okadaate is an active compound that shares a pharmacological target with OA that may be a Ser/Thr phosphatase, probably different from PP1 and PP2A.     

The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzyme-coupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosine-specific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C alpha, and lambda PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.     

Subchronic microcystin-LR exposure increased hepatic apoptosis and induced compensatory mechanisms in mice

Acute lethal cytotoxicity of microcystin-LR (MC-LR), a toxin produced by fresh-water cyanobacteria, has been attributed to protein phosphatases type 1 and type 2A (PP1/PP2A) inhibition and reactive oxygen species (ROS) generation. However, the effects and molecular mechanisms of prolonged, sublethal MC-LR exposure are less known. We studied mice intraperitonealy injected with saline or 25 μg MC-LR/kg for 28 days (every 2 days). MC-LR induced apoptosis in liver and not in kidneys or heart of treated animals. Liver also showed decreased α-tubulin levels (45.56% ± 7.65% of controls) and activation of p38-MAPK and CaMKII pathways (137.93% ± 11.64% and 419.35% ± 67.83% of the control group, respectively). PP1/PP2A activity decreased from 1.82 ± 0.23 (controls) to 0.91 ± 0.98 mU/mg (MC-LR-treated mice); however, no difference in total Ser/Thr phosphatase activity was found between both the groups. The results demonstrated that apoptosis and cytoskeleton disruption contributed to the hepatic cytotoxic effects of subchronic MC-LR administration. These effects occurred in association with sustained activation of signaling cascades and development of compensatory mechanisms to maintain total Ser/Thr phosphatase activity.     

Inhibition of B56-containing protein phosphatase 2As by the early response gene IEX-1 leads to control of Akt activity

The importance of PP2A in the regulation of Akt/PKB activity has long been recognized but the nature of the holoenzyme involved and the mechanisms controlling dephosphorylation are not yet known. We identified IEX-1, an early gene product with proliferative and survival activities, as a specific inhibitor of B56 regulatory subunit-containing PP2A. IEX-1 inhibits B56-PP2A activity by allowing the phosphorylation of B56 by ERK. This leads to sustained ERK activation. IEX-1 has no effect on PP2A containing other B family subunits. Thus, studying IEX-1 contribution to signaling should help the discovery of new pathways controlled by B56-PP2A. By using overexpression and RNA interference, we show here that IEX-1 increases Akt/PKB activity in response to various growth factors by preventing Akt dephosphorylation on both Thr(308) and Ser(473) residues. PP2A-B56beta and gamma subunits have the opposite effect and reverse IEX-1-mediated Akt activation. The effect of IEX-1 on Akt is ERK-dependent. Indeed: (i) a IEX-1 mutant deficient in ERK binding had no effect on Akt; (ii) ERK dominant-negative mutants reduced IEX-1-mediated increase in pAkt; (iii) a B56beta mutant that cannot be phosphorylated in the ERK.IEX-1 complex showed an enhanced ability to compete with IEX-1. These results identify B56-containing PP2A holoenzymes as Akt phosphatases. They suggest that IEX-1 behaves as a general inhibitor of B56 activity, enabling the control of both ERK and Akt signaling downstream of ERK.     

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.     

Mitochondrial Src tyrosine kinase plays a role in the cardioprotective effect of ischemic preconditioning by modulating complex I activity and mitochondrial ROS generation

Abstract

While ischemic preconditioning (IPC) and other cardioprotective interventions have been proposed to protect the heart from ischemia/reperfusion (I/R) injury by inhibiting mitochondrial complex I activity upon reperfusion, the exact mechanism underlying the modulation of complex I activity remains elusive. This study was aimed to test the hypothesis that IPC modulates complex I activity at reperfusion by activating mitochondrial Src tyrosine kinase, and induces cardioprotection against I/R injury. Isolated rat hearts were preconditioned by three cycles of 5-min ischemia and 5-min reperfusion prior to 30-min index ischemia followed by 2 h of reperfusion. Mitochondrial Src phosphorylation (Tyr⁴¹⁶) was dramatically decreased during I/R, implying inactivation of Src tyrosine kinase by I/R. IPC increased mitochondrial Src phosphorylation upon reperfusion and this was inhibited by the selective Src tyrosine kinase inhibitor PP2. IPC's anti-infarct effect was inhibited by the selective Src tyrosine kinase inhibitor PP2. Complex I activity was significantly increased upon reperfusion, an effect that was prevented by IPC in a Src tyrosine kinase-dependent manner. In support, Src and phospho-Src were found in complex I. Furthermore, IPC prevented hypoxia/reoxygenation-induced mitochondrial reactive oxygen species (ROS) generation and cellular injury in rat cardiomyocytes, which was revoked by PP2. Finally, IPC reduced LDH release induced by both hypoxia/reoxygenation and simulated ischemia/reperfusion, an effect that was reversed by PP2 and Src siRNA. These data suggest that mitochondrial Src tyrosine kinase accounts for the inhibitory action of IPC on complex I and mitochondrial ROS generation, and thereby plays a role in the cardioprotective effect of IPC.     

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.     

The predicted beta12-beta13 loop is important for inhibition of PP2Acalpha by the antitumor drug fostriecin

The potential anticancer agent fostriecin (FOS) is a potent inhibitor of the protein Ser/Thr phosphatases PP2A and PP4 and a weaker inhibitor of PP1. Random mutagenesis and automated screening in yeast identified residues in human PP2Acalpha important for inhibitory FOS binding. A C269S substitution in the predicted beta12-beta13 loop decreased the FOS sensitivity of intact cells and increased the IC(50) of PP2Acalpha by 10-fold in vitro. Changing PP2Acalpha Cys-269 to phenylalanine, the equivalent residue in PP1, and the Y267G and G270D substitutions caused a similar effect. The results provide information relevant to the design of novel protein Ser/Thr phosphatase inhibitory drugs.     

Type 2C protein phosphatases in fungi

Type 2C Ser/Thr phosphatases are a remarkable class of protein phosphatases, which are conserved in eukaryotes and involved in a large variety of functional processes. Unlike in other Ser/Thr phosphatases, the catalytic polypeptide is not usually associated with regulatory subunits, and functional specificity is achieved by encoding multiple isoforms. For fungi, most information comes from the study of type 2C protein phosphatase (PP2C) enzymes in Saccharomyces cerevisiae, where seven PP2C-encoding genes (PTC1 to -7) with diverse functions can be found. More recently, data on several Candida albicans PP2C proteins became available, suggesting that some of them can be involved in virulence. In this work we review the available literature on fungal PP2Cs and explore sequence databases to provide a comprehensive overview of these enzymes in fungi.     

FTDP-17 missense mutations site-specifically inhibit as well as promote dephosphorylation of microtubule-associated protein tau by protein phosphatases of HEK-293 cell extract

FTDP-17 missense tau mutations: G272V, P301L, V337M and R406W promote tau phosphorylation in human and transgenic mice brains by interfering with the tau phosphorylation/dephosphorylation balance. The effect of FTDP-17 mutations on tau phosphorylation by different kinases has been studied previously. However, it is not known how various FTDP-17 mutations affect tau dephosphorylation by phosphoprotein phosphatases. In this study we have observed that when transfected into HEK-293 cells, tau is phosphorylated on various sites that are also phosphorylated in diseased human brains. When transfected cells are lysed and incubated, endogenously phosphorylated tau is dephosphorylated by cellular protein phosphatase 1 (PP1), phosphatase 2A (PP2A) and phosphatase 2B (PP2B), which are also present in the lysate. By using this assay and specific inhibitors of PP1, PP2A and PP2B, we have observed that the G272V mutation promotes tau dephosphorylation by PP2A at Ser(396/404), Ser(235), Thr(231), Ser(202/205) and Ser(214) and by PP2B at Ser(214) but inhibits dephosphorylation by PP2B at Ser(396/404). The P301L mutation promotes tau dephosphorylation at Thr(231) by PP1 and at Ser(396/404), Thr(231), Ser(235) and Ser(202/205) by PP2A but inhibits dephosphorylation at Ser(214) by PP2B. The V337M mutation promotes tau dephosphorylation at Ser(235), Thr(231) and Ser(202/205) by PP2A and at Ser(202/205) by PP2B whereas the R406W mutation promotes tau dephosphorylation at Ser(396/404) by PP1, PP2A and PP2B but inhibits dephosphorylation at Ser(202/205) and Ser(235) by PP1 and PP2A, respectively. Our results indicate that each FTDP-17 tau mutation not only site-specifically inhibits tau dephosphorylation on some sites but also promotes dephosphorylation by phosphatases on other sites.     

人参皂甙Rb1减轻冈田酸诱导的大鼠海马神经元Tau蛋白过度磷酸化

为研究人参皂甙Rb1(ginsenoside Rb1)对冈田酸(okadaic acid,OA)诱导的大鼠海马神经元Tau蛋白过度磷酸化的影响及其可能机制,实验随机分为正常组、溶媒对照组、OA模型组和Rb1预处理组。正常组不作任何处理;Rb1预处理组大鼠分别用5、10、20 mg/kg的Rb1预处理,每天一次,共14 d,于第13天向海马背侧注射1.5μl OA[0.483 μl,溶于10% 二甲基亚砜(dimethysulphoxide,DMSO)];OA模型组大鼠于第13天时海马背侧注射OA,溶媒对照组则注射等体积的生理盐水。各组均于第15天收取标本。通过Biescbowski’s染色、免疫组化和Western blot,分别观察大鼠海马神经元胞体和突起内神经原纤维的改变和磷酸化Tau蛋白的表达水平,同时检测蛋白磷酸酯酶2A(protein phosphatase-2A,PP2A)活性以探讨其作用机制。结果显示:(1)OA模型组与溶媒对照组及正常组比较,海马神经元胞体和突起着色较深,染色不均匀;神经元中Thr231和Sei396位点磷酸化的Tau蛋白和总Tau含量增多;PP2A活性则明显下降(P<0.01):(2)Rb1预处理组大鼠海马神经元胞体和突起染色均匀,神经原纤维走行规则;海马神经元中Thr231和Ser396位点磷酸化的Tau蛋白和总Tau 含量较OA模型组减少,而PP2A活性明显增高(P<0.01)。以上观察结果表明,人参皂甙Rb1可以减轻OA诱导的大鼠海马神经元Tau蛋白过度磷酸化,其机制可能与提高PP2A活性有关。     

Ser/Thr-phosphatases from bovine retina: detection of cDNA coding for the catalytic subunit of the gamma-isoform PP2B and two homologs of rdgC/PPEF]

The expression of genes for Ser/Thr phosphatases of the PP1/2A/2B class in the bovine retina was analyzed using PCR on cDNA. The mRNAs were found for the following phosphatases: PP1 (alpha- and gamma-isoforms), PP2A (alpha- and beta-isoforms), PPV, and PP2B (beta- and gamma-isoforms). Earlier, PP2B gamma-isoform was considered testis-specific. cDNAs of two phosphatases related to rdgC/PPEF were also detected. Thus, there are mRNAs of at least four phosphatases in retina that may be regulated by Ca2+.     

Plasmodium falciparum protein phosphatase type 1 functionally complements a glc7 mutant in Saccharomyces cerevisiae

We have identified a new homologue of protein phosphatase type 1 from Plasmodium falciparum, designated PfPP1, which shows 83-87% sequence identity with yeast and mammalian PP1s at the amino acid level. The PfPP1 sequence is strikingly different from all other P. falciparum Ser/Thr phosphatases cloned so far. The deduced 304 amino acid sequence revealed the signature sequence of Ser/Thr phosphatase LRGNHE, and two putative protein kinase C and five putative casein kinase II phosphorylation sites. Calyculin A, a potent inhibitor of Ser/Thr phosphatase 1 and 2A showed hyperphosphorylation of a 51kDa protein among other parasite proteins. Okadaic acid on the other hand, was without any effect suggesting that PP1 activity might predominate over PP2A activity in intra-erythrocytic P. falciparum. Complementation studies showed that PfPP1 could rescue low glycogen phenotype of Saccharomyces cerevisiae glc7 (PP1) mutant, strongly suggesting functional interaction of PfPP1 and yeast proteins involved in glycogen metabolism.     

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.     

Akt phosphorylation at Thr308 and Ser473 is required for CHIP-mediated ubiquitination of the kinase

Phosphorylation at Thr308 and Ser473 is known to activate Akt, a major kinase in mammalian cells. Once activated to turn on downstream signaling pathways, Akt returns to an inactive pool via PP2A-mediated dephosphorylation. We show here that Thr308 and Ser473 phosphorylations prompt Akt to enter the CHIP-mediated ubiquitin-proteasome pathway. Mutation at either Thr308 or Ser473 dampened its ability to bind to the U-box E3 ligase CHIP (C-terminal Hsp70 -interacting protein), and the Akt mutants revealed decreased rate of ubiquitination by CHIP. Our study unveils that the well-known phosphorylations responsible for Akt activation turn out to transduce recognition signals for Akt-CHIP binding and ensuing degradation.     

Protein phosphatase 2B inhibitor potentiates endothelial PKC activity and barrier dysfunction

Serine/threonine (Ser/Thr) protein phosphatases (PPs) are implicated in the recovery from endothelial barrier dysfunction caused by inflammatory mediators. We hypothesized that Ser/Thr PPs may regulate protein kinase C (PKC), a critical signaling molecule in barrier dysfunction, in the promotion of barrier recovery. Western analysis indicated that bovine pulmonary microvascular endothelial cells (BPMECs) expressed the three major Ser/Thr PPs, PP1, PP2A, and PP2B. Pretreatment with 100 ng/ml of FK506 (a PP2B inhibitor) but not with the PP1 and PP2A inhibitors calyculin A or okadaic acid potentiated the thrombin-induced increase in PKC phosphotransferase activity. FK506 also potentiated thrombin-induced PKC-alpha but not PKC-beta phosphorylation. FK506 but not calyculin A or okadaic acid inhibited recovery from the thrombin-induced decrease in transendothelial resistance. Neither FK506 nor okadaic acid altered the thrombin-induced resistance decrease, whereas calyculin A potentiated the decrease. Downregulation of PKC with phorbol 12-myristate 13-acetate rescued the FK506-mediated inhibition of recovery, which was consistent with the finding that the thrombin-induced phosphorylation of PKC-alpha was reduced during the recovery phase. These results indicated that PP2B may play a physiologically important role in returning endothelial barrier dysfunction to normal through the regulation of PKC.