Methionine is a signal of amino acid sufficiency that inhibits autophagy through the methylation of PP2A

Cells respond to the deprivation of nutrients by inducing autophagy. However, mechanisms through which cells coordinately regulate autophagy with metabolic state remain incompletely understood. We previously observed that prototrophic strains of yeast induce autophagy upon switch from a rich to minimal medium in the absence of severe nitrogen starvation. We determined that the sulfur-containing amino acid methionine and its downstream metabolite S-adenosylmethionine (SAM) are sufficient to strongly inhibit such autophagy. These metabolites function through Ppm1, an enzyme that methylates the catalytic subunit of the protein phosphatase PP2A. As such, methionine and SAM act as critical signals of amino acid sufficiency that reciprocally regulate autophagy and cell growth by modulating the methylation status of PP2A.     

PP2A regulates autophagy in two alternative ways in Drosophila

Protein phosphatase 2A (PP2A) holoenzyme is a heterotrimeric complex, consisting of A, B and C subunits. The catalytic subunit PP2A-C (microtubule star/mts) binds to the C-terminal part of the scaffold protein PP2A-A (PP2A-29B). In Drosophila, there are three different forms of B subunits (widerborst/wdb, twins/tws and PP2A-B'), which determine the subcellular localization and substrate specificity of the holoenzyme. Previous studies demonstrated that PP2A is involved in the control of TOR-dependent autophagy both in yeast and mammals. Furthermore, in Drosophila, wdb genetically interacts with the PtdIns3K/PTEN/Akt signaling cascade, which is a main upstream regulatory system of dTOR. Here we demonstrate that in Drosophila, two different PP2A complexes (containing B' or wdb subunit) play essential roles in the regulation of starvation-induced autophagy. The PP2A-A/wdb/C complex acts upstream of dTOR, whereas the PP2A-A/B'/C complex functions as a target of dTOR and may regulate the elongation of autophagosomes and their subsequent fusion with lysosomes. We also identified three Drosophila Atg orthologs (Atg14, Atg17 and Atg101), which represent potential targets of the PP2A-A/B'/C complex during autophagy.     

Endogenous PP2A inhibitor CIP2A degradation by chaperone-mediated autophagy contributes to the antitumor effect of mitochondrial complex I inhibition

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Greatwall maintains mitosis through regulation of PP2A

Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B–Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B–Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B–Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B–Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A‐dependent dephosphorylation of cyclin B–Cdc2 substrates is increased in GW‐depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B–Cdc2 but also by the regulation of PP2A by GW.     

甲硫氨酸腺苷转移酶1A/2A平衡与肝细胞癌

肝细胞癌是一种死亡率极高的癌症,大多数病人发现时已属晚期.甲硫氨酸腺苷转移酶(MAT)是细胞生命活动的关键酶,可以通过催化甲酼氨酸和三磷酸腺苷(ATP)结合,促进生物甲基供体S-腺苷甲酼氨酸(SAMe)的生物合成.正常肝细胞中MAT1A与MAT2A存在动态平衡,共同维持细胞内SAMe稳态;肝细胞癌中MAT1A转变成MAT2A,会使SAMe生物合成减少,为癌细胞生长提供有利条件,故MAT1A表达降低而MAT2A增高.因此,促进MAT2A向MAT1A转化,进而提高MAT1A/MAT2A的比值可能成为治疗肝细胞癌的关键靶点之一.本文就MAT1A/MAT2A平衡在肝细胞癌中的重要作用作一综述,旨在为寻找肝细胞癌防治靶点提供新的思路.     

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)     

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.     

Protein phosphatase 2A family members (PP2A and PP6) associate with U1 snRNP and the spliceosome during pre-mRNA splicing

Protein phosphorylation and dephosphorylation are both important for multiple steps in the splicing pathway. Members of the PP1 and PP2A subfamilies of phospho-serine/threonine phosphatases play essential but redundant roles in the second step of the splicing reaction. PP6, a member of the PP2A subfamily, is the mammalian homolog of yeast Sit4p and ppe1, which are involved in cell cycle regulation; however, the involvement of PP6 in the splicing pathway remains unclear. Here we show that PP2A family members physically associate with the spliceosome throughout the splicing reaction. PP2A holoenzyme and PP6 were found stably associated with U1 snRNP. Together our findings indicate that these phosphatases regulate splicing catalysis involving U1 snRNP and suggest an important evolutionary conserved role of PP2A family phosphatases in pre-mRNA splicing.     

Regulation of Endosome Sorting by a Specific PP2A Isoform

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869–5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205–216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.     

PP2A as a master regulator of the cell cycle

Protein phosphatase 2A (PP2A) plays a critical multi-faceted role in the regulation of the cell cycle. It is known to dephosphorylate over 300 substrates involved in the cell cycle, regulating almost all major pathways and cell cycle checkpoints. PP2A is involved in such diverse processes by the formation of structurally distinct families of holoenzymes, which are regulated spatially and temporally by specific regulators. Here, we review the involvement of PP2A in the regulation of three cell signaling pathways: wnt, mTOR and MAP kinase, as well as the G1→S transition, DNA synthesis and mitotic initiation. These processes are all crucial for proper cell survival and proliferation and are often deregulated in cancer and other diseases.     

Fam72a functions as a cell-cycle-controlled gene during proliferation and antagonizes apoptosis through reprogramming PP2A substrates

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TLR2 ligation induces corticosteroid insensitivity in A549 lung epithelial cells: Anti-inflammatory impact of PP2A activators

Corticosteroids are effective anti-inflammatory therapies widely utilized in chronic respiratory diseases. But these medicines can lose their efficacy during respiratory infection resulting in disease exacerbation. Further in vitro research is required to understand how infection worsens lung function control in order to advance therapeutic options to treat infectious exacerbation in the future. In this study, we utilize a cellular model of bacterial exacerbation where we pretreat A549 lung epithelial cells with the synthetic bacterial lipoprotein Pam3CSK4 (a TLR2 ligand) to mimic bacterial infection and tumor necrosis factor α (TNFα) to simulate inflammation. Under these conditions, Pam3CSK4 induces corticosteroid insensitivity; demonstrated by substantially reduced ability of the corticosteroid dexamethasone to repress TNFα-induced interleukin 6 secretion. We then explored the molecular mechanism responsible and found that corticosteroid insensitivity induced by bacterial mimics was not due to altered translocation of the glucocorticoid receptor into the nucleus, nor an impact on the NF-κB pathway. Moreover, Pam3CSK4 did not affect corticosteroid-induced upregulation of anti-inflammatory MAPK deactivating phosphatase—MKP-1. However, Pam3CSK4 can induce oxidative stress and we show that a proportion of the MKP-1 produced in response to corticosteroid in the context of TLR2 ligation was rendered inactive by oxidation. Thus to combat inflammation in the context of bacterial exacerbation we sought to discover effective strategies that bypassed this road-block. We show for the first time that known (FTY720) and novel (theophylline) activators of the phosphatase PP2A can serve as non-steroidal anti-inflammatory alternatives and/or corticosteroid-sparing approaches in respiratory inflammation where corticosteroid insensitivity exists.     

PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing

Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A–B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A–B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death.     

Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization

Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.     

Expression of the ErbB4 receptor causes reversal regulation of PP2A in the Shc signal transduction pathway in human cancer cells

Expression of ErbB4 receptor is correlated with the incidence of non-metastatic types of human cancers, whereas the overexpression of other ErbB receptor families (ErbB1/EGFR, ErbB2 and ErbB3) is correlated to the formation of metastatic tumors. However, the molecular mechanism underlying this phenomenon has been unclear. Earlier, we demonstrated that okadaic acid (OA), an inhibitor of a serine/threonine phosphatase PP2A, stimulated the growth hormone-induced ERK phosphorylation in the wild type Chinese hamster ovary (CHO) cells and the cells expressing ErbB1 receptor, but suppressed ERK activation in CHO cells that express ErbB4 receptor. PP2A had been understood as a negative regulator of the growth hormone-stimulated signal transduction pathways, however, this observation suggested that expression of ErbB4 receptor reversed the regulation of PP2A in the ErbB4 signalling pathway. In this study, we found that OA suppressed phosphorylation of Shc at Tyr317, therefore it down-regulated ERK phosphorylation in the ErbB4 expressing CHO cells. Accordingly, basal PP2A contributed to the phosphorylation of Shc Tyr317 in ErbB4 expressing CHO cells, nevertheless it had been reported that PP2A negatively regulates Shc tyrosine phosphorylation in the EGF- or IGF-I-induced signalling pathways. By testing OA for human cancer cell lines that express different types of ErbB receptors, we found that ErbB4 receptor expression was accompanied with positive regulation of PP2A for phosphorylation of Shc Tyr317 and its downstream ERK phosphorylation in MCF-7 and SK-OV-3 cell lines, but not in LNCaP and PC-3 cells. Thus, PP2A regulates the ERK activity in a cell-specific manner, and it is speculated that distinct regulation of PP2A in the ErbB4 receptor signalling pathway may cause a difference in progression of cancer phenotypes.     

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.     

Increased activity of phosphatase PP2A in the presence of the PlA2 polymorphism of alphaIIbbeta3

Polymorphisms in αIIbβ3 are important genetic factors that alter platelet biology and have been associated with susceptibility to thromboembolic disorders. To define the molecular mechanisms that lead to variance in thrombotic diathesis dictated by the β3 polymorphism, we examined regulation of intracellular signaling by αIIbβ3, and studied the effects of a common β subunit PlA2 polymorphism. We found that PP2A regulates αIIbβ3 control of the ERK signaling in a polymorphism specific fashion. In CHO cells, exogenous expression of αIIbβ3 reduced ATP-stimulated ERK phosphorylation and more so for PlA2 than PlA1. Interestingly, reduced level of ERK phosphorylation correlated with an increase in PP2A activity, with higher activity associated with PlA2 than PlA1. We tested the effect of PP2A on αIIbβ3-dependent adhesion, and found that PP2A overexpression increased cell adhesion, while phosphatase inhibitors decreased cell adhesion. We propose that PlA2 alters cell signaling at least in part by increasing β3-associated PP2A activity.     

PP2A:B56epsilon is required for eye induction and eye field separation

Eye induction and eye field separation are the earliest events during vertebrate eye development. Both of these processes occur much earlier than the formation of optic vesicles. The insulin-like growth factor (IGF) pathway appears to be essential for eye induction, yet it remains unclear how IGF downstream pathways are involved in eye induction. As a consequence of eye induction, a single eye anlage is specified in the anterior neural plate. Subsequently, this single eye anlage is divided into two symmetric eye fields in response to Sonic Hedgehog (Shh) secreted from the prechordal mesoderm. Here, we report that B56epsilon regulatory subunit of protein phosphatase 2A (PP2A) is involved in Xenopus eye induction and subsequent eye field separation. We provide evidence that B56epsilon is required for the IGF/PI3K/Akt pathway and that interfering with the PI3K/Akt pathway inhibits eye induction. In addition, we show that B56epsilon regulates the Hedgehog (Hh) pathway during eye field separation. Thus, B56epsilon is involved in multiple signaling pathways and plays critical roles during early development.