At least three major mitotic processes are regulated by the PP2A-B56 phosphatase complex: the Spindle Assembly Checkpoint (SAC), kinetochore-microtubule attachments and sister chromatid cohesion. We show here that these key functions of PP2A-B56, which require its localization to either the kinetochore or centromere, are split between distinct subsets of B56 isoforms. PP2A-B56γ and PP2A-B56δ localize to the outer kinetochore (via BUBR1), whereas PP2A-B56α and PP2A-B56ε localize to the centromere (via Sgo2). The differential localization observed is due to a difference in affinity for the receptors: PP2A-B56γ has a reduced affinity for Sgo2 compared to PP2A-B56α and, vice versa, PP2A-B56α has a reduced affinity for BUBR1 compared to PP2A-B56γ. Given that the known binding interfaces for both BUBR1 and Sgo2 are highly conserved in all B56 isoform, we generated a series of chimeras between B56α and B56γ to uncover isoform specific interactions. This led to the identifications of two distinct regions within B56α and B56γ that regulate the binding to Sgo2 and BUBR1. Furthermore, site directed mutagenesis has revealed that proper holoenzyme assembly has a role in regulating the localization of B56: it is needed for centromeric accumulation and it interferes with kinetochore accumulation of B56α. We will present a model to explain how this differential localization could be linked to post-translational modifications of PP2AC. Together, these results help to clarify how individual PP2A-B56 isoforms achieve subcellular specificity during mitosis.

Protein Phosphatase 2A (PP2A) is a holoenzyme consisting of three subunits – a scaffold subunit (A), a catalytic subunit (C), and a regulatory subunit (B). One of its functions is to oppose hyperphosphorylation of pocket proteins in the Rb family, including p107, allowing them to form a suppressor complex with E2F that silences E2F-dependent genes required for cell cycle progression. B55α, a regulatory subunit of PP2A, binds p107 mediating its dephosphorylation by the trimeric B55α /PP2A holoenzyme. Our lab has previously shown that binding of B55α/PP2A is dependent on two regions in the intrinsically disordered spacer region of p107, which contain three potential CDK phosphorylated sites. However, it is not known which of these sites are direct B55α/PP2A substrates and how individual phosphorylation sites are presented to the catalytic active site. In order to perform these studies, we first need to purify the B55α/PP2A holoenzyme. We tested various conditions for purification, and found that the most efficient method for purification of complete holoenzymes is to use stably transfected HEK293T cells expressing flag-B55α, which can be pulled down using α-flag beads and eluted using a high concentration of flag peptide which competes with the flag tag for binding to the beads. Once the enzyme had been successfully purified we tested enzymatic activity, and found that purified enzyme was able to dephosphorylate PP2A substrates at a rate equal to or higher commercially available enzyme. We also showed that it was responsive to PP2A inhibitors. Our lab has previously created several GST-p107 fusion proteins allowing the identification of the p107 spacer binding sites for B55α/PP2A complexes using pull downs from cellular lysates. Our results using purified enzyme show that the B55a/PP2A holoenzyme binds p107 directly, and confirms the binding properties previously determined using crude lysates. We plan to use a series of p107 mutants to determine whether the CDK phosphorylated sites in these mutants are direct targets of this holoenzyme. / Biomedical Sciences

Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is limited. It was previously demonstrated that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. Typically, a structural A subunit, a catalytic C subunit, and a regulatory B subunit form PP2A heterotrimer complex. Significantly, there are two PP2A regulatory subunits in F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. Based on preliminary data, I hypothesized that Ppr1 and Ppr2 are independently involved in the regulation of fumonisin biosynthesis and/or cell development, and to test this hypothesis I generated gene-deletion mutants of PPR1 and PPR2. The ppr1 deletion strain (Δppr1) resulted in drastic growth defect, but with increased microconidia production. The ppr2 deletion mutant strain (Δppr2) showed elevated fumonisin production similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cell with central septation. Δppr2 showed early hyphal branching during conidia germination. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out unique roles in regulating fumonisin biosynthesis and fungal development.

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (A scaffolding and C catalytic subunits) bound to a variable (B) regulatory subunit of either the B, B' or B" families. Further genetic expansion and alternative splicing within each B subunit family affords the enzyme tremendous functional heterogeneity as PP2A contributes dozens of heterotrimers with varying subcellular locations and cellular substrates dictated by the variable B subunit. B'β is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca2+/calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B'β interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of more than 40 Kelch-like proteins, many of which have been defined as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. KLHL15/Cul3 specifically targets B'β to promote protein turnover via ubiquitination and proteasomal degradation. Comparison of KLHL15 and B'β expression profiles suggest that the E3 ligase adaptor contributes to selective expression of the PP2A/B'β holoenzyme in the brain. Mapping of KLHL15 residues critical for Cul3 binding and protein dimerization indicate two distinct and independent functions of KLHL15's N-terminal BTB domain while similar analysis of the C-terminal kelch domain identifies a B'β-specific binding core. While B' regulatory subunit association with the AC dimer is mediated by a highly conserved inner core of roughly 400 amino acids, the divergent N-terminus of B'β is found to be both necessary and sufficient for KLHL15-mediated degradation, with Tyr52 having an obligatory role, underlying the selective association of KLHL15 with the B'β regulatory subunit only. KLHL15 can interact with not only the monomeric version of B'β but also the more stable, PP2A/B'β heterotrimer. However, proteasomal targeting is reserved for the B subunit only. The loss of B'β promotes an exchange of B subunits and a reciprocal upregulation of alternative B subunit-containing heterotrimers. That is, excess KLHL15 may not only downregulate B'β-guided PP2A dephosphorylation activity, but moreover, may upregulate PP2A activity dictated by alternative B subunits. Taken together, these data suggest regulatory subunit-specific ubiquitination and proteasomal degradation as a novel mechanism for controlling total cellular PP2A activity.

Plants possess unique features in many aspects of development. One of these features is seen in cell wall placement during cytokinesis, which is determined by the position of the preprophase band (PPB) and the subsequent expansion of the phragmoplast that deposits the new cell wall. During phragmoplast expansion, the phragmoplast tracks to the cortical division site, which was delineated by the PPB. Thus the position of the PPB determines the orientation of the division plane. In Arabidopsis thaliana, TONNEAU2 (TON2) is required for PPB formation and has been shown to interact with a type A subunit of the PP2A phosphatase in the yeast two-hybrid system. In Arabidopsis tonneau2 (ton2) mutants, abnormalities of the cortical microtubule cytoskeleton, such as disorganization of the interphase microtubule array and lack of PPB formation before mitosis markedly affects cell shape and arrangement as well as overall plant morphology. Loss of dcd1/add1, the maize ton2 homologues gives rise to a similar phenotype in Zea mays. The TON2 protein has two EF hand domains which are calcium-binding sites. Since calcium has been known to play key roles in several areas of plant functioning, the following question was raised: “Does calcium binding contribute to the localization and function of TONNEAU at the PPB?” To address this question, a series of constructs were generated to determine if TON2 binds calcium. Additionally, Ca2+ binding sites were mutated in constructs containing the TON2 gene fused to GFP or YPF. These constructs were then transformed into ton2 mutant plants and the localization of TON2 fusion protein and whether the construct is capable of rescuing the mutant phenotype were observed. Although, localization of TON2 to the PPB was not observed, the presence of the constructs were confirmed in the transformed plants using selection markers and by observing fluorescence under a confocal microscope.

Au cours de la division cellulaire, l'information génétique doit être transmise de façon précise et identique de la cellule mère aux cellules filles. Le génome est répliqué au cours de la phase S tandis que la distribution des deux copies entre les cellules filles se fait au cours de la mitose. L'initiation et le maintien de la mitose nécessite un équilibre contrôlé entre les activités des kinases et des phosphatases. La protéine kinase Greatwall est requise pour l'entrée et le maintien de la mitose à travers l'inhibition de la PP2A, la principale phosphatase qui déphosphoryle les substrats du complexe Cdk1-cycline B. Au cours de ce travail, nous avons entrepris l'étude structure/fonction de la protéine kinase Greatwall qui nous a permis de caractériser ses mécanismes d'activation. Nos résultats montrent que Greatwall appartient à la famille des AGC kinases mais qu'elle présente la particularité d'être contrôlée par des mécanismes qui lui sont propres: l'activation de la protéine, qui se fait en deux étapes, est différente de celle décrite pour les autres membres de cette famille de kinases. Par la suite, nous avons identifié deux substrats de la protéine kinase Greatwall, Arpp19 (cAMP-Regulated Phosphoprotein 19) et l'alpha-Endosulfine (ENSA). Nous avons montré qu'une fois phosphorylées par Greatwall, ces deux protéines s'associent à la PP2A et inhibent cette phosphatase. Malgré le fait que ces deux substrats soient capables d'inhiber la PP2A, seul Arpp19 endogène est responsable de l'inhibition de la phosphatase pour promouvoir l'entrée en mitose dans le modèle des extraits d'ovocytes de xénope. Nous nous intéressons à présent à l'étude du rôle d'ENSA. / During cell division, genetic information must be transmitted from the mother cell to the daughter cell in an accurate and identical way. During the S phase the genome is replicated while an equal distribution of two copies of DNA between the daughter cells is made during mitosis. Initiation and maintenance of mitosis require a controlled balance between kinase and phosphatase activities. Greatwall kinase is essential for mitotic entry and maintenance through the inhibition of PP2A, the main phosphatase that dephosphorylates Cdk1/cycline B mitotic substrates. Here we investigate the mechanisms regulating Greatwall. Our results show that Greatwall is a member of the AGC family of kinases that appears to be regulated by a unique two-step mechanism that differs from the other members of this family. Furthermore we identified Arpp19 (cAMP-Regulated Phosphoprotein 19) and alpha-Endosulfine (ENSA) as two substrates of Greatwall that, when phosphorylated by this kinase, associate with and inhibit PP2A. Despite the fact that these two substrates are able to inhibit PP2A, only endogenous Arpp19 is responsible for the phosphatase inhibition at mitotic entry in xenopus egg extratcs. Roles of ENSA are currently under investigation.

O câncer é a segunda doença com maior índice de mortalidade no Brasil e ainda é responsável por um elevado número de óbitos em todo o mundo. Durante a tumorigênese ocorrem diversas alterações no genoma, transcriptoma, proteoma, e interatoma que permitem o desenvolvimento da célula maligna. Alterações descritas na via de sinalização PI3K-Akt, tais como ganho de função da quinase PI3K ou perda de função da fosfatase PTEN, levam ao aumento de PIP3 com ativação constitutiva dos alvos downstream, como da quinase Akt. A regulação negativa da Akt pode ser realizada pela fosfatase PP2A, que é inibida pela proteína SET (ou inibidor 2 da PP2A). Existem diversos mecanismos que podem contribuir para a desregulação da sinalização celular e o aumento na quantidade de uma única proteína pode levar ao desequilíbrio no processo. Recentemente, nosso grupo identificou o aumento da proteína SET em diversas amostras de pacientes com carcinoma bucal, que foi associado à ativação da Akt. Sendo assim, o objetivo geral deste trabalho foi caracterizar os potenciais mecanismos de regulação da fosfatase PTEN pelas proteínas SET e PP2A e o papel de PTEN na predisposição ao carcinoma bucal. Para isso, através de vetores de expressão foi identificada dentre outras, a subunidade B56? da PP2A capaz de reduzir os níveis de PTEN fosforilado no resíduo de S380; a interação das proteínas PTEN e PP2A foi confirmada por co-imunoprecipitação (co-IP) e imunofluorescência; a atividade de PP2A e PTEN foram avaliadas frente a expressão de SET e regiões da SET na presença ou não de mutações sítio-específicas; e os níveis de expressão de PTEN foram relacionados ao acúmulo ou silenciamento (siRNA e shRNA) de SET em CECPs e no tratamento com agente hiperacetilante (TSA) e desmetilante (5aza-deoxicitidina). Também foi avaliado o papel de PTEN na expressão de BMAL1 in vitro e in vivo, utilizando animais geneticamente modificados com deleção de PTEN tecido-condicional ao epitélio. Os resultados obtidos sugerem a participação da SET em mecanismos de controle da expressão gênica de PTEN e a participação de PTEN no controle da expressão de BMAL. / Cancer is the second cause of death in Brazil and the oral cancer is among the most predominant cancers worldwide. During tumorigenesis several changes occur in the genome, transcriptome, proteome and interatoma leading to malignant cells development. Some of the more important modifications occur in the PI3K-Akt pathway, such as the loss of PTEN phosphatase function, which increase PIP3 and results in the constitutive activation of downstream targets, including the kinase Akt. PP2A is responsible for the negative regulation of Akt and is inhibited by SET (or Inhibitor 2 of PP2A). Many mechanisms can lead to deregulation of these signaling pathways and the increase in one protein can result in pathway loss of balance. Recently Leopoldino et.al. (2009) identified SET levels increased in oral cancer tissue samples, associate to Akt activation. The main objective of this project is evaluating how PP2A and SET regulate PTEN and its relation to cancer predisposition. For this, expression vectors were used to identify, among others, B56? subunit of PP2A reducing levels of p-PTEN S380; the interaction between PP2A and PTEN was confirmed by co-immunoprecipitation (co-IP) and immunofluorescence; PP2A and PTEN activity were evaluated against expression of SET and SET regions in the presence or not of site-specific mutations; and PTEN expression levels were related to the accumulation or silencing (siRNA and shRNA) of SET in CECPs and the treatment with agents for hyperacetylation (TSA) and demethylation (5-aza-deoxycytidine). The role of PTEN on BMAL1 expression was evaluated in vitro and in vivo, using transgenic animals with tissue-specific deletion of PTEN for epithelium. The results suggest the involvement of SET in control of PTEN gene expression and participation of PTEN in the control of BMAL expression.

La phosphorylation/déphosphorylation des protéines est une modification clé dans les mécanismes qui contrôlent les évènements mitotiques.Classiquement, l’entrée en mitose requiert l’activation de Cdk1. Pour se faire, les phosphorylations inhibitrices sur Cdk1 par Myt1 et Wee1 doivent être éliminées par Cdc25. Le complexe Cdk1-Cycline B (MPF) est ainsi actif, les kinases inhibitrices inactivées.Dernièrement, une nouvelle protéine kinase clé pour l’entrée en mitose a été mise en évidence : Greatwall (Gwl). Les récents résultats publiés par notre équipe montrent que Gwl permet l’entrée et le maintien en mitose en inhibant l’activité de la phosphatase PP2A, la phosphatase responsable de la déphosphorylation des substrats de la protéine kinase Cdk1-Cycline B, via son substrat Arpp19. Gwl phosphoryle Arpp19 sur la sérine 71 lui conférant ainsi la capacité d’inhiber l’activité de la phosphatase PP2A.Une étude sur les modifications post traductionnelles d’Arpp19 a été initiée dans l’équipe et met en évidence plusieurs sites de phosphorylation : <br>• La sérine 71, site de phosphorylation par Gwl <br>• La sérine 28, dont la phosphorylation est attribuée à Cdk1 (vérifié in vitro) <br>• La sérine 113, site de phosphorylation par pKA <br>Ce projet de thèse s’inscrit dans la suite logique du travail déjà effectué dans l’équipe et a pour objectif de caractériser les modifications post traductionnelles d’Arpp19, leurs rôles dans la progression mitotique, leurs incidences sur la liaison et l’inhibition de la cible d’Arpp19, PP2A.Cette partie du projet repose sur la synthèse de mutants d’Arpp19Xe, mutants phosphomimétiques d’une part (sérine transformée en acide aspartique par mutagenèse dirigée) ou mutants dont la phosphorylation est impossible (sérine en alanine). Ces mutants nous ont permis de travailler sur l’impact de ces différentes phosphorylations dans l’extrait d’œufs de Xénope.Ce projet s’attache également à mettre en lumière l’ensemble de la voie de signalisation aboutissant aux différentes modifications post traductionnelles d’Arpp19, leurs chronologies au cours du cycle et ainsi identifier les protéines effectrices de ces phosphorylations sur Arpp19 qui sont autant de leviers potentiels sur lesquels les thérapies anti-tumorales pourraient s’appuyer. / Proteins phosphorylation and dephosphorylation are key post translational modifications controlling mitotic events.Traditionally, mitotic entry requires Cdk1 activation. To allow this to occur, inhibitory phosphorylations on Cdk1 by Myt1 and Wee1 kinases must be removed by phosphatase Cdc25. Thus, the Cdk1-Cyclin B complex, also called MPF (Mitotic Promoting Factor), is active and inhibitory kinases inactivated.Along this canonic scheme, another key kinase has been shown to play a critical role: the Greatwall (Gwl) kinase also called MAST-L for MAST like. Results published by our team show that in Xenopus laevis, Gwl allows entry and maintains mitosis by inhibiting the activity of the phosphatase responsible for dephosphorylation of Cdk1/Cycline B substrates: PP2A. This activity is driven by Gwl target: Arpp19. Gwl phosphorylates Arpp19 on its 71st residue turning it into a potent inhibitor of PP2A.A study of Arpp19 post translational modifications of Arpp19 has been initiated in the team which will allow the further study of several phosphosites: <br>• Serine 71, Gwl phosphosite, the best documented site. <br>• Serine 28, shown in vitro to be a Cdk1-CycB phosphosite. <br>• Serine 113, assigned to PKA. <br>This thesis project joins logically after the work already made in the team and has for objective to characterize the post translational modifications of Arpp19, their roles in mitotic progress, their incidences on binding and inhibition of Arpp19’s target, PP2A.This part of the project relies on mutants' synthesis of Arpp19Xe, phosphomimetics’ mutants on one hand (serine transformed into aspartic acid by mutagenesis) or mutants unable to be phosphorylated (serine into alanine). These mutants allowed us to work on the impact of these various phosphorylations in Xenopus eggs extracts.This project also attempts to highlight the whole signalization pathway ending in the various post translational modifications of Arpp19, their timelines during the cycle and thus to identify effector proteins of these phosphorylations on Arpp19 which are as much as potential levers on which can serve as targets for cancer therapy.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and major regulator of the DNA damage response (DDR). One critical ATM target is protein phosphatase 2A (PP2A) known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylation of many proteins during the recovery from the DDR while returning the cell to normalcy. Interestingly, ATM and PP2A are known to form an auto-regulatory yin-yang kinase-phosphatase relationship. Herein, we show that the phosphorylation of the PP2A-Aα structural subunit at S401 by ATM results in nuclear export, which regulates the DDR at multiple levels and affects genomic stability and cell growth. We generated PP2A-Aα conditional knockout mouse embryonic fibroblasts expressing PP2A-Aα-WT, S401A (cannot be phosphorylated), or S401D phosphomimetic) transgenes by floxing out the endogenous PP2A-Aα alleles with Cre. The S401D mutant cells displayed increased ERK and AKT signaling, resulting in an enhanced growth rate. Phosphorylation of PP2A-Aα at S401 caused the dissociation of ATM with the holoenzyme, an effect that could be recapitulated with S401D. Additionally, the S401A and S401D mutants exhibited significantly more chromosomal aberrations and underwent increased mitotic catastrophe after radiation. Both the S401A and the S401D cells showed impaired DSB repair (Non-homologous end joining and Homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Time-lapse video and cellular localization experiments showed that the PP2A-Aα subunit was exported to the cytoplasm after radiation possibly by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects seen. In conclusion, our study demonstrates using a genetically defined system that ATM phosphorylation of a single, critical amino acid S401 is essential for regulating DDR. To study how the interplay between ATM and PP2A affects DDR in the brain, we are in the process of generating a brain specific PP2A-Aα conditional knockout mouse. Loss of many DDR related proteins like ATM and PP2A can lead to severe neuropathological effects. This model will be helpful in dissecting the PP2A-Aα/ATM regulatory circuit in the brain in response to DDR.