Phosphatase Function Research Articles

Long noncoding RNA MAGI2-AS3 inhibits bladder cancer progression through MAGI2/PTEN/epithelial-mesenchymal transition (EMT) axis.

Long noncoding RNA (lncRNA) are critical regulators of tumor progression. To determine how the lncRNA membrane associated guanylate kinase, WW and PDZ domain-containing 2 (MAG12) antisense RNA 3 (MAGI2-AS3) and the phosphatase and tensin homolog (PTEN) gene function in regulating bladder cancer (Bca) progression. Total RNA from 80 Bca tissues and 30 paired para-cancerous tissues from patients was sequentially extracted, quantified, purified, and reverse transcribed using RT-PCR. A library was constructed and sequenced. Four Bca cell lines and a normal urothelial cell line were transfected with lentiviral plasmids, and cell migration and invasion were assayed in vitro. An orthotopic mouse model of Bca was created for in vivo studies. MAGI2-AS3 expression was significantly downregulated in Bca, compared with normal tissues, and negatively associated with tumor stage and a poor prognosis. MAGI2-AS3 and its sense RNA MAGI2 showed significant and positive correlation. The expression of MAGI2 and its downstream gene, PTEN, increased in Bca cells overexpressing MAGI2-AS3, and interference by MAGI2 expression reversed the migration and invasion inhibited by MAGI2-AS3 overexpression. MAGI2-AS3 overexpression inhibited Bca cell progression by regulating the MAGI2/PTEN/epithelial-mesenchymal transition, offering novel insights into the mechanism of Bca progression.

S2288 Nausea and Hepatocellular Injury Associated With Tofacitinib 10 mg Tablets but Not 5 mg: A Case Report

INTRODUCTION: Tofacitinib (tofa) is a non-selective Janus kinase inhibitor, approved for treatment of moderately to severely active ulcerative colitis (UC), and is available in 5mg and 10mg tablets (tabs). We present a case of a patient with UC who transitioned from tofa 5mg tabs to 10mg tabs with subsequent nausea and liver transaminase elevation and propose it is due to distinct ingredients in the 10mg tab formulation. CASE DESCRIPTION/METHODS: Our patient is a 42-year-old woman with a 12-year history of left-sided UC. She was previously treated with oral and rectal 5-ASA, infliximab, certolizumab, and vedolizumab. While on vedolizumab she developed a new synovitis of her fingers. In 2017, she was transitioned to tofa 5mg tab twice daily (BID) and achieved remission from her arthropathy and colitis. Two years later, she relapsed and subsequently dose escalated to tofacitinib 10mg BID, taken as two 5mg tabs BID. She was well with this formulation and dose for a year. A recent refill was transitioned to the 10mg tab formulation. After 3 doses of this formulation, she developed nausea. She continued the therapy for 5 additional doses. At this point, we advised stopping the therapy; the nausea resolved within 24 hours. On re-challenge with the 10mg formulation, nausea recurred after 2 doses, so liver enzymes were obtained. Her transaminases were double from baseline (AST = 45, ALT = 55, GGT = 25). Bilirubin, alkaline phosphatase and synthetic function remained normal. She denied any new drugs, herbal supplements or alcohol use. Her therapy was discontinued for two days; repeat labs were normal. After re-challenge with two 5 mg tabs BID and one week of follow-up, the patient’s transaminases remain normal and she is asymptomatic. DISCUSSION: We describe a UC patient who developed drug induced liver injury from the 10 mg formulation of tofa. We believe the drug-induced liver injury occurred from the excipient dyes (Brilliant Blue FCF Aluminum Lake and Indigo Carmine Aluminum Lake) found in the 10 mg formulation (and in the 22 mg XR formulation) but are not in the 5 mg formulation (or in the 11 mg XR formulation) (Table). Both dyes are triarylmethanes, which have been previously implicated in hepatocellular injury. Our suspicion was further confirmed by a positive de-challenge (discontinuation of the 10mg tabs) and successful re-challenge with the 5 mg tabs without recurrence of symptoms or chemistry abnormalities. Clinicians should be aware of this reaction and when it is suspected, consider the alternate formulation.Table.: Ingredients in tofacitinib (Xeljanz) tablets.

Correction: Neurogenic decisions require a cell cycle independent function of the CDC25B phosphatase.

Correction: Neurogenic decisions require a cell cycle independent function of the CDC25B phosphatase.

MAPK pathway-related tyrosine phosphatases regulate development, secondary metabolism and pathogenicity in fungus Aspergillus flavus.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved in eukaryotic cells and are known to play crucial roles in the regulation of various cellular processes. However, compared with kinase-mediated phosphorylation, dephosphorylation catalysed by phosphatases has not been well characterized in filamentous fungi. In this study, we identified five MAPK pathway-related phosphatases (Msg5, Yvh1, Ptp1, Ptp2 and Oca2) and characterized their functions in Aspergillus flavus, which produces aflatoxin B1 (AFB1 ), one of the most toxic and carcinogenic secondary metabolites. These five phosphatases were identified as negative regulators of MAPK (Slt2, Fus3 and Hog1) pathways. Deletion of Msg5 and Yvh1 resulted in significant defects in conidiation, sclerotia formation, aflatoxin production and crop infection. Additionally, double knockout mutants (ΔMsg5/ΔPtp1, ΔMsg5/ΔPtp2 and ΔMsg5/ΔOca2) displayed similar defects to those observed in the ΔMsg5 single mutant, indicating that Msg5 plays a major role in the regulation of development and pathogenicity in A. flavus. Importantly, we found that the active site at C439 is essential for the function of the Msg5 phosphatase. Furthermore, the MAP kinase Fus3 was found to be involved in the regulation of development, aflatoxin biosynthesis and pathogenicity, and its conserved phosphorylation residues (Thr and Tyr) were critical for the full range of its functions in A. flavus. Overall, our results reveal that MAPK related tyrosine phosphatases play important roles in the regulation of development, secondary metabolism and pathogenicity in A. flavus, and could be developed as potential targets for preventing damage caused by this fungal pathogen.

Abstract 5314: A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway

Abstract Objectives: Poly ADP-ribose polymerase (PARP) inhibitors are effective therapies for patients with homologous recombination (HR) deficient tumors. While HR mutations are rare in endometrial cancers (ECs), emerging data suggests that loss of function of the tumor suppressor gene Phosphatase and Tensin homolog (PTEN), which is mutated in 80% of endometrioid ECs, may also sensitize tumors to PARP inhibition. The imipridone ONC206, a chemical derivative of ONC201, is an orally bioavailable dopamine receptor D2 (DRD2) antagonist that has anti-tumorigenic effects in EC via induction of apoptosis and activation of the integrated stress response. ONC206 exhibits enhanced non-competitive DRD2 antagonism, potency, and bioavailability relative to ONC201. Both olaparib and imipridones (i.e. ONC201) are being evaluated in EC clinical trials, but have yet to be explored in combination. Thus, we sought to examine the effects of olaparib in combination with ONC206 in human endometrioid EC cell lines. Methods: The ECC-1 and HEC-1 EC cell lines were used. Cell proliferation was assessed after exposure to varying doses of olaparib (MedChemExpress) and ONC206 (Oncoceutics) via the MTT assay. Synergy between olaparib and ONC206 was assessed by the combination index (CI) method of Chou-Talalay. Cellular stress was evaluated using the DCFH-DA assay. Apoptosis was assessed using the cleaved caspase-3 assay. Western immunoblotting was performed to determine effects of olaparib and ONC206 on apoptosis and the mTOR pathway. Results: Both drugs inhibited cell proliferation in a dose-dependent manner (IC50 for olaparib of 15 uM for ECC-1 and 25 uM for HEC-1; IC50 for ONC206 of 3.3 uM for ECC-1 and 0.9 uM for HEC-1). Simultaneous exposure of OC cells to ONC206 and olaparib at low doses resulted in synergistic anti-proliferative effects (CI<1). Combination treatment with ONC206 and olaparib at low doses increased cellular stress and apoptosis in both cell lines (p=0.05-0.001), compared to either drug alone. Dual therapy with olaparib and ONC206 decreased expression of the anti-apoptotic protein Bcl-2 and decreased phosphorylation of S6, a downstream target of the mTOR pathway, with greater effects compared to either drug alone. Conclusions: Olaparib and ONC206 had synergistic anti-proliferative effects in human endometrioid EC cell lines, suggesting that this novel dual therapy may be worthy of further exploration in clinical trials in EC. Citation Format: Sarah E. Paraghamian, Gabrielle M. Hawkins, Wenchuan Sun, Yali Fan, Xin Zhang, Hongyan Suo, Varun Vijay Prabhu, Joshua E. Allen, Chunxiao Zhou, Victoria Bae-Jump. A novel dopamine receptor D2 antagonist (ONC206) potentiates the effects of olaparib in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5314.

Zingerone protects liver and kidney tissues by preventing oxidative stress, inflammation, and apoptosis in methotrexate-treated rats

The clinical use of drugs used in the treatment of diseases is limited due to the toxic side effects, and many studies have been conducted to benefit from herbal adjuvant therapies recently to eliminate these effects. In this study, the protective effect of zingerone against liver and kidney damage generated in rats through methotrexate (MTX). Histopathological investigations were performed to determine tissue damage caused by MTX and the healing effect of zingone and liver function markers such as serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and renal function markers such as urea, creatine, and aquaporin-1 (AQP-1) were measured. The effects of MTX and protective properties of zingerone on oxidative stress were investigated through the measurement of malondialdehyde and reduced glutathione (GSH) levels, catalase (CAT), and glutathione peroxidase (GPx) enzyme activities. The anti-inflammatory effect of zingerone was determined by measuring the cytokine levels causing inflammation such as nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), and its effects on apoptosis were determined by immunohistochemical analysis of caspase-3 and B-cell lymphoma-2 (Bcl-2) expression levels. According to the results obtained within the scope of the study, it was determined that zingerone treatment prevented the increase in MTX-induced liver and kidney function markers, showed healing effects on antioxidant parameters degraded in both tissues, and decreased the inflammation parameters. It was determined that it also prevented apoptosis and possessed a protective effect on disrupted tissue architecture by decreasing the increased caspase-3 expression and increasing the decreased Bcl-2 level.

Unveiling the Heterogenous Dephosphorylation of DNA Using an Aerolysin Nanopore.

The simultaneous occurrence of multiple heterogeneous DNA phosphorylation statuses, which include 5' end phosphorylation, 5' end dephosphorylation, 3' end phosphorylation, and 3' end dephosphorylation, is crucial for regulating numerous cellular processes. Although there are many methods for detecting a single type of DNA phosphorylation, the direct and simultaneous identification of DNA phosphorylation/dephosphorylation on the 5' and/or 3' ends remains a challenge, let alone the unveiling of the heterogeneous catalysis processes of related phosphatases and kinases. Taking advantage of the charge-sensitive aerolysin nanopore interface, herein, an orientation-dependent sensing strategy is developed to enhance phosphorylation-site-dependent interaction with the nanopore sensing interface, enabling the direct and simultaneous electric identification of four heterogeneous phosphorylation statuses of a single DNA. By using this strategy, we can directly evaluate the heterogeneous dephosphorylation process of alkaline phosphatase (ALP) at the single-molecule level. Our results demonstrate that the ALP in fetal bovine serum preferentially catalyzes the 3' phosphate rather than both ends. The quantification of endogenous ALP activity in fetal bovine serum could reach the submilli-IU/L level. Our aerolysin measurements provide a direct look at the heterogeneous phosphorylation status of DNA, allowing the unveiling of the dynamic single-molecule functions of kinase and phosphatase.

The Spo7 sequence LLI is required for Nem1-Spo7/Pah1 phosphatase cascade function in yeast lipid metabolism

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphory-lation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.

Controlling Phosphate Removal with Light: The Development of Optochemical Tools to Probe Protein Phosphatase Function.

Protein phosphatases play an essential role in cell signaling; however, they remain understudied compared with protein kinases, in part due to a lack of appropriate tools. In order to provide conditional control over phosphatase function, we developed two different approaches for rendering MKP3 (a dual-specific phosphatase, also termed DUSP6) activatable by light. Specifically, we expressed the protein with strategically placed light-removable protecting groups in cells with an expanded genetic code. This allowed for the acute perturbation of the Ras/MAPK signaling pathway upon photoactivation in live cells. In doing so, we confirmed that MKP3 does not act as a thresholding gate for growth factor stimulation of the extracellular signal-regulated kinase (ESRK) pathway.

Plants mitigate restrictions to phosphatase activity in metal contaminated soils

Soil anthropogenic contaminants can limit enzymatic nutrient mineralization, either by direct regulation or via impacts on the microbial community, thus affecting plant growth in agricultural and non-agricultural soils. The impact on phosphatase activity of mixing two contaminated, post-industrial rail yard soils was investigated; one was vegetated and had high phosphatase function, the other was barren and had low enzymatic function. The two soils had different abiotic properties, including contaminant load, vegetation cover, soil aggregate size distribution, and phosphatase potential. An experimental gradient was established between the two soils to systematically vary the abiotic properties and microbial community composition of the two soils, creating a gradient of novel ecosystems. The time dependence of extracellular phosphatase activity, soil moisture, and organic matter content was assessed along this gradient in the presence and absence of plants. Initially, mixtures with higher percentages of functional, vegetated soil had higher phosphatase activities. Phosphatase activity remained unchanged through time (65 days) in all soil mixtures in unplanted pots, but it increased in planted pots. For example, in the presence of plants, phosphatase activity increased from 0.6 ± 0.1 to 2.4 ± 0.3 μmol•h−1•gdry soil−1 from day one to day 65 in the 1:1 functional:barren soil mixture. The presence of plants also promoted moisture retention. Inoculation of poorly functioning soil with 10% of the functional soil with its microbial community did not, over 65 days, revitalize the poorly functioning soil. The findings showed that abiotic limitations to enzymatic activity in barren brownfield soils could be mitigated by establishing primary production but not by the addition of enzymatically active microbial communities alone.

Phosphatase-defective DevS sensor kinase mutants permit constitutive expression of DevR-regulated dormancy genes in Mycobacterium tuberculosis.

The DevR-DevS/DosR-DosS two-component system of Mycobacterium tuberculosis, that comprises of DevS sensor kinase and DevR response regulator, is essential for bacterial adaptation to hypoxia by inducing dormancy regulon expression. The dominant phosphatase activity of DevS under aerobic conditions enables tight negative control, whereas its kinase function activates DevR under hypoxia to induce the dormancy regulon. A net balance in these opposing kinase and phosphatase activities of DevS calibrates the response output of DevR. To gain mechanistic insights into the kinase-phosphatase balance of DevS, we generated alanine substitution mutants of five residues located in DHp α1 helix of DevS, namely Phe-403, Gly-406, Leu-407, Gly-411 and His-415. For the first time, we have identified kinase positive phosphatase negative (K+P-) mutants in DevS by a single-site mutation in either Gly-406 or Leu-407. M. tuberculosis Gly-406A and Leu-407A mutant strains constitutively expressed the DevR regulon under aerobic conditions despite the presence of negative signal, oxygen. These mutant proteins exhibited ∼2-fold interaction defect with DevR. We conclude that Gly-406 and Leu-407 residues are individually essential for the phosphatase function of DevS. Our study provides new insights into the negative control mechanism of DevS by demonstrating the importance of an optimal interaction between DevR and DevS, and local changes associated with individual residues, Gly-406 and Leu-407, which mimic ligand-free DevS. These K+P- mutant strains are expected to facilitate the rapid aerobic screening of DevR antagonists in M. tuberculosis, thereby eliminating the requirement for hypoxic culture conditions.

Structure and function of the co-chaperone protein phosphatase 5 in cancer.

Protein phosphatase 5 (PP5) is a serine/threonine protein phosphatase that regulates many cellular functions including steroid hormone signaling, stress response, proliferation, apoptosis, and DNA repair. PP5 is also a co-chaperone of the heat shock protein 90 molecular chaperone machinery that assists in regulation of cellular signaling pathways essential for cell survival and growth. PP5 plays a significant role in survival and propagation of multiple cancers, which makes it a promising target for cancer therapy. Though there are several naturally occurring PP5 inhibitors, none is specific for PP5. Here, we review the roles of PP5 in cancer progression and survival and discuss the unique features of the PP5 structure that differentiate it from other phosphoprotein phosphatase (PPP) family members and make it an attractive therapeutic target.

Effect of a Combination of Ethanol Extract of Ficus capensis and Cnidoscolus aconitifolius on Liver and Kidney Function Parameters of Phenylhydrazine-Induced Anemic Rats

Background: Anemia is a condition in which there is a reduced number of red blood cells or haemoglobin and iron concentration in the body thereby leading to a decreased capacity of the blood to carry oxygen to the body tissues.
 Objectives: The study was designed to investigate the effect of a combination of ethanol extract of Ficus capensis and Cnidoscolus aconitifolius in phenylhydrazine-induced anemic rats.
 Methods: The animals were divided into five groups of five rats each. Group A served as normal control, Group B as anemic control, group C as standard drug control, groups D and E were treated with 200 mg/kg and 400 mg/kg of combined ethanol leaf extract of F. capensis and C. aconitifolius respectively. Phenylhydrazine was administered intraperitoneally at a dose of 20 mg/kg b.w. for two days to induce anemia in rats. The administration of the extract lasted for 14 days after which the animals were sacrificed and blood obtained through cardiac puncture for kidney and liver biomarkers analyses.
 Results: Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Total bilirubin, Direct bilirubin and Kidney function parameters assayed in the anemic untreated group showed significant increases (p<0.05) compared to the normal rats which may be attributed to toxicity induced by phenylhydrazine. The groups treated with the ethanol extract at a dose of 200 and 400 mg/kg body weight revealed a significant (p<0.05) decrease in the liver function parameters compared to the anemic untreated group. The creatinine and urea levels of the anemic untreated group increased more than the that of the test groups although the increase was not statistically significant.
 Conclusion: This study suggests that combined ethanol leaf extract of F. capensis and C. aconitifolius is safe and can be effective in the treatment and management of anemia.

Effect of imatinib on DOCA-induced myocardial fibrosis in rats through P38 MAPK signaling pathway.

To explore the role of imatinib in desoxycorticosterone acetate (DOCA)-induced myocardial fibrosis in rats by the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Normal group (n=20), DOCA induction group (n=20), and imatinib treatment group (treatment group, n=20) were set up. Then, the cardiac function was examined via magnetic resonance imaging (MRI) and echocardiography (ECG) on the 21st d after modeling. Alkaline phosphatase (ALP) and myocardial function index creatine kinase-MB (CK-MB) were detected. The enzyme-linked immunosorbent assay (ELISA) was performed to measure tumor necrosis factor-gamma (TNF-γ) and interleukin-6 (IL-6). Hematoxylin-eosin (HE) staining assay was carried out to observe the pathological changes in myocardial tissues. Quantitative Polymerase Chain Reaction (qPCR) and Western blotting were employed to measure the expression levels of important myocardial fibrosis-related genes [checkpoint kinase 1 (Chek1) and alpha-smooth muscle actin (α-SMA)], as well as genes and proteins of the p38 MAPK signaling pathway. In comparison with the normal group, DOCA induction group had significantly lowered fractional shortening (FS, %) and ejection fraction (EF, %), but overtly increased left ventricular end-diastolic dimension (LVEDd) and left ventricular end-systolic dimension (LVESd), as well as levels of serum ALP, alanine aminotransferase (ALT), and CK-MB. Besides, the levels of TNF-γ, IL-6, and IL-1β were notably raised in the DOCA induction group. HE staining results showed that myocardial injury was more severe in DOCA induction group. The results of the gene detection revealed that the expression levels of Chek1, α-SMA, p38 MAPK, and JNK were evidently higher in DOCA induction group than those in the imatinib treatment group (p<0.05), and the expression of p38 MAPK protein in the rat myocardial tissues was remarkably lower in the treatment group than that in the DOCA induction group (p<0.05). Imatinib can regulate the repair of myocardial injury caused by DOCA-induced myocardial fibrosis in rats by repressing the p38 MAPK signaling pathway.

Structural insights into selective interaction between type IIa receptor protein tyrosine phosphatases and Liprin-\u03b1

Synapse formation is induced by transsynaptic interaction of neuronal cell-adhesion molecules termed synaptic organizers. Type IIa receptor protein tyrosine phosphatases (IIa RPTPs) function as presynaptic organizers. The cytoplasmic domain of IIa RPTPs consists of two phosphatase domains, and the membrane-distal one (D2) is essential for synapse formation. Liprin-α, which is an active zone protein critical for synapse formation, interacts with D2 via its C-terminal domain composed of three tandem sterile alpha motifs (tSAM). Structural mechanisms of this critical interaction for synapse formation remain elusive. Here, we report the crystal structure of the complex between mouse PTPδ D2 and Liprin-α3 tSAM at 1.91 Å resolution. PTPδ D2 interacts with the N-terminal helix and the first and second SAMs (SAM1 and SAM2, respectively) of Liprin-α3. Structure-based mutational analyses in vitro and in cellulo demonstrate that the interactions with Liprin-α SAM1 and SAM2 are essential for the binding and synaptogenic activity.

Controlling Ser/Thr protein phosphatase PP1 activity and function through interaction with regulatory subunits.

Protein phosphatase 1 is a major Ser/Thr protein phosphatase activity in eukaryotic cells. It is composed of a catalytic polypeptide (PP1C), with little substrate specificity, that interacts with a large variety of proteins of diverse structure (regulatory subunits). The diversity of holoenzymes that can be formed explain the multiplicity of cellular functions under the control of this phosphatase. In quite a few cases, regulatory subunits have an inhibitory role, downregulating the activity of the phosphatase. In this chapter we shall introduce PP1C and review the most relevant families of PP1C regulatory subunits, with particular emphasis in describing the structural basis for their interaction.

PP2A Functions during Mitosis and Cytokinesis in Yeasts.

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.

The Opposing Functions of Protein Kinases and Phosphatases in Chromosome Bipolar Attachment.

Accurate chromosome segregation during cell division is essential to maintain genome integrity in all eukaryotic cells, and chromosome missegregation leads to aneuploidy and therefore represents a hallmark of many cancers. Accurate segregation requires sister kinetochores to attach to microtubules emanating from opposite spindle poles, known as bipolar attachment or biorientation. Recent studies have uncovered several mechanisms critical to chromosome bipolar attachment. First, a mechanism exists to ensure that the conformation of sister centromeres is biased toward bipolar attachment. Second, the phosphorylation of some kinetochore proteins destabilizes kinetochore attachment to facilitate error correction, but a protein phosphatase reverses this phosphorylation. Moreover, the activity of the spindle assembly checkpoint is regulated by kinases and phosphatases at the kinetochore, and this checkpoint prevents anaphase entry in response to faulty kinetochore attachment. The fine-tuned kinase/phosphatase balance at kinetochores is crucial for faithful chromosome segregation during both mitosis and meiosis. Here, we discuss the function and regulation of protein phosphatases in the establishment of chromosome bipolar attachment with a focus on the model organism budding yeast.

Investigation of Proteomic and Phosphoproteomic Responses to Signaling Network Perturbations Reveals Functional Pathway Organizations in Yeast

SUMMARYGovernance of protein phosphorylation by kinases and phosphatases constitutes an essential regulatory network in eukaryotic cells. Network dysregulation leads to severe consequences and is often a key factor in disease pathogenesis. Previous studies revealed multiple roles for protein phosphorylation and pathway structures in cellular functions from different perspectives. We seek to understand the roles of kinases and phosphatases from a protein homeostasis point of view. Using a streamlined tandem mass tag (SL-TMT) strategy, we systematically measure proteomic and phosphoproteomic responses to perturbations of phosphorylation signaling networks in yeast deletion strains. Our results emphasize the requirement for protein normalization for more complete interpretation of phosphorylation data. Functional relationships between kinases and phosphatases were characterized at both proteome and phosphoproteome levels in three ways: (1) Gene Ontology enrichment analysis, (2) Δgene-Δgene correlation networks, and (3) molecule covariance networks. This resource illuminates kinase and phosphatase functions and pathway organizations.

Adipose PTEN regulates adult adipose tissue homeostasis and redistribution via a PTEN-leptin-sympathetic loop

ObjectiveDespite the large body of work describing the tumor suppressor functions of Phosphatase and tensin homologue deleted on chromosome ten (PTEN), its roles in adipose homeostasis of adult animals are not yet fully understood. Here, we sought to determine the role of PTEN in whole-body adipose homeostasis. MethodsWe genetically manipulated PTEN in specific fat depots through recombinant adeno-associated viral vector (rAAV)-based gene transfer of Cre recombinase to adult PTENflox mice. Additionally, we used a denervation agent, 6OHDA, to assess the role of sympathetic signaling in PTEN-related adipose remodeling. Furthermore, we chemically manipulated AKT signaling via a pan-AKT inhibitor, MK-2206, to assess the role of AKT in PTEN-related adipose remodeling. Finally, to understand the role of leptin and central signaling on peripheral tissues, we knocked down hypothalamic leptin receptor with a microRNA delivered by a rAAV vector. ResultsKnockdown PTEN in individual fat depot resulted in massive expansion of the affected fat depot through activation of AKT signaling associated with suppression of lipolysis and induction of leptin. This hypertrophic expansion of the affected fat depot led to upregulation of PTEN level, higher lipolysis, and induction of white fat browning in other fat depots, and the compensatory reduced fat mass to maintain a set point of whole-body adiposity. Administration of AKT inhibitor MK-2206 prevented the adipose PTEN knockdown-associated effects. 6OHDA-mediated denervation demonstrated that sympathetic innervation was required for the PTEN knockdown-induced adipose redistribution. Knockdown hypothalamic leptin receptor attenuated the adipose redistribution induced by PTEN deficiency in individual fat depot. ConclusionsOur results demonstrate the essential role of PTEN in adipose homeostasis, including mass and distribution in adulthood, and reveal an “adipose PTEN-leptin-sympathetic nervous system” feedback loop to maintain a set point of adipose PTEN and whole-body adiposity.