Xenopus embryos begin development by undergoing a series of extremely rapid cell divisions that occur without growth, gap phases, or cell cycle checkpoints. This cell cycle program, which allows the fertilized egg to rapidly subdivide its contents into many separate cells, is made possible by the extraordinary ability of these embryos to replicate DNA quickly. After a dozen such divisions, the time required to complete S phase and complete the cell cycle increases sharply amidst other embryonic changes during the midblastula transition (MBT). Successful completion of the MBT is essential for viability, but the mechanism responsible for actuating these changes remains unknown. Previous work has shown that the onset of the MBT is dependent upon the embryo reaching a critical nucleus to cytoplasm (N/C) ratio, but it is unclear how this controls cell cycle lengthening. Here, we use Xenopus egg extracts to investigate the mechanism responsible for S phase lengthening at the MBT. As in embryos, high N/C extracts exhibit lengthened S phases, and this is due to both reduced utilization of origins of replication and reduced replication fork progression. Although recent work has suggested that developmental activation of the ATR/Chk1 pathway may provide the stimulus for cell cycle remodeling at the MBT, we find that this pathway is not activated more efficiently at high N/C ratio. Rather, the Chk1 phosphorylation observed at high N/C is simply the aggregated, basal checkpoint activity associated with normal replication in a large number of nuclei. Instead, we provide evidence that the reduced replication rates at high N/C ratio are the result of the depletion of maternal factors by the increased number of nuclei, and these factors are involved in both the initiation of replication and replication fork progression. We provide evidence that protein phosphatase 2A (PP2A) activity is the limiting factor for origin firing in high N/C extracts. Likewise, partial depletion of PP2A is sufficient to prevent the high levels of origin firing observed in low N/C extracts. These results suggest a mechanism by which PP2A levels control the rate of origin firing in Xenopus egg extracts and in Xenopus embryos at the MBT.

Centrosomes are organelles that promote microtubule growth. Normally, a single centrosome duplicates once each cell cycle to guide assembly of a bipolar mitotic spindle, ensuring that each daughter cell inherits an equal complement of the genome and a single centrosome. Centrosomes are composed of a pair of ‘mother-daughter’ centrioles and, during duplication, each mother centriole assembles one daughter at a single site. However, mother centrioles can inappropriately assemble multiple daughters, thereby generating centriole amplification (or overduplication), resulting in multipolar spindle assembly and, consequently, chromosome missegration - a driving force for chromosomal instability/aneuploidy which induces birth defects, miscarriage, and tumorigenesis. We have elucidated how the cell cycle control program regulates the centriole duplication machinery to limit centriole duplication to one event per cell cycle via the cell cycle-dependent regulation of Ana2/STIL and PLK4 degradation. In the case of the centrosome licensing factor Plk4, we found that autophosphorylation promotes its own destruction during interphase, which is then counteracted by the Protein Phosphatase 2A (PP2A) in complex with its Twins (tws) regulatory subunit during mitosis. This promotes stabilization of Plk4 and thus allows for the licensing of the mother centriole, making it competent to duplicate during the proceeding S-phase. While PP2Atws plays a positive role in regulating Plk4 to promote centriole duplication, we found that PP2A complexed with the Well-rounded (wrd) and Widerborst (wdb) regulatory subunits negatively regulates Ana2 by promoting its degradation to limit centriole duplication. PP2Awrd/wdb dephosphorylates numerous serine/threonine residues residing in Ana2, including several CDK phosphorylation consensus motifs. We found that CDK1/cycA and CDK2/cycE phosphorylate these residues to promote Ana2 stabilization from S-phase, the start of centriole duplication, to M-phase, the start of centriole duplication licensing. Interestingly, we found that the tumorigenic SV40 virus protein Small Tumor Antigen (ST) amplifies centrioles by targeting the PP2A complex to stabilize Plk4 as well as Ana2, underscoring the oncogenic importance of these newly discovered centriole duplication pathways. Finally, we shed insight into the mechanism for centriole amplification upon Ana2 stabilization by showing that Ana2 associates with Plk4 to promote Plk4 kinase activity as well as Plk4 stabilization.

T-cells are lymphocytes that make up part of the adaptive arm of the immune system, and are essential for efficient protection from and eradication of viruses and pathogens. T-cells not only play an important role in protection from external agents, but also regulate and prevent activation towards self-peptides and detect and remove erratically growing cells. Alterations in T-cell activation and suppression contribute to auto-immunity, immunocompromised disorders, and cancer progression. The immune system, and T-cells in particular, provides daily surveillance, recognition and destruction of aberrant cells. Although the immune system is proficient at suppressing malignant progression, tumor cells acquire various methods of immune evasion. Myelodysplastic Syndrome (MDS) is a pre-malignant dysplastic disorder of the bone marrow characterized by ineffective hematopoiesis and clonality in the myeloid lineage, where lack of immune response has been implicated in the propensity for progression to acute myeloid leukemia (AML). Leukemia progression is associated with the acquisition of complex genetic abnormalities. Alterations in immune system regulation have been implicated in various stages of the disease process, although the role of the immune system in response to several therapies in MDS has not been fully discovered. Lenalidomide is a small molecule therapeutic preferentially effective in MDS patients with an interstitial chromosome 5q deletion (del(5q)). Improved erythropoiesis has also been reported to occur in 20-30% of low-risk, non-del(5q) patients. Although lenalidomide is a potent immunomodulatory drug that potentiates T-cell and NK-cell responses, the T-cell compartment in MDS is highly deregulated by aberrant repertoire skewing, decreased function and abnormal naïve and memory cell homeostasis. The presence of lymphoid infiltrates in the bone marrow of lenalidomide-responsive patients suggests that T-cells may participate in the hematopoietic response, but it is unclear if lenalidomide is capable of reversing these functional T-cell defects. We therefore assessed immunological changes in low-risk MDS patients before and after 16-weeks of lenalidomide therapy, and assessed the relationship to erythroid response. Although MDS T-cells were anergic prior to treatment, we have shown that T-cells in responders have a significant increase in antigen-induced proliferative response and T helper type-1 (Th1) cytokine production (IL-2, IFN-γ, TNF-α) compared to non-responders. The change in function positively correlated with an increase in naïve T-cells and a decrease in memory cells, indicating that lenalidomide has immunomodulatory activity to reverse anergy in MDS. Although it is known that lenalidomide may increase T-cell activation and proliferation in the absence of co-stimulatory signals, a direct mechanism of action has yet to be elucidated. Since CD28 is one of the most important co-stimulatory molecules deregulated in cancer, we therefore set out to determine if the expression of CD28 was essential for lenalidomide's mechanism in T-cells. We knocked out CD28 expression in healthy donor T-cells, and sorted on inherently deficient, CD28null, T-cells that accumulate in older healthy donors and found that lenalidomide-induced proliferation and function were completely ablated within the CD28null subset. These data indicate the immunotyrosine-based activation motifs (ITAMs) on the intracellular domain of the CD28 receptor are necessary for lenalidomide action. Interestingly, during the natural aging process, repeated exposure to antigens results in the accumulation of CD28null T-cells that are phenotypically distinct and functionally deficient due to excessive proliferative history in vivo. We therefore examined whether CD28 expression on MDS patient T-cells affected responses to lenalidomide, and if this could be used as a predictive biomarker of responsiveness. We found that patients who fail lenalidomide therapy had higher CD8+ Terminal Effector Memory (TEM), which are inherently CD28null, and that non-responders had an overall increase in CD4+ and CD8+CD28null T-cells, as well as an increase in CD28null cells within the TEM compartment compared to hematologic responders. We then sought to determine the particular protein target of lenalidomide responsible for increased CD28 receptor signaling in T-cells. Several targets in a variety of cell types have been postulated, although the direct mechanism in T-cells is unclear. Our group has previously shown that lenalidomide inhibits the activity of two haplodeficient phosphatases located within the commonly deleted region (CDR) on chromosome 5q in the MDS myeloid clone, Protein Phosphatase 2A (PP2A) and Cdc25c. PP2Ac is known to bind CD28 and is hypothesized to inhibit T-cell co-stimulation. Therefore, it is plausible that lenalidomide and other IMiDs inhibit the phosphatase activity of PP2A which leads to increased activation of T-cell proximal signals dependent on CD28 expression. We examined this hypothesis using molecular modeling and virtual screening and found that all of the IMiDs (lenalidomide, pomalidomide, and thalidomide) can theoretically interact with the catalytic pocket of the PP2A heterotrimer, potentially inhibiting PP2Ac activity. In vitro phosphatase activity assays supported these findings as lenalidomide-inhibition of PP2Ac activity was seen in both ad293 and Jurkat cell lines, and in primary T-cells. Mutations of theorized lenalidomide hydrogen-bond sites within the catalytic pocket of PP2A rendered the enzyme catalytically dead, indicating that these are important residues for enzymatic activity, but unfortunately could not be used to determine if lenalidomide activity was disrupted by mutation of those sites. These data together suggest that the ability of lenalidomide to augment immune activation in vivo in MDS patients, and potentially other diseases, is extremely important to patient response. Also, that CD28 expression on T-cells is essential for lenalidomide immune-mediated tumor eradication through CD28 downstream signaling, and potentially through inhibition of PP2A. These results are useful in designing future lenalidomide-combination therapy trials in other hematologic and solid malignancies, and could be used to help stratify patients for future therapeutic decisions in MDS and other malignancies.

L'obésité et le diabète de type 2 sont associés à la sédentarité et à une alimentation riche en graisses. En effet, les acides gras saturés s'accumulent dans les tissus non adipeux, comme les muscles squelettiques pour générer des lipides appelés céramides (CER). Mon projet de thèse s'est articulé en deux parties dont l'objectif est d'empêcher les CER d'agir. Nous avons montré que, selon la structure de la membrane plasmique, les CER altèrent la voie de signalisation insulinique en ciblant la PKB, protéine clef de la voie insulinique, via la voie PKC? dans les myotubes L6 et la voie PP2A dans les myotubes C2C12. Nous avons aussi mis en évidence que les CER altèrent la sensibilité à l'insuline via la voie PKC? dans les cellules musculaires humaines. Une fois les CER produits au niveau du réticulum endoplasmique (RE), ils sont transportés au Golgi par un transporteur CERT pour y être métabolisés en sphingomyéline (SM) et des études ont montré que la transformation des CER en SM pouvait être une étape cruciale pour empêcher les CER d'agir. Dans plusieurs modèles d'insulino-résistance musculaire, l'expression de CERT est diminuée et nous avons démontré l'importance du transport des céramides du RE vers le Golgi en inhibant artificiellement l'activité ou l'expression de CERT. A l'opposé, la surexpression de CERT améliore la sensibilité à l'insuline dans les cellules musculaires dans des conditions lipotoxiques. Nos résultats montrent que CERT joue un rôle crucial dans les mécanismes conduisant au développement de l'insulinorésistance musculaire puisque sa présence est essentielle pour le maintien d'un trafic normal des CER entre le RE et le golgi. / Obesity and type 2 diabetes are associated with a sedentary lifestyle and a diet rich in fat. Indeed, saturated fatty acids accumulate in non-adipose tissue such as skeletal muscle to generate lipids called ceramides (CER). My thesis project was divided into two parts with the objective to prevent CER to act. We have shown that, depending on the structure of the plasma membrane, CER alter the insulin signaling pathway by targeting PKB, a key insulin signalling protein, via a PKCζ pathway in L6 myotubes and a PP2A pathway in C2C12 myotubes. We also demonstrated that CER affect insulin sensitivity via the PKCζ pathway in human muscle cells. Once CER generated in the endoplasmic reticulum (ER), they are transported to the Golgi by a carrier called CERT to be metabolized into sphingomyelin (SM). Studies have shown that the transformation of CER into SM could be a crucial step to prevent CER to act. In several muscle insulin resistance models, expression of CERT is decreased and we demonstrated the importance of the transport of ceramide from the ER to the Golgi by inhibiting artificially the activity or the expression of CERT. In contrast, overexpression of CERT enhances insulin sensitivity in muscle cells in lipotoxiques conditions. Our results show that CERT plays a crucial role in mechanisms leading to the development of muscle insulin resistance since its presence is essential for maintaining normal traffic of CER between the ER and the Golgi.

No description available.

Ataxia telangiectasia and Rad3-related protein (ATR) is a serine/threonine-protein kinase of the PI3K family and is well known for its key role in regulating DNA damage responses in the nucleus. In addition to its nuclear functions, ATR also was found to be a substrate of the prolyl isomerase Pin1 in the cytoplasm where Pin1 isomerizes cis ATR at the Ser428-Pro429 motif, leading to formation of trans ATR. Cis ATR is an antiapoptotic protein at mitochondria upon UV damage. Here we report that Pin1’s activity on cis ATR requires the phosphorylation of the S428 residue of ATR and describe the molecular mechanism by which Pin1-mediated ATR isomerization in the cytoplasm is regulated. We identified protein phosphatase 2A (PP2A) as the phosphatase that dephosphorylates Ser428 following DNA damage. The dephosphorylation led to an increased level of the antiapoptotic cis ATR (ATR-H) in the cytoplasm and, thus, its accumulation at mitochondria via binding with tBid. Inhibition or depletion of PP2A promoted the isomerization by Pin1, resulting in a reduction of cis ATR with an increased level of trans ATR. We conclude that PP2A plays an important role in regulating ATR’s anti-apoptotic activity at mitochondria in response to DNA damage. Our results also imply a potential strategy in enhancing cancer therapies via selective moderation of cis ATR levels.

L'entrée en mitose est initiée par le complexe cycline B – Cdk1. La phosphorylation de ses substrats déclenche des transformations incluant la condensation des chromosomes, le bris de l'enveloppe nucléaire et la formation d'un fuseau mitotique. Ces transformations permettent à la cellule de se diviser. La protéine phosphatase 2A (PP2A) en complexe avec sa sous-unité B55/Twins (Tws) reconnaît et déphosphoryle les substrats de cycline B – Cdk1. Pour éviter la déphosphorylation précoce des phosphoprotéines mitotiques, PP2A-B55/Tws est inhibée en entrée de mitose. Cette inhibition de la phosphatase est attribuable au module Greatwall (Gwl) – endosulfines. Activée en entrée de mitose, la kinase Gwl phosphoryle les endosulfines, qui inhibent alors de manière spécifique PP2A-B55/Tws. Gwl est exportée du noyau vers le cytoplasme en prophase, avant le bris de l’enveloppe nucléaire. Les mécanismes de régulation spatiotemporelle du module Gwl – endosulfines – PP2AB55/Tws ne sont pas entièrement élucidés. De plus, les substrats ciblés par PP2AB55/Tws en sortie mitose ne sont pas tous identifiés à ce jour. Dans mon travail de thèse, j’ai trouvé que Tws peut transiter par le noyau via un signal de localisation nucléaire (NLS), mais que ses fonctions essentielles sont au cytoplasme. De plus, j’ai trouvé que l’unique endosulfine présente chez Drosophila melanogaster, Endos, a une localisation cytoplasmique. Cette localisation est requise pour qu’Endos soit efficacement phosphorylée par la forme active et cytoplasmique de Gwl. Endos phosphorylée lie ensuite PP2A-Tws pour l’inhiber. Empêcher la localisation cytoplasmique d’Endos avant le bris de l’enveloppe nucléaire entraîne des défauts mitotiques dépendants de l’activité de PP2A-Tws. Les substrats mitotiques de PP2A-Tws ne sont pas tous connus. Par des cribles de phosphoprotéomique, j’ai identifié des substrats mitotiques potentiels de PP2A-Tws. L’un des candidats hyperphosphorylés suite à la déplétion de Tws, Otefin (Ote), est une protéine de l’enveloppe nucléaire. Les sites de phosphorylation d’Otefin identifiés dans mes cribles sont adjacents à son domaine d’interaction avec BAF, une protéine liant l’ADN et certaines protéines de l’enveloppe nucléaire. L’introduction de mutations phosphomimétiques à ces sites abolit l’association d’Otefin avec BAF, en plus de retarder le recrutement d’Otefin à l’enveloppe nucléaire en sortie de mitose. Par ailleurs, l’association Otefin – BAF dépend de l’activité de PP2A-Tws. Enfin, la perte d’Otefin dans l’embryon syncytial de mouche affecte le développement. En somme, mes travaux ont permis d’approfondir notre compréhension mécanistique de la régulation spatiotemporelle du module Gwl – endosulfines – PP2A et d’identifier de nouveaux substrats potentiels de PP2A-Tws. / Mitosis is triggered by the cyclin B – Cdk1 complex that phosphorylates multiple substrates to promote transformations such as chromosome condensation, nuclear envelope breakdown and mitotic spindle formation. These transformations are required for cell division. The protein phosphatase 2A (PP2A) in complex with its B55/Twins (Tws) subunit dephosphorylates cyclin B – Cdk1 substrates. To prevent premature dephosphorylation of the mitotic phosphoproteins, PP2A-B55/Tws is inhibited upon mitotic entry. The Greatwall (Gwl) – endosulfines pathway is responsible for PP2AB55/Tws inhibition. Activated upon mitotic entry, the Gwl kinase phosphorylates small proteins called endosulfines to turn them into specific inhibitors of PP2A-B55/Tws. Gwl is exported from the nucleus to the cytoplasm before nuclear envelope breakdown. However, the mechanisms of spatiotemporal regulation of the Gwl – endosulfines – PP2A module are not entirely elucidated. Moreover, the identity of the proteins targeted by PP2A-Tws during mitotic exit is still unclear. During my PhD training, I found that Tws can transit through the nucleus via a nuclear localization signal (NLS), but its essential functions are cytoplasmic. Moreover, the sole endosulfine present in Drosophila melanogaster, Endos, has a cytoplasmic localization. Such localization is required for efficient phosphorylation of Endos by active and cytoplasmic Gwl. Once phosphorylated, Endos binds PP2A-Tws to inhibit its activity. Preventing the cytoplasmic localization of Endos prior to nuclear envelope breakdown causes mitotic defects that are PP2A-Tws-dependent. The mitotic substrates of PP2A-Tws are not all identified. By phosphoproteomic screening, I identified potential novel PP2A-Tws substrates. Among the hits that are hyperphosphorylated following Tws depletion, there is the nuclear envelope protein Otefin (Ote). The identified phosphosites on Otefin are adjacent to its domain of interaction with BAF, a protein binding DNA and nuclear envelope proteins. Introducing phosphomimetic mutations at these sites abolishes the Otefin – BAF association and delays Otefin recruitment at the reforming nuclear envelope during mitotic exit. Moreover, the Otefin – BAF association is PP2A-Tws-dependent. Finally, loss of Otefin in the syncytial embryo of the fly impairs development. Altogether, my results deepen our understanding of the spatiotemporal coordination of the Gwl – endosulfines – PP2A module and provide potential novel PP2A-Tws substrates.

Protein phosphorylation is a reversible post-translational modification that is critical for the regulation of key cellular processes. It is estimated that two-thirds of all cellular proteins are phosphorylated, with more than 98% of those phosphorylation events occurring on Ser/Thr residues. Protein phosphorylation is mediated by protein kinases and reversed via dephosphorylation by protein phosphatases. Two protein phosphatases, phosphatase 1 (PP1) and Protein phosphatase 2A (PP2A), are thought to account for more than 90% of the total phosphatase activity in eukaryotic cells. PP2A is a highly conserved assortment of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). Substrate recruitment of PP2A/B55α, the most abundant PP2A holoenzyme, was thought to be mediated by charge-charge interactions between the surface of B55α and its substrates. Challenging this view, we recently discovered a conserved SLiM (Short Linear Motif) [RK]-V-x-x-[VI]-R in a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowle et al. eLife 2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55α/PP2A, and analysis of the associated proteomic datasets that aided in identifying FAM122A, which can assist in the further identification of potential substrates and cellular pathways regulated by this phosphatase. The newly identified conserved SLiM is necessary for FAM122A binding to B55α in vitro and in cells. Computational structure prediction with AlphaFold2 predicts an interaction consistent with the mutational data and supports a mechanism whereby FAM122A uses the ‘SLiM’ in the form of a short α-helix to dock to the B55α top groove. In this model, FAM122A spatially constrains substrate access by occluding the catalytic subunit with a second α-helix immediately adjacent to helix-1. Consistently, FAM122A functions as a competitive inhibitor as it prevents the binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. Ablation of FAM122A in human cell lines reduces the rate of proliferation, the progression through cell cycle transitions, and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in the attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a ‘SLiM’-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase. In agreement with these findings, ectopic expression of B55α results in the downregulation of 14-3-3σ signaling mediated by ATM and ATR as determined by pathway analysis of phosphoproteomic datasets and a reduction of ATM signaling within the total proteome. Altogether, this work has significantly expanded our understanding of the PP2A/B55 SLiM, resulting from the characterization of FAM122A, a high-affinity substrate inhibitor, and enables future interrogation of novel substrates and signaling networks regulated by PP2A/B55α. / Biomedical Sciences

Protein phosphorylation is a reversible post-translation modification that is essential in cell signaling. It is estimated that a third of all cellular proteins are phosphorylated (reviewed in Ficarro et al., 2002), with more than 98% of those phosphorylation events occurring on serine and threonine residues (Olsen et al., 2006). Kinases are the necessary enzymes for phosphorylation and protein phosphatases dynamically reverse this action. While the mechanisms of substrate recognition for kinases have been well-characterized to date, the same is not true for phosphatases that play an equally important role in opposing kinase function and determining global phosphorylation levels in cells. This dichotomy has also translated into the clinic, where there has been a persistently narrow research focus on the development of small-molecule kinase inhibitors for use as chemotherapeutic agents, without an equal effort being placed into the generation of the analogous phosphatase activators (reviewed in Westermarck, 2018). Members of the phosphoprotein phosphatase (PPP) family of serine/threonine phosphatases are responsible for the majority of dephosphorylation in eukaryotic cells, with protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) accounting for more than 90% of the total phosphatase activity (Moorhead et al., 2007; Virshup and Shenolikar, 2009). Structurally, PP2A is a trimeric holoenzyme consisting of a scaffold (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. B55α is a ubiquitous regulatory subunit that is reported to target many substrates with critical functions in processes including cell division. A long-standing question that has persisted in the field of cellular signaling is as to how the most abundant serine/threonine PP2A holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site for subsequent dephosphorylation. Such critical data have only recently become well understood for the B56 family of ‘B’ regulatory subunits, where an LxxIxE short linear motif (or SLiM) has been identified in a subset of protein targets and shown via crystal structure analysis to dock into a 100% conserved binding pocket on the B56 surface (Hertz et al., 2016; Wang et al., 2016a; Wang et al., 2016b; Wu et al., 2017). Here, we show how B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an “HxRVxxV619-625” SLiM in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous additional PP2A/B55α substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, allowing us to propose a consensus SLiM sequence, “p[ST]-P-x(5-10)-[RK]-V-x-x-[VI]-R”. In support of this, mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. Moreover, a data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation to the PP2A/C active site. Altogether, these data provide key insights into PP2A/B55α mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding the role of PP2A/B55α in many key cellular processes. As a parallel continuation of our efforts to identify novel B55α substrates/regulators, we generated mutant B55α constructs that occlude PP2A/A-C dimer engagement but retain substrate binding to the β-propeller structure (allowing us to interrogate direct interactors). Our preliminary AP-MS data led to the identification of several proteins that bound better to our “monomeric B55α” mutant compared to wild-type B55α in the context of the PP2A/B55α heterotrimer, including the centrosomal proteins HAUS6 and CEP170 (two substrates previously validated in a phosphoproteomic screen by our lab), suggesting that these mutants trap substrates as they cannot be dephosphorylated by PP2A/C. These analyses also identified an enrichment of T-complex protein 1 subunits in the “monomeric B55α” mutant elutions, further supporting the notion that these mutants may function as dominant negatives. Several additional proteins of interest were identified in the two independent rounds of mass spectrometry, including subunits of the DNA-directed RNA polymerases I, II, and IV, as well as the double-strand break repair protein MRE11, which can be followed up as potential novel B55α substrates. These studies can contribute to significant advances in our understanding of the network of proteins that B55α interacts with, and thus the signaling pathways that can be modulated by PP2A/B55α complexes in cells. Moreover, these advances can also provide translational benefits as has been demonstrated through the study of PP2A activators termed SMAPs, which demonstrate selective stabilization of PP2A/B56α complexes in cells that result in selective dephosphorylation of substrates including the oncogenic target c-MYC. / Biomedical Sciences

Les cellules cancéreuses sont sujettes à des défauts de reformation de l'enveloppe nucléaire (EN) après la mitose. BAF est l'une des premières protéines recrutées sur les chromosomes pour initier la reformation de l’EN. Chez l'humain, le recrutement de BAF nécessite sa déphosphorylation par la phosphatase PP2A et Ankle2, une protéine du réticulum endoplasmique (RE) interagissant avec PP2A. Cependant, les fonctions d’Ankle2 dans la reformation de l’EN ne sont pas complètement comprises. Pour les étudier, notre laboratoire utilise la drosophile comme organisme modèle. On ne sait pas si Ankle2 de drosophile fonctionne dans le NER. Nous avons constaté qu’Ankle2 est nécessaire au recrutement de BAF pour le réassemblage du noyau après la mitose chez la drosophile. Pour mieux comprendre son fonctionnement, nous avons identifié des protéines avec lesquelles BAF interagit : PP2A, Vap33 (une protéine du RE) et certaines Kinases Dépendantes des Cyclines (CDK). Nous avons cartographié les régions d’Ankle2 impliquées dans ces interactions protéiques grâce à une analyse mutationnelle, des co-purifications par affinité et des pulldowns GST. Nous avons ensuite généré des mutants d’Ankle2 spécifiquement déficients pour des interactions et testé leur capacité à sauver la prolifération et la reformation de l’EN dans des cellules où Ankle2 endogène est déplété. Nos résultats indiquent que l'interaction entre Ankle2 et PP2A est essentielle pour sa fonction dans la reformation de l’EN. Une analyse biochimique suggère qu’Ankle2 fonctionne comme une sous-unité régulatrice de PP2A. En utilisant une approche phosphoprotéomique, nous avons confirmé que la déphosphorylation de BAF dépend d’Ankle2 et nous avons aussi identifié de nouveaux substrats potentiels du complexe PP2A-Ankle2. Nous concluons que le complexe PP2A-Ankle2 est nécessaire à la déphosphorylation de BAF et à son recrutement pour le réassemblage du noyau. Les expériences en cours permettront de déterminer les exigences d'autres interactions d’Ankle2 pour ses fonctions dans la reformation de l’EN. La suite de ces travaux impliquera l’étude de la régulation de nouveaux substrats de PP2A-Ankle2 impliqués dans ce processus. Une reformation de l’EN défectueuse peut provoquer une 4 micronucléation, ce qui peut déclencher une réponse immunitaire innée. La perturbation de la reformation de l’EN dans les cellules cancéreuses pourrait donc être bénéfique dans le contexte de l’immunothérapie. / Cancer cells are prone to defects in Nuclear Envelope Reformation (NER) after mitosis. BAF is one of the first proteins recruited on chromosomes to initiate NER. In humans, BAF recruitment requires its dephosphorylation by PP2A and Ankle2, a PP2A-interacting protein of the endoplasmic reticulum (ER). However, the functions of Ankle2 in NER are incompletely understood. Our lab uses Drosophila as a model system. Whether Drosophila Ankle2 functions in NER is unknown. We found that Ankle2 is required for BAF recruitment to reassembling nuclei in Drosophila. To better understand how it functions, we identified its interactors, which include PP2A, Vap33 (an ER protein) and Cyclin-Dependent Kinases (CDKs). We mapped the regions of Ankle2 involved in these protein-protein interactions through a mutational analysis, affinity co-purifications and GST pulldowns. We then generated mutant forms of Ankle2 defective in individual interactions and tested their ability to rescue proliferation and NER in cells depleted from endogenous Ankle2. Our results indicate that the interaction of Ankle2 with PP2A is essential for its function in NER. A biochemical analysis suggests that Ankle2 functions as a regulatory subunit of PP2A. Using a phosphoproteomic approach, we confirmed that BAF dephosphorylation depends on Ankle2 and also identified novel candidate substrates of the PP2A-Ankle2 complex. We conclude that PP2A-Ankle2 complex is required for BAF dephosphorylation and recruitment to reassembling nuclei. Ongoing experiments will determine the requirements of other interactions of Ankle2 for its functions in NER. Future work will explore the regulation of novel PP2A-Ankle2 substrates in this process. Defective NER can cause micronucleation, which can elicit an innate immune response. Disrupting NER in cancer cells could be beneficial in the context of immunotherapy.