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PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, TRUE, pub

Post-translational modifications govern almost every aspect of cell biology. The addition and removal of functional groups on proteins lead to complex signal transduction pathways. This dissertation focuses on elucidating regulatory mechanisms of two enzymes whose activity and signaling are interlocked in the serine/threonine phosphorylation realm: Protein Kinase C (PKC) and Protein Phosphatase 2A (PP2A). The conventional class of PKC isozymes (cPKC) is transiently activated by Ca2+ and diacylglycerol (DG) to allow phosphorylation of downstream substrates. Importantly, in the absence of these second messengers, PKC adopts an autoinhibited conformation where it has low activity and is quite stable in the cell. We first address the need for a structure of autoinhibited PKC by building a model of this conformation to generate testable hypotheses as to important residues that are involved in interdomain contacts. The second part of this dissertation utilizes our model to assess the effect of two disease-associated mutations at the same position (R42H from cancer and R42P from spinocerebellar ataxia) that are both predicted to disrupt an interaction that contributes to autoinhibition. Mutations that disrupt autoinhibitory contacts typically result in quality-control degradation. We use biochemical, cellular, and structural approaches to demonstrate that these mutants have higher basal activity and are in an open conformation as our model predicts. Whereas histidine at this position allows canonical downregulation both basally and in response to activators, R42P bypasses the ubiquitination associated with activator-induced degradation. These studies highlight the fine-tuning of PKC levels in the cell and provide examples of how two diseases manipulate this regulation to exert opposing effects on PKC. Finally, we examined potential mechanisms by which PP2A regulates PKC dephosphorylation and levels. We show that a novel motif identified on PKC may contribute to its rate of

Autophagy is a catabolic pathway capable of degrading cellular components ranging from individual molecules to organelles. Autophagy helps cells cope with stress by removing superfluous or hazardous material. In a previous work, we demonstrated that transcriptional upregulation of two autophagy-related genes, ATG5 and ATG7, in Arabidopsis thaliana positively affected agronomically important traits: biomass, seed yield, tolerance to pathogens and oxidative stress. Although the occurrence of these traits correlated with enhanced autophagic activity, it is possible that autophagy-independent roles of ATG5 and ATG7 also contributed to the phenotypes. In this study, we employed affinity purification and LC-MS/MS to identify the interactome of wild-type ATG5 and its autophagy-inactive substitution mutant, ATG5K128R Here we present the first interactome of plant ATG5, encompassing not only known autophagy regulators but also stress-response factors, components of the ubiquitin-proteasome system, proteins involved in endomembrane trafficking, and potential partners of the nuclear fraction of ATG5. Furthermore, we discovered post-translational modifications, such as phosphorylation and acetylation present on ATG5 complex components that are likely to play regulatory functions. These results strongly indicate that plant ATG5 complex proteins have roles beyond autophagy itself, opening avenues for further investigations on the complex roles of autophagy in plant growth and stress responses.

DNA damage elicits a checkpoint response depending on the Mec1/ATR kinase, which detects the presence of single-stranded DNA and activates the effector kinase Rad53/CHK2. In Saccharomyces cerevisiae, one of the signaling circuits leading to Rad53 activation involves the evolutionarily conserved 9-1-1 complex, which acts as a platform for the binding of Dpb11 and Rad9 (referred to as the 9-1-1 axis) to generate a protein complex that allows Mec1 activation. By examining the effects of both loss-of-function and hypermorphic mutations, here, we show that the Cdc55 and Tpd3 subunits of the PP2A phosphatase counteract activation of the 9-1-1 axis. The lack of this inhibitory function results in DNA-damage sensitivity, sustained checkpoint-mediated cell-cycle arrest, and impaired resection of DNA double-strand breaks. This PP2A anti-checkpoint role depends on the capacity of Cdc55 to interact with Ddc1 and to counteract Ddc1-Dpb11 complex formation by preventing Dpb11 recognition of Ddc1 phosphorylated on Thr602

Autophagy is a catabolic pathway capable of degrading cellular components ranging from individual molecules to organelles. Autophagy helps cells cope with stress by removing superfluous or hazardous material. In a previous work, we demonstrated that transcriptional upregulation of two autophagy-related genes, ATG5 and ATG7, in Arabidopsis thaliana positively affected agronomically important traits: biomass, seed yield, tolerance to pathogens and oxidative stress. Although the occurrence of these traits correlated with enhanced autophagic activity, it is possible that autophagy-independent roles of ATG5 and ATG7 also contributed to the phenotypes. In this study, we employed affinity purification and LC-MS/MS to identify the interactome of wild-type ATG5 and its autophagy-inactive substitution mutant, ATG5K128R Here we present the first interactome of plant ATG5, encompassing not only known autophagy regulators but also stress-response factors, components of the ubiquitin-proteasome system, proteins involved in endomembrane trafficking, and potential partners of the nuclear fraction of ATG5. Furthermore, we discovered post-translational modifications, such as phosphorylation and acetylation present on ATG5 complex components that are likely to play regulatory functions. These results strongly indicate that plant ATG5 complex proteins have roles beyond autophagy itself, opening avenues for further investigations on the complex roles of autophagy in plant growth and stress responses.

Autophagy is a catabolic pathway capable of degrading cellular components ranging from individual molecules to organelles. Autophagy helps cells cope with stress by removing superfluous or hazardous material. In a previous work, we demonstrated that transcriptional upregulation of two autophagy-related genes, ATG5 and ATG7, in Arabidopsis thaliana positively affected agronomically important traits: biomass, seed yield, tolerance to pathogens and oxidative stress. Although the occurrence of these traits correlated with enhanced autophagic activity, it is possible that autophagy-independent roles of ATG5 and ATG7 also contributed to the phenotypes. In this study, we employed affinity purification and LC-MS/MS to identify the interactome of wild-type ATG5 and its autophagy-inactive substitution mutant, ATG5K128R Here we present the first interactome of plant ATG5, encompassing not only known autophagy regulators but also stress-response factors, components of the ubiquitin-proteasome system, proteins involved in endomembrane trafficking, and potential partners of the nuclear fraction of ATG5. Furthermore, we discovered post-translational modifications, such as phosphorylation and acetylation present on ATG5 complex components that are likely to play regulatory functions. These results strongly indicate that plant ATG5 complex proteins have roles beyond autophagy itself, opening avenues for further investigations on the complex roles of autophagy in plant growth and stress responses.

Entry into quiescence in the fission yeast Schizosaccharomyces pombe is induced by nitrogen starvation. In the absence of nitrogen, proliferating fission yeast cells divide twice without cell growth and undergo cell cycle arrest in G1 before becoming G0 quiescent cells. Under these conditions, autophagy is induced to produce enough nitrogen for the two successive cell divisions that take place before the G1 arrest. In parallel to the induction of autophagy, the Greatwall–Endosulfine switch is activated upon nitrogen starvation to down-regulate protein phosphatase PP2A/B55 activity, which is essential for cell cycle arrest in G1 and implementation of the quiescent program. Here we show that, although inactivation of PP2A/B55 by the Greatwall–Endosulfine switch is not required to promote autophagy initiation, it increases autophagic flux at least in part by upregulating the expression of a number of autophagy-related genes.

Mixed Lineage Leukaemia (MLL) is an aggressive form of blood cancer which predominantly affects children as results of rearrangements of the MLL (KMT2A) gene at 11q23. MLL accounts for more than 70% of infant Acute Lymphoblastic Leukaemia (ALL) and between 35-50% of infant Acute Myeloid Leukaemia (AML) and, presents a very poor survival. Indeed, only 40% of the MLL patients approaches 5-years survival rate. Although new treatments have been devised to target the activity of KMT2A fusion proteins, none of these have been translated into new therapies for patients and MLL is a leukaemia of unmet clinical need. Several recent papers have identified protein kinases as hyperactivated in MLL leukaemia and potential target for treatment. Our group is investigating the contribution of Protein Phosphatase 2A (PP2A), one of the upstream regulators of protein kinases, in MLL. Our results showed that the downstream targets of this phosphatase were constitutively deregulated and hyperactivated in MLL, suggesting a potential inactivation of PP2A in this disease. SET and CIP2A are the best well-known endogenous inhibitors of PP2A. SET directly interacts with the catalytic subunit of PP2A and impairs its activity towards the phosphorylation of AKT, ERK and GSK3-ß, whereas CIP2A modulates the activity of the phosphatase by direct interaction with the structural and regulatory subunit PP2A/B56 and by preventing the dephosphorylation of a PP2A downstream target and potent oncoprotein known as c-Myc. Published studies showed that therapeutic targeting of SET and CIP2A, rescued PP2A activity and blocked malignant proliferation in solid tumours and haematological malignancies. In this study I investigated for the first time the molecular profile of SET and CIP2A in MLL. Importantly, my findings showed that SET and CIP2A are essential proteins involved in MLL self renewal of MLL and that their pharmacological inhibition blocks MLL proliferation. The impact of these data open new avenu

Viruses must effectively remodel host cellular pathways to replicate and evade immune defenses, and they must do so with limited genomic coding capacity. Targeting post-translational modification (PTM) pathways provides a mechanism by which viruses can broadly and rapidly transform a hostile host environment into a hospitable one. We use mass spectrometry-based proteomics to quantify changes in protein abundance and two PTM types-phosphorylation and ubiquitination-in response to HIV-1 infection with viruses harboring targeted deletions of a subset of HIV-1 genes. PTM analysis reveals a requirement for Aurora kinase activity in HIV-1 infection and identified putative substrates of a phosphatase that is degraded during infection. Finally, we demonstrate that the HIV-1 Vpr protein inhibits histone H1 ubiquitination, leading to defects in DNA repair.

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.

Mixed Lineage Leukaemia (MLL) is an aggressive form of blood cancer which predominantly affects children as results of rearrangements of the MLL (KMT2A) gene at 11q23. MLL accounts for more than 70% of infant Acute Lymphoblastic Leukaemia (ALL) and between 35-50% of infant Acute Myeloid Leukaemia (AML) and, presents a very poor survival. Indeed, only 40% of the MLL patients approaches 5-years survival rate. Although new treatments have been devised to target the activity of KMT2A fusion proteins, none of these have been translated into new therapies for patients and MLL is a leukaemia of unmet clinical need. Several recent papers have identified protein kinases as hyperactivated in MLL leukaemia and potential target for treatment. Our group is investigating the contribution of Protein Phosphatase 2A (PP2A), one of the upstream regulators of protein kinases, in MLL. Our results showed that the downstream targets of this phosphatase were constitutively deregulated and hyperactivated in MLL, suggesting a potential inactivation of PP2A in this disease. SET and CIP2A are the best well-known endogenous inhibitors of PP2A. SET directly interacts with the catalytic subunit of PP2A and impairs its activity towards the phosphorylation of AKT, ERK and GSK3-ß, whereas CIP2A modulates the activity of the phosphatase by direct interaction with the structural and regulatory subunit PP2A/B56 and by preventing the dephosphorylation of a PP2A downstream target and potent oncoprotein known as c-Myc. Published studies showed that therapeutic targeting of SET and CIP2A, rescued PP2A activity and blocked malignant proliferation in solid tumours and haematological malignancies. In this study I investigated for the first time the molecular profile of SET and CIP2A in MLL. Importantly, my findings showed that SET and CIP2A are essential proteins involved in MLL self renewal of MLL and that their pharmacological inhibition blocks MLL proliferation. The impact of these data open new avenu

The serine/threonine phosphatase PP2A and the cysteine protease Caspase 9 are two proteins involved in physiological and pathological processes, including cancer and apoptosis. We previously demonstrated the interaction between Caspase 9 and PP2A and identified the C9h peptide, corresponding to the binding site of Caspase 9 to PP2A. This interfering peptide can modulate Caspase 9/PP2A interaction leading to a strong therapeutic effect in vitro and in vivo in mouse models of tumor progression. In this manuscript, we investigate (I) the peptide binding to PP2A combining docking with molecular dynamics and (II) the secondary structure of the peptide using CD spectroscopy. Additionally, we compare the binding affinity, using biolayer interferometry, of the wild-type protein PP2A with Caspase 9 and vice versa to that observed between the PP2A protein and the interfering peptide C9h. This result strongly encourages the use of peptides as new therapeutics against cancer, as shown for the C9h peptide already in clinical trial.

Mutations in the KRAS gene are associated with approximately 15% of all human cancers. This makes it one of the most frequently mutated oncogenes known. Whilst recent breakthroughs in Ras drug discovery have led to the FDA approval of the first direct and covalent inhibitor of the KRAS-G12C mutant, the majority of KRAS driven cancers are not G12C mutated. Furthermore, recent studies have identified resistance mechanisms against the new inhibitors. Consequently, research into other direct and indirect Ras inhibition strategies, as well as synergistic drug combination efforts are being vigorously pursued. In the first part of this thesis, I describe my contributions to the development and characterization of novel PDE6D inhibitors with activity against KRAS driven cancers. PDE6D is a trafficking chaperone of K-Ras that facilitates its dynamic localization to the plasma membrane. Although some progress had been made in identifying lead drug candidates against this protein, an Arl2 dependent PDE6D cargo ejection mechanism continues to hamper progress. We describe the development of Deltaflexin 1 and Deltaflexin 2, into which we engineered a ‘molecular spring’ to improve resilience to Arl2 ejection of PDE6D cargo. We show that these compounds selectively inhibit K-Ras membrane organization and exhibited K-Ras selective anti-proliferative effects against cancer cell lines from colon and breast tissues whilst blocking the 3D spheroid growth of lung and breast cancer cell lines. In the second part, I describe my main project, the identification of a novel covalent inhibitor of calmodulin (CaM) with anti-cancer activity in K-Ras mutated cancers named Calmirasone1. A relevance of the K-Ras/ CaM interaction for the promotion of cancer cell stemness has been previously suggested. We previously showed that the natural product Ophiobolin A (OphA) blocked K-Ras membrane organization in a CaM dependent manner and cancer cell spheroid formation. However, because of the broad toxicit

Mutations in the KRAS gene are associated with approximately 15% of all human cancers. This makes it one of the most frequently mutated oncogenes known. Whilst recent breakthroughs in Ras drug discovery have led to the FDA approval of the first direct and covalent inhibitor of the KRAS-G12C mutant, the majority of KRAS driven cancers are not G12C mutated. Furthermore, recent studies have identified resistance mechanisms against the new inhibitors. Consequently, research into other direct and indirect Ras inhibition strategies, as well as synergistic drug combination efforts are being vigorously pursued. In the first part of this thesis, I describe my contributions to the development and characterization of novel PDE6D inhibitors with activity against KRAS driven cancers. PDE6D is a trafficking chaperone of K-Ras that facilitates its dynamic localization to the plasma membrane. Although some progress had been made in identifying lead drug candidates against this protein, an Arl2 dependent PDE6D cargo ejection mechanism continues to hamper progress. We describe the development of Deltaflexin 1 and Deltaflexin 2, into which we engineered a ‘molecular spring’ to improve resilience to Arl2 ejection of PDE6D cargo. We show that these compounds selectively inhibit K-Ras membrane organization and exhibited K-Ras selective anti-proliferative effects against cancer cell lines from colon and breast tissues whilst blocking the 3D spheroid growth of lung and breast cancer cell lines. In the second part, I describe my main project, the identification of a novel covalent inhibitor of calmodulin (CaM) with anti-cancer activity in K-Ras mutated cancers named Calmirasone1. A relevance of the K-Ras/ CaM interaction for the promotion of cancer cell stemness has been previously suggested. We previously showed that the natural product Ophiobolin A (OphA) blocked K-Ras membrane organization in a CaM dependent manner and cancer cell spheroid formation. However, because of the broad toxicit

HIV-1 Vif forms an E3 ubiquitin ligase complex with host proteins to counteract host restrictive APOBEC3, and is also known to accumulate infected cells at the G2 phase to promote viral replication. However, the underlying mechanism of how Vif induces G2 arrest is not fully understood, and more specifically, direct target molecules of G2 arrest have not been identified. Here we show that degradation of B56 family proteins (PP2A-B56), one of the regulatory subunits of protein phosphatase 2A, is critical for the Vif-induced G2 arrest. NL4-3 Vif caused degradation of PP2A-B56, and complementation of PP2A-B56 overcome the Vif-induced arrest. Supportively, knockdown of PPP2R5D, one of PP2A-B56, by siRNA itself induced cell cycle arrest of non-infected cells. We also identified Vif residues I31 and R or K33 are determinants for inducing G2 arrest, and Vif variants that did not cause G2 arrest did not induce PPP2R5D degradation, although it maintain the ability to induce APOBEC3G degradation, showing strong correlation between Vif-induced arrest and PP2A-B56 degradation. In a sequence database of HIV-1 isolates, Vif strains harboring residues that presumably induce cell cycle arrest are approximately 43%, suggesting Vif-induced G2 arrest contributes to HIV-1 infection in vivo and spread. Our data help understand the mechanism of Vif-mediated arrest, and gain insights into general cell cycle regulation.

Protein Phosphatase 2 Regulatory Subunit B' Delta (PPP2R5D)-related intellectual disability (ID) and neurodevelopmental delay results from germline de novo mutations in the PPP2R5D gene. This gene encodes the protein PPP2R5D (also known as the B56 delta subunit), which is an isoform of the subunit family B56 of the enzyme serine/threonine-protein phosphatase 2A (PP2A). Clinical signs include intellectual disability (ID); autism spectrum disorder (ASD); epilepsy; speech problems; behavioral challenges; and ophthalmologic, skeletal, endocrine, cardiac, and genital malformations. The association of defective PP2A activity in the brain with a wide range of severity of ID, along with its role in ASD, Alzheimer's disease, and Parkinson's-like symptoms, have recently generated the impetus for further research into mutations within this gene. PP2A, together with protein phosphatase 1 (PP1), accounts for more than 90% of all phospho-serine/threonine dephosphorylations in different tissues. The specificity for a wide variety of substrates is determined through nearly 100 different PP2A holoenzymes that are formed by at least 23 types of regulatory B subunits, and two isoforms each of the catalytic subunit C and the structural subunit A. In the mammalian brain, PP2A-mediated protein dephosphorylation plays an important role in learning and memory. The PPP2R5D subunit is highly expressed in the brain and the PPP2A-PPP2R5D holoenzyme plays an important role in maintaining neurons and regulating neuronal signaling. From 2015 to 2017, 25 individuals with PPP2R5D-related developmental disorder were diagnosed. Since then, Whole-Exome Sequencing (WES) has helped to identify more unrelated individuals clinically diagnosed with a neurodevelopmental disorder with pathological variants of PPP2R5D. In this review, we discuss the current understanding of the clinical and genetic aspects of the disorder in the context of the known functions of the PP2A-PPP2R5D holoenzyme in the brain, as we

This thesis details efforts to generate chemical probes for the interrogation of important protein targets: the CLK and DYRK kinases, ceramide synthases (CerS), and the PP2A-inhibitory protein, SBDS. Modulation of activity with chemical probes and protein degradation with PROTACs were investigated. Chapter 1 provides an introduction to chemical probes and PROTACs, introduces the relevant protein targets, and provides background on previous work by the Morris group. Chapters 2-4 are focused on the development of chemical probes and PROTACs for the CLK and DYRK kinases. In Chapter 2, an analysis of crystal structures and inhibitors of CLK1 and DYRK1a resulted in elucidation of the factors that determine selective inhibition. This analysis led to the investigation of 6-azaindole 2.3—previously reported as a DYRK1a-selective inhibitor— and it was found to be a potent inhibitor of CLK1, CLK2, and DYRK1a. Insights gained from the structural analysis were used to design quinoline 2.36, a CLK1 inhibitor with 15-fold selectivity over DYRK1a.Work in Chapter 3 led to improved pharmacophore models for selectivity and potency, which were used to develop novel potent CLK/DYRK inhibitors, including 3-quinoline 3.44, a 1.2 nM inhibitor of DYRK1a with 5.4-fold selectivity over CLK1. Chapter 4 describes a preliminary study of 6-azaindole PROTACs targeted towards CLK1/DYRK1a. A small library of PROTACs based on amide 4.5 were synthesised and initial biological evaluation by our collaborators did not indicate degradation of CLK1 or DYRK1a at 1 µM across multiple treatment times, possibly due to insufficient formation of stable ternary complexes. Chapter 5 outlined investigations with the AAL(S) scaffold. A preliminary investigation of PROTACs for the degradation of CerSs was carried out, with a small library of CerS PROTACs synthesised. These influenced cellular ceramide concentrations, via a yet-to-be-determined mechanism. The second part of Chapter 5 was focused on the elucidation of

Protein Phosphatase 2 Regulatory Subunit B' Delta (PPP2R5D)-related intellectual disability (ID) and neurodevelopmental delay results from germline de novo mutations in the PPP2R5D gene. This gene encodes the protein PPP2R5D (also known as the B56 delta subunit), which is an isoform of the subunit family B56 of the enzyme serine/threonine-protein phosphatase 2A (PP2A). Clinical signs include intellectual disability (ID); autism spectrum disorder (ASD); epilepsy; speech problems; behavioral challenges; and ophthalmologic, skeletal, endocrine, cardiac, and genital malformations. The association of defective PP2A activity in the brain with a wide range of severity of ID, along with its role in ASD, Alzheimer's disease, and Parkinson's-like symptoms, have recently generated the impetus for further research into mutations within this gene. PP2A, together with protein phosphatase 1 (PP1), accounts for more than 90% of all phospho-serine/threonine dephosphorylations in different tissues. The specificity for a wide variety of substrates is determined through nearly 100 different PP2A holoenzymes that are formed by at least 23 types of regulatory B subunits, and two isoforms each of the catalytic subunit C and the structural subunit A. In the mammalian brain, PP2A-mediated protein dephosphorylation plays an important role in learning and memory. The PPP2R5D subunit is highly expressed in the brain and the PPP2A-PPP2R5D holoenzyme plays an important role in maintaining neurons and regulating neuronal signaling. From 2015 to 2017, 25 individuals with PPP2R5D-related developmental disorder were diagnosed. Since then, Whole-Exome Sequencing (WES) has helped to identify more unrelated individuals clinically diagnosed with a neurodevelopmental disorder with pathological variants of PPP2R5D. In this review, we discuss the current understanding of the clinical and genetic aspects of the disorder in the context of the known functions of the PP2A-PPP2R5D holoenzyme in the brain, as we

Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. Only a limited number of PP2A-regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site-specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. Strikingly, we find that B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A-B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A-B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.

Plant growth-promoting rhizobacteria (PGPR) stimulate plant growth, but the underlying mechanism is poorly understood. In this study, we asked whether PROTEIN PHOSPHATASE 2A (PP2A), a regulatory molecular component of stress, growth, and developmental signaling networks in plants, contributes to the plant growth responses induced by the PGPR Azospirillum brasilense (wild type strain Sp245 and auxin deficient strain FAJ0009) and Pseudomonas simiae (WCS417r). The PGPR were co-cultivated with Arabidopsis wild type (WT) and PP2A (related) mutants. These plants had mutations in the PP2A catalytic subunits (C), and the PP2A activity-modulating genes LEUCINE CARBOXYL METHYL TRANSFERASE 1 (LCMT1) and PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (PTPA). When exposed to the three PGPR, WT and all mutant Arabidopsis revealed the typical phenotype of PGPR-treated plants with shortened primary root and increased lateral root density. Fresh weight of plants generally increased when the seedlings were exposed to the bacteria strains, with the exception of catalytic subunit double mutant c2c5. The positive effect on root and shoot fresh weight was especially pronounced in Arabidopsis mutants with low PP2A activity. Comparison of different mutants indicated a significant role of the PP2A catalytic subunits C2 and C5 for a positive response to PGPR.

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. Only a limited number of PP2A-regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site-specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. Strikingly, we find that B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A-B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A-B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.

Cell size is proportional to growth rate. Thus, cells growing rapidly in rich nutrients can be nearly twice the size of cells growing slowly in poor nutrients. This proportional relationship appears to hold across all orders of life, yet the underlying mechanisms are unknown. In budding yeast, most growth occurs during mitosis, and the proportional relationship between cell size and growth rate is therefore enforced primarily by modulating growth in mitosis. When growth is slow, the duration of mitosis is increased to allow more time for growth, yet the amount of growth required to complete mitosis is reduced, which leads to the birth of small daughter cells. Previous studies have found that Rts1, a member of the conserved B56 family of protein phosphatase 2A regulatory subunits, works in a TORC2 signaling network that influences cell size and growth rate. However, it was unclear whether Rts1 influences cell growth and size in mitosis. Here, we show that Rts1 is required for the proportional relationship between cell size and growth rate during mitosis. Moreover, nutrients and Rts1 influence the duration and extent of growth in mitosis via Wee1 and Pds1/securin, two conserved regulators of mitotic progression. Together, the data are consistent with a model in which global signals that set growth rate also set the critical amount of growth required for cell cycle progression, which would provide a simple mechanistic explanation for the proportional relationship between cell size and growth rate.

Protein phosphorylation is a common mechanism for the regulation of cell cycle progression. The opposing functions of cell cycle kinases and phosphatases are crucial for accurate chromosome segregation and exit from mitosis. Protein phosphatases 2A are heterotrimeric complexes that play essential roles in cell growth, proliferation, and regulation of the cell cycle. Here, we review the function of the protein phosphatase 2A family as the counteracting force for the mitotic kinases. We focus on recent findings in the regulation of mitotic exit and cytokinesis by PP2A phosphatases in S. cerevisiae and other fungal species.

Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.

Maintenance of genome integrity is fundamental for cellular physiology. Our hereditary information encoded in the DNA is intrinsically susceptible to suffer variations, mostly due to the constant presence of endogenous and environmental genotoxic stresses. Genomic insults must be repaired to avoid loss or inappropriate transmission of the genetic information, a situation that could lead to the appearance of developmental anomalies and tumorigenesis. To safeguard our genome, cells have evolved a series of mechanisms collectively known as the DNA damage response (DDR). This surveillance system regulates multiple features of the cellular response, including the detection of the lesion, a transient cell cycle arrest and the restoration of the broken DNA molecule. While the role of multiple kinases in the DDR has been well documented over the last years, the intricate roles of protein dephosphorylation have only recently begun to be addressed. In this review, we have compiled recent information about the function of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DDR, focusing mainly on their capacity to regulate the DNA damage checkpoint and the repair mechanism encompassed in the restoration of a DNA lesion.

The Golgi is the main hub for the modification, sorting, and transporting of proteins to various intra- and extracellular destinations. Against conventional belief that most of Golgi trafficking was constitutive, our data show regulation of Golgi morphology and trafficking in response to cell density, and extracellular cue.Studies in our lab showed that the Golgi phosphoprotein 3 (GOLPH3) functions at the Golgi in the GOLPH3 complex to regulate Golgi trafficking. GOLPH3 is recruited to the Golgi by binding to phosphatidylInositol-4-phosphate (PI(4)P) which is enriched at the trans-Golgi. GOLPH3 also interacts with Myosin 18A (MYO18A), which in turn binds to F-actin. The linkages of PI(4)P-GOLPH3-MYO18A-F-actin make the basis of the GOLPH3 complex, which provides a tensile force applied onto Golgi membrane to aid in vesicles budding for protein transport. The consequence of that process is Golgi being stretched like an extended ribbon that surrounds the nucleus. We have shown that interfering with the GOLPH3 complex reduces cellular transport and alters Golgi morphology. Here, we identify a signaling pathway that transduces cell density signal through the Hippo proteins MST1/2 and the STRIPAK complex to the GOLPH3 complex to regulate Golgi forward trafficking. In sparsely growing cells, MST1/2, independently of their kinase activity and the canonical Hippo pathway, act to promote the localization of Striatin 3 (STRN3) to the Golgi where STRN3 directly interacts with GOLPH3 bridging it with other members of the STRIPAK complex. The PP2A phosphatase activity of STRIPAK promotes GOLPH3’s interaction with MYO18A, which together drive Golgi forward trafficking and impart the Golgi’s normal, extended ribbon shape. On the other hand, high cell density inhibits interaction between STRIPAK complex to GOLPH3 taking away the effect of PP2A, which results in a weaken GOLPH3-MYO18A interaction. Inhibition of GOLPH3-MYO18A interaction results in compact Golgi and reduced Golgi tra

peer-reviewed, Conflicting reports implicate the scaffolding protein RACK1 in the progression of breast cancer. RACK1 has been identified as a key regulator downstream of growth factor and adhesion signalling and as a direct binding partner of PP2A. Our objective was to further characterise the interaction between PP2A and RACK1 and to advance our understanding of this complex in breast cancer cells. We examined how the PP2A holoenzyme is assembled on the RACK1 scaffold in MCF-7 cells. We used immobilized peptide arrays representing the entire PP2A-catalytic subunit to identify candidate amino acids on the C subunit of PP2A that might be involved in binding of RACK1. We identified the RACK1 interaction sites on PP2A. Stable cell lines expressing PP2A with FR69/70AA, R214A and Y218F substitutions were generated and it was confirmed that the RACK1/PP2A interaction is essential to stabilise PP2A activity. We used Real-Time Cell Analysis and a series of assays to demonstrate that disruption of the RACK1/PP2A complex also reduces the adhesion, proliferation, migration and invasion of breast cancer cells and plays a role in maintenance of the cancer phenotype. This work has significantly advanced our understanding of the RACK1/PP2A complex and suggests a pro-carcinogenic role for the RACK1/PP2A interaction. This work suggests that approaches to target the RACK1/PP2A complex are a viable option to regulate PP2A activity and identifies a novel potential therapeutic target in the treatment of breast cancer. (C) 2016 Elsevier Inc All rights reserved., ACCEPTED, peer-reviewed

The Golgi is the main hub for the modification, sorting, and transporting of proteins to various intra- and extracellular destinations. Against conventional belief that most of Golgi trafficking was constitutive, our data show regulation of Golgi morphology and trafficking in response to cell density, and extracellular cue.Studies in our lab showed that the Golgi phosphoprotein 3 (GOLPH3) functions at the Golgi in the GOLPH3 complex to regulate Golgi trafficking. GOLPH3 is recruited to the Golgi by binding to phosphatidylInositol-4-phosphate (PI(4)P) which is enriched at the trans-Golgi. GOLPH3 also interacts with Myosin 18A (MYO18A), which in turn binds to F-actin. The linkages of PI(4)P-GOLPH3-MYO18A-F-actin make the basis of the GOLPH3 complex, which provides a tensile force applied onto Golgi membrane to aid in vesicles budding for protein transport. The consequence of that process is Golgi being stretched like an extended ribbon that surrounds the nucleus. We have shown that interfering with the GOLPH3 complex reduces cellular transport and alters Golgi morphology. Here, we identify a signaling pathway that transduces cell density signal through the Hippo proteins MST1/2 and the STRIPAK complex to the GOLPH3 complex to regulate Golgi forward trafficking. In sparsely growing cells, MST1/2, independently of their kinase activity and the canonical Hippo pathway, act to promote the localization of Striatin 3 (STRN3) to the Golgi where STRN3 directly interacts with GOLPH3 bridging it with other members of the STRIPAK complex. The PP2A phosphatase activity of STRIPAK promotes GOLPH3’s interaction with MYO18A, which together drive Golgi forward trafficking and impart the Golgi’s normal, extended ribbon shape. On the other hand, high cell density inhibits interaction between STRIPAK complex to GOLPH3 taking away the effect of PP2A, which results in a weaken GOLPH3-MYO18A interaction. Inhibition of GOLPH3-MYO18A interaction results in compact Golgi and reduced Golgi tra

The size of all cells, from bacteria to vertebrates, is proportional to the growth rate set by nutrient availability, but the underlying mechanisms are unknown. Here, we show that nutrients modulate cell size and growth rate via the TORC2 signaling network in budding yeast. An important function of the TORC2 network is to modulate synthesis of ceramide lipids, which play roles in signaling. TORC2-dependent control of ceramide signaling strongly influences both cell size and growth rate. Thus, cells that cannot make ceramides fail to modulate their growth rate or size in response to changes in nutrients. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is embedded in a feedback loop that controls TORC2 signaling and helps set the level of TORC2 signaling to match nutrient availability. Together, the data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals arising from the TORC2 network.

B cell activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as a tumor suppressor in multiple types of cancer, our genetic studies revealed an essential role of PP2A in B cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway (PPP) to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B cells and transcriptional repression of G6PD and other key PPP enzymes by the B cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional PPP-repression, glucose carbon utilization in B cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a gatekeeper function of the PPP in a broad range of B cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD.

Regulatory T cells (T reg ) are necessary to maintain immunological tolerance and are key players in the control of autoimmune disease susceptibility. Expression of the transcription factor FOXP3 is essential for differentiation of T reg cells and indispensable for their suppressive function. However, there is still a lack of knowledge about the mechanisms underlying its regulation. Here, we demonstrate that pro-autophagy protein AMBRA1 is also a key modulator of T cells, regulating the complex network that leads to human T reg differentiation and maintenance. Indeed, through its ability to interact with the phosphatase PP2A, AMBRA1 promotes the stability of the transcriptional activator FOXO3, which, in turn, triggers FOXP3 transcription. Furthermore, we found that AMBRA1 plays a significant role in vivo by regulating T reg cell induction in mouse models of both tumor growth and multiple sclerosis, thus highlighting the role of AMBRA1 in the control of immune homeostasis. Regulatory T cells (T reg ) maintain immunological tolerance and help control autoimmune disease susceptibility. Becher et al. show pro-autophagy factor AMBRA1 regulates human and mouse T reg differentiation and maintenance. AMBRA1 is upregulated in stimulated T cells to stabilize FOXO3 and has a protective effect in a mouse model of multiple sclerosis.

Many natural products that show therapeutic activities are often difficult to synthesize or isolate and have unknown targets, hindering their development as drugs. Identifying druggable hotspots targeted by covalently acting anti-cancer natural products can enable pharmacological interrogation of these sites with more synthetically tractable compounds. Here, we used chemoproteomic platforms to discover that the anti-cancer natural product withaferin A targets C377 on the regulatory subunit PPP2R1A of the tumor-suppressor protein phosphatase 2A (PP2A) complex leading to activation of PP2A activity, inactivation of AKT, and impaired breast cancer cell proliferation. We developed a more synthetically tractable cysteine-reactive covalent ligand, JNS 1-40, that selectively targets C377 of PPP2R1A to impair breast cancer signaling, proliferation, and in vivo tumor growth. Our study highlights the utility of using chemoproteomics to map druggable hotspots targeted by complex natural products and subsequently interrogating these sites with more synthetically tractable covalent ligands for cancer therapy.

Recent efforts in cancer therapy investigations have been focused on developing a rational strategy to specifically target cancer cells to reduce toxic side effects. Uncontrolled cell division (mitosis) is a hallmark of cancer cells. Classical anti-mitotic drugs such as Taxanes and Vinca alkaloids work by disrupting microtubule dynamics, which results in a block in cell division and induction of mitotic arrest. This prolonged mitotic arrest can trigger cell death, which is often curative in some cancer patients. However, despite the success of these chemotherapies, many patients have innate or develop acquired resistance. Furthermore, higher doses of these drugs cause toxicities, which limit the efficacy of these compounds. To overcome these limitations research has focused on finding novel drug combinations that enhance the effects of mitotic chemotherapies. Recently, blocking mitotic exit has emerged as a promising strategy for killing cancer cells and enhancing mitotic chemotherapy response. In order to find novel regulators of mitotic exit, we analysed published genome wide RNA interference screens and online-databases to identify novel genes whose knockdown increased mitotic index and raised the sensitivity of cells to Taxol. In this thesis, we explored two of these potential new target genes, STK16 (serine/threonine kinase 16) and Protein phosphatase 2 A (PP2A). The effects of targeting STK16 and PP2A (using specific siRNA or chemical inhibitors) were examined in combinations with Taxol. HeLa cells stably expressing histone H2B fused to red fluorescent protein (H2B-mCherry) were treated with combination of drugs and monitored by live cell imaging systems. Phenotypes of the cells were tracked during the treatment to measure duration of mitotic arrest and percentage of dead cells. Knockdown of STK16 failed to affect mitotic progression or the duration of mitotic arrest in combination with Taxol. In contrast, siRNA knockdown of the B55 subunit of PP2A and inhibit

The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A ( CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2A(HOZ)) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2A(HOZ) mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4(+) T-cells and CD8(+) effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2A(HOZ) as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects.

Proliferating cells adjust their cell size depending on the nutritional environment. Cells are large in rich media and small in poor media. This physiological response has been demonstrated in both unicellular and multicellular organisms. Here we show that the greatwall-endosulfine (Ppk18-Igo1 in fission yeast) pathway couples the nutritional environment to the cell-cycle machinery by regulating the activity of PP2A·B55. In the presence of nutrients, greatwall (Ppk18) protein kinase is inhibited by TORC1 and PP2A·B55 is active. High levels of PP2A·B55 prevent the activation of mitotic Cdk1·Cyclin B, and cells increase in size in G2 before they undergo mitosis. When nutrients are limiting, TORC1 activity falls off, and the activation of greatwall (Ppk18) leads to the phosphorylation of endosulfine (Igo1) and inhibition of PP2A·B55, which in turn allows full activation of Cdk1·CyclinB and entry into mitosis with a smaller cell size. Given the conservation of this pathway, it is reasonable to assume that this mechanism operates in higher eukaryotes, as well.

We utilized three tiers of screening to identify novel therapeutic agents for pancreatic cancers. First, we analyzed 14 pancreatic cancer cell lines against a panel of 66 small-molecule kinase inhibitors and dasatinib was the most potent. Second, we performed RNA expression analysis on 3 dasatinib-resistant and 3 dasatinib-sensitive pancreatic cancer cell lines to profile their gene expression. Third, gene profiling data was integrated with the Connectivity Map database to search for potential drugs. Thioridazine was one of the top ranking small molecules with highly negative enrichment. Thioridazine and its family members of phenothiazine including penfluridol caused pancreatic cancer cell death and affected protein expression levels of molecules involved in cell cycle regulation, apoptosis, and multiple kinase activities. This family of drugs causes activation of protein phosphatase 2 (PP2A). The drug FTY-720 (activator of PP2A) induced apoptosis of pancreatic cancer cells. Silencing catalytic unit of PP2A rendered pancreatic cancer cells resistant to penfluridol. Our observations suggest potential therapeutic use of penfluridol or similar agent associated with activation of PP2A in pancreatic cancers.

Development of a transgenic animal model always is followed by extensive characterization steps. Here, we intended to test and optimize a fast screening method to get general findings about the differential protein composition in the heart of wild type (WT) and transgenic (TG) mice. Therefore, we developed a protocol for magnetic bead-based separation (MB) combined with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDITOF MS) for protein profiling in plasma and cardiac tissue from TG and WT mice. We studied tissues from mice with cardiac specific overexpression of the catalytic subunit of PP2A, a model system for cardiac hypertrophy. EDTA-plasma or an extract of homogenized cardiac tissue (or skeletal muscle as control) were fractionated by hydrophobic interaction (MB-HIC C8) or weak cation exchange (MB-WCX) chromatography. MALDI-TOF MS spectra were generated in the mass range of m/z 1000-80000 using different matrices. The number of mass signals in the cardiac tissue extract was critically dependent on the use of the homogenization buffer, the residual blood contamination, and the surface modification of the magnetic beads. We noted different profiles in cardiac homogenates from WT compared to TG. As a control, the profile in skeletal muscle was not different between WT and TG. The results indicate that proteome profiling using MB-based sample preparation combined with MALDI-TOF MS is suitable for the proteome profiling in cardiac tissue of transgenic mice.

PP2A (protein phosphatase 2A) is a major phosphatase in eukaryotic cells that plays an essential role in many processes. PP2A mutations in Schizosaccharomyces pombe result in defects of cell cycle control, cytokinesis and morphogenesis. Which PP2A substrates are responsible for these changes is not known. In this work, we searched for PP2A substrates in S. pombe using two approaches, 2D-DIGE analysis of PP2A complex mutants and identification of PP2A interacting proteins. In both cases, we used MS to identify proteins of interest. In the DIGE experiment, we compared proteomes of wild-type S. pombe, deletion of pta2, the phosphoactivator of the PP2A catalytic subunit, and pab1-4, a mutant of B-type PP2A regulatory subunit. A total of 1742 protein spots were reproducibly resolved by 2D-DIGE and 51 spots demonstrated significant changes between PP2A mutants and the wild-type control. MS analysis of these spots identified 27 proteins that include key regulators of glycerol synthesis, carbon metabolism, amino acid biosyntesis, vitamin production, and protein folding. Importantly, we independently identified a subset of these proteins as PP2A binding partners by affinity precipitation, suggesting they may be direct targets of PP2A. We have validated our approach by demonstrating that phosphorylation of Gpd1, a key enzyme in glycerol biogenesis, is regulated by PP2A and that ability of cells to respond to osmotic stress by synthesizing glycerol is compromised in the PP2A mutants. Our work contributes to a better understanding of PP2A function and identifies potential PP2A substrates.

Dictyostelium discoideum is a simple model widely used to study many cellular functions, including differentiation, gene regulation, cellular trafficking and directional migration. Adaptation mechanisms are essential in the regulation of these cellular processes. The misregulation of adaptation components often results in persistent activation of signaling pathways and aberrant cellular responses. Studying adaptation mechanisms regulating cellular migration will be crucial in the treatment of many pathological conditions in which motility plays a central role, such as tumor metastasis and acute inflammation. I will describe two adaptation mechanisms regulating directional migration in Dictyostelium cells. The Extracellular signal Regulated Kinase 2 (ERK2) plays an essential role in Dictyostelium cellular migration. ERK2 stimulates intracellular cAMP accumulation in chemotaxing cells. Aberrant ERK2 regulation results in aberrant cAMP levels and defective directional migration. The MAP Phosphatase with Leucine-rich repeats (MPL1) is crucial for ERK2 adaptation. Cells lacking, MPL1 (mpl1- cells) displayed higher pre-stimulus and persistent post-stimulus ERK2 phosphorylation, defective cAMP production and reduced cellular migration. Reintroduction of a full length Mpl1 into mpl1- cells restored aggregation, ERK2 regulation, random and directional motility, and cAMP production similar to wild type cells (Wt). These results suggest Mpl1 is essential for proper regulation of ERK2 phosphorylation and optimal motility in Dictyostelium cells. Cellular polarization in Dictyostelium cells in part is regulated by the activation of the AGC-related kinase Protein Kinase Related B1 (PKBR1). The PP2A regulatory subunit, B56, and the Glycogen Synthase Kinase 3 (GSK3) are necessary for PKBR1 adaptation in Dictyostelium cells. Cells lacking B56, psrA-cells, exhibited high basal and post-stimulus persistent phosphorylation of PKBR1, increased phosphorylation of PKBR1 substrates, and abe

The heterotrimeric PP2A holoenzyme consists of a scaffolding A, a catalytic C, and a variable regulatory B subunit. Previous study has reported that B56gamma subunit mediates the tumor suppressive function of PP2A. B56gamma specific PP2A inhibits cell transformation by dephosphorylating and activating p53. In addition, B56gamma specific PP2A possesses the p53 independent tumor suppressive function although its mechanism is unclear. In the present study, we demonstrate the mechanism of B56gamma tumor suppressive function inactivation through characterizing B56gamma mutations identified in human cancer. First, we demonstrate that the A383G and F395C mutations disrupt B56gamma from interacting and activating p53. The mutants are unable to dephosphorylate p53 and thus lack the p53 dependent tumor suppressive function. Moreover, we identify the region adjacent to these mutations as the p53 binding domain. This finding implies that the bridging interaction between B56gamma and p53 is required for p53 dephosphorylation by PP2A. Second, we show that the C39R mutation within the HEAT repeat 1 disrupts B56gamma binding with the AC core and thus abolishes the B56gamma tumor suppressive function completely. We further demonstrate that the intact HEAT repeat 1 is required for B56gamma to bind the AC core, providing structural insights into the B56gamma specific PP2A holoenzyme assembly. Further characterization of additional B56gamma mutations reveals two classes of mutations with different mechanisms of tumor suppressive function inactivation. Mutations in the first class disrupt the interaction between B56gamma and the AC core and thus fail to regulate all substrates and completely lose the tumor suppressive function. Mutations in the second class prevent specific substrates from binding B56gamma and partially reduce the tumor suppressive function. Although it remains to be investigated how frequently these mutations occur, our results underline the importance of B56gamma tumo

The heterotrimeric PP2A holoenzyme consists of a scaffolding A, a catalytic C, and a variable regulatory B subunit. Previous study has reported that B56gamma subunit mediates the tumor suppressive function of PP2A. B56gamma specific PP2A inhibits cell transformation by dephosphorylating and activating p53. In addition, B56gamma specific PP2A possesses the p53 independent tumor suppressive function although its mechanism is unclear. In the present study, we demonstrate the mechanism of B56gamma tumor suppressive function inactivation through characterizing B56gamma mutations identified in human cancer. First, we demonstrate that the A383G and F395C mutations disrupt B56gamma from interacting and activating p53. The mutants are unable to dephosphorylate p53 and thus lack the p53 dependent tumor suppressive function. Moreover, we identify the region adjacent to these mutations as the p53 binding domain. This finding implies that the bridging interaction between B56gamma and p53 is required for p53 dephosphorylation by PP2A. Second, we show that the C39R mutation within the HEAT repeat 1 disrupts B56gamma binding with the AC core and thus abolishes the B56gamma tumor suppressive function completely. We further demonstrate that the intact HEAT repeat 1 is required for B56gamma to bind the AC core, providing structural insights into the B56gamma specific PP2A holoenzyme assembly. Further characterization of additional B56gamma mutations reveals two classes of mutations with different mechanisms of tumor suppressive function inactivation. Mutations in the first class disrupt the interaction between B56gamma and the AC core and thus fail to regulate all substrates and completely lose the tumor suppressive function. Mutations in the second class prevent specific substrates from binding B56gamma and partially reduce the tumor suppressive function. Although it remains to be investigated how frequently these mutations occur, our results underline the importance of B56gamma tumo

ITH12246 (ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8] naphthyridine-3-carboxylate) is a 1,8-naphthyridine described to feature an interesting neuroprotective profile in in vitro models of Alzheimer's disease. These effects were proposed to be due in part to a regulatory action on protein phosphatase 2A inhibition, as it prevented binding of its inhibitor okadaic acid. We decided to investigate the pharmacological properties of ITH12246, evaluating its ability to counteract the memory impairment evoked by scopolamine, a muscarinic antagonist described to promote memory loss, as well as to reduce the infarct volume in mice suffering phototrombosis. Prior to conducting these experiments, we confirmed its in vitro neuroprotective activity against both oxidative stress and Ca2+ overload-derived excitotoxicity, using SH-SY5Y neuroblastoma cells and rat hippocampal slices. Using a predictive model of blood-brain barrier crossing, it seems that the passage of ITH12246 is not hindered. Its potential hepatotoxicity was observed only at very high concentrations, from 0.1 mM. ITH12246, at the concentration of 10 mg/kg i.p., was able to improve the memory index of mice treated with scopolamine, from 0.22 to 0.35, in a similar fashion to the well-known Alzheimer's disease drug galantamine 2.5 mg/kg. On the other hand, ITH12246, at the concentration of 2.5 mg/kg, reduced the phototrombosis-triggered infarct volume by 67%. In the same experimental conditions, 15 mg/kg melatonin, used as control standard, reduced the infarct volume by 30%. All of these findings allow us to consider ITH12246 as a new potential drug for the treatment of neurodegenerative diseases, which would act as a multifactorial neuroprotectant.

Protein phosphorylation is involved in the regulation of a wide variety of physiological processes and is the result of a balance between protein kinase and phosphatase activities. Biologically active marine derived compounds have been shown to represent an interesting source of novel compounds that could modify that balance. Among them, the marine toxin and tumor promoter, okadaic acid (OA), has been shown as an inhibitor of two of the main cytosolic, broad-specificity protein phosphatases, PP1 and PP2A, thus providing an excellent cell-permeable probe for examining the role of protein phosphorylation, and PP1 and PP2A in particular, in any physiological or pathological process. In the present work, we review the use of okadaic acid to identify specific phosphoepitopes mainly in proteins relevant for neurodegeneration. We will specifically highlight those cases of highly dynamic phosphorylation-dephosphorylation events and the ability of OA to block the high turnover phosphorylation, thus allowing the detection of modified residues that could be otherwise difficult to identify. Finally, its effect on tau hyperhosphorylation and its relevance in neurodegenerative pathologies such as Alzheimer's disease and related dementia will be discussed. © 2013 by the authors; licensee MDPI.

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, t

Endoplasmic reticulum (ER) stress triggers apoptosis by activating Bim in diverse types of cells, which involves dephosphorylation of Bim(EL) by protein phosphatase 2A (PP2A). However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that Bim activation is suppressed in melanoma cells undergoing ER stress. We show here that ER stress reduces PP2A activity leading to increased ERK activation and subsequent phosphorylation and proteasomal degradation of Bim(EL). Despite sustained upregulation of Bim at the transcriptional level, the Bim(EL) protein expression was downregulated after an initial increase in melanoma cells subjected to pharmacological ER stress. This was mediated by increased activity of ERK, whereas the phosphatase activity of PP2A was reduced by ER stress in melanoma cells. The increase in ERK activation was, at least in part, due to reduced dephosphorylation by PP2A, which was associated with downregulation of the PP2A catalytic C subunit. Notably, instead of direct dephosphorylation of Bim(EL), PP2A inhibited its phosphorylation indirectly through dephosphorylation of ERK in melanoma cells. Taken together, these results identify downregualtion of PP2A activity as an important protective mechanism of melanoma cells against ER stress-induced apoptosis.