Egon Ogris

Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I

Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesins Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres.

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABαC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.

Protein Phosphatase 2A Methyltransferase Links Homocysteine Metabolism with Tau and Amyloid Precursor Protein Regulation

Alzheimers disease (AD) neuropathology is characterized by the accumulation of phosphorylated tau and amyloid-β peptides derived from the amyloid precursor protein (APP). Elevated blood levels of homocysteine are a significant risk factor for many age-related diseases, including AD. Impaired homocysteine metabolism favors the formation of S-adenosylhomocysteine, leading to inhibition of methyltransferase-dependent reactions. Here, we show that incubation of neuroblastoma cells with S-adenosylhomocysteine results in reduced methylation of protein phosphatase 2A (PP2A), a major brain Ser/Thr phosphatase, most likely by inhibiting PP2A methyltransferase (PPMT). PP2A methylation levels are also decreased after ectopic expression of PP2A methylesterase in Neuro-2a (N2a) cells. Reduced PP2A methylation promotes the downregulation of Bα-containing holoenzymes, thereby affecting PP2A substrate specificity. It is associated with the accumulation of both phosphorylated tau and APP isoforms and increased secretion of β-secretase-cleaved APP fragments and amyloid-β peptides. Conversely, incubation of N2a cells with S-adenosylmethionine and expression of PPMT enhance PP2A methylation. This leads to the accumulation of dephosphorylated tau and APP species and increased secretion of neuroprotective α-secretase-cleaved APP fragments. Remarkably, hyperhomocysteinemia induced in wild-type and cystathionine-β-synthase +/− mice by feeding a high-methionine, low-folate diet is associated with increased brain S-adenosylhomocysteine levels, PPMT downregulation, reduced PP2A methylation levels, and tau and APP phosphorylation. We reported previously that downregulation of neuronal PPMT and PP2A methylation occur in affected brain regions from AD patients. The link between homocysteine, PPMT, PP2A methylation, and key CNS proteins involved in AD pathogenesis provides new mechanistic insights into this disorder.

Mechanism and functions of membrane binding by the Atg5–Atg12/Atg16 complex during autophagosome formation

Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane‐bound vesicles termed autophagosomes. The conserved Atg5–Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5–Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5–Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre‐autophagosomal structure but is essential for autophagy and cytoplasm‐to‐vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5–Atg12/Atg16 complex during autophagosome formation.

A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A.

Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.

Protein phosphatase 2A subunit assembly : the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen

The carboxy terminus of protein phosphatase 2A (PP2A) catalytic subunit is highly conserved. Seven out of the last nine residues, including two potential in vivo phosphorylation sites, threonine 304 and tyrosine 307, are completely invariant in all known PP2As. Mutational analysis of the carboxy terminus in vivo was facilitated by efficient immunoprecipitation of trimeric PP2A holoenzyme via an epitope-tagged catalytic subunit. The results indicate that the catalytic subunit carboxy terminus is important for complex formation with the PP2A 55 kDa regulatory B subunit, but not with polyomavirus oncogene, middle tumor antigen (MT), a viral B-type regulatory subunit. Replacing catalytic subunit threonine 304 or tyrosine 307 with a negatively charged amino acid abolished binding of the B subunit to the dimeric enzyme core and altered substrate specificity. Certain other amino acid substitutions of different size and/or charge also abolished or greatly reduced B subunit binding. Substitution of alanine at position 304 or phenylalanine at position 307 did not dramatically reduce B subunit binding or phosphatase activity in vitro, yet the latter substitutions are not found in naturally occurring PP2As. Thus, the wild-type residues are important for a yet unknown function in vivo. Additionally, deleting the carboxy terminal nine amino acids inhibited binding of the B subunit to the dimeric enzyme core, indicating a requirement for one or more of these amino acids for complex formation. MT interaction with the dimeric PP2A enzyme core was not inhibited by any of these mutations. Finally, unlike B subunit, MT does not activate the phosphatase activity of the PP2A heterodimer towards cdc2-phosphorylated histone H1.

Expression of protein phosphatase 2A mutants and silencing of the regulatory Bα subunit induce a selective loss of acetylated and detyrosinated microtubules

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down‐regulation of PP2A methylation and PP2A enzymes containing the Bα regulatory subunit occur in Alzheimer’s disease. In this study, we show that expressed wild‐type and methylation‐ (L309Δ) and phosphorylation‐ (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B′, and B′′‐type regulatory subunits in NIH 3T3 fibroblasts and neuro‐2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild‐type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309Δ, and T304D mutants, which are impaired in their ability to associate with the Bα subunit, induces their loss. Silencing of Bα subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole‐induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal Bα‐containing PP2A heterotrimers contribute to MT destabilization in Alzheimer’s disease.

A Phosphorylation Switch Regulates the Transcriptional Activation of Cell Cycle Regulator p21 by Histone Deacetylase Inhibitors

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells.

Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.

J. Neurochem. (2010) 115, 1455–1465.

Budding yeast greatwall and endosulfines control activity and spatial regulation of PP2A(Cdc55) for timely mitotic progression.

Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2ACdc55 phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to PP2ACdc55 in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2ACdc55 activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2ACdc55, timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2ACdc55, which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms.