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Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A subunits, 17 B subunits, and 5 C subunits that are encoded by the Arabidopsis genome allow 255 possible PP2A holoenzyme combinations. The regulatory subunits are crucial for substrate specificity and PP2A complex localization and are classified into the B, B’, and B” non-related families in land plants. In Arabidopsis, the close homologs B’η, B’θ, B’γ, and B’ζ are further classified into a subfamily of B’ called B’η. Previous studies have suggested that mitochondrial targeted PP2A subunits (B’ζ) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B’ζ holoenzyme is involved in the regulation of the mitochondrial succinate/fumarate translocator, and it may affect the enzymes involved in energy metabolism. To investigate this hypothesis, the interactions between PP2A-B’ζ and the enzymes involved in the mitochondrial energy flow were investigated using bimolecular fluorescence complementation in tobacco and onion cells. Interactions were confirmed between the B’ζ subunit and the Krebs cycle proteins succinate/fumarate translocator (mSFC1), malate dehydrogenase (mMDH2), and aconitase (ACO3). Additional putative interacting candidates were deduced by comparing the enriched phosphoproteomes of wild type and B’ζ mutants: the mitochondrial regulator Arabidopsis pentatricopeptide repeat 6 (PPR6) and the two metabolic enzymes phosphoenolpyruvate carboxylase (PPC3) and phosphoenolpyruvate carboxykinase (PCK1). Overall, this study identifies potential PP2A substrates and highlights the role of PP2A in regulating energy metabolism in mitochondria.

Hyponatremia (hypo-osmolality) is a disorder of water homeostasis due to abnormal renal diluting capacity. The body limits the degree to which serum sodium concentration falls through a mechanism called “vasopressin escape”. Vasopressin escape is a process that prevents the continuous decrease in serum sodium concentration even under conditions of sustained high plasma vasopressin levels. Previous reports suggest that aldosterone may be involved in the vasopressin escape mechanism. The abilities of aldosterone synthase (Cyp11b2) knockout and wild-type mice to escape from vasopressin were compared. Wild-type mice escaped while the aldosterone synthase knockout mice did not. Both the water channel aquaporin 2 (AQP2) and the urea transporter UT-A1 protein abundances were higher in aldosterone synthase knockout than in wild-type mice at the end of the escape period. Vasopressin escape was also blunted in rats given spironolactone, a mineralocorticoid receptor blocker. Next, the role of the phosphatase, calcineurin (protein phosphatase 2B, PP2B), in vasopressin escape was studied since aldosterone activates calcineurin in rat cortical collecting ducts. Tacrolimus, a calcineurin inhibitor, blunted vasopressin escape in rats compared with the control rats, increased UT-A1, AQP2, and pS256-AQP2, and decreased pS261-AQP2 protein abundances. Our results indicate that aldosterone regulates vasopressin escape through calcineurin-mediated protein changes in UT-A1 and AQP2.

Brazilian semi-arid tropical is responsible for the production and export of fine table grapes, but the demand for phosphate fertilizers and other agricultural inputs increases the cost of production and threatens local water bodies by leaching of phosphorus (P). The objectives of this study were to quantify the organic and inorganic fractions of P and to determine chemical and microbiological attributes related to its mineralization in soils cultivated with vines in São Francisco Valley. It was hypothesized that P fractions and microbial activity would increase in areas fertilized with phosphate. Soil samples from vines were collected in cultivated areas and in fallow systems. P fractionation, macro-elements analysis, microbial biomass phosphorus (Pmic), and phosphatase enzyme activity were determined and correlated with each other and with the total proportion of synthesized glomalin. In both treatments, the inorganic P fractions exceeded the organic ones, and the total phosphorus available in those soils was associated with the most recalcitrant fractions, reducing risks of leaching of this element and contamination of water bodies. Pmic was higher in the winery areas due to the presence of phosphate fertilizers and organic matter, hence the activity of alkaline phosphatase enzymes and the production of total glomalin increased respectively in three and four evaluated areas, indicating that P-mineralized bacteria and arbuscular mycorrhizal fungi have been positively affected by the current agricultural system.

The Human Genome Project was the first step in a scientific revolution that unleashed the full potential of the field of genomics (Durmaz et al., 2015). The project launched in the 1990s and the first draft of the human genome was published in 2003. Since this development researchers have been able to harness genetic data from patients using methods such as next-generation sequencing (NGS) and RNA-seq to draw meaningful conclusions. Two ways this data has been used are in the analysis of disease mechanisms and the development of genetic tests. The technical report will focus on using genetic expressions to study disease mechanisms. Specifically, this technical report observes the pathogenic signaling mechanisms in Systemic Lupus Erythematosus (SLE). SLE is a chronic, heterogeneous autoimmune disease that causes damage to numerous organs (COJOCARU et al., 2011). The current understanding of disease pathogenesis is incomplete and subsequently, treatments for SLE are inadequate (Mok & Lau, 2003). The approach to this project is based on current knowledge of autoimmune diseases. Previous research has identified that dysregulated immune signaling in autoimmune disease often arises because of an improper balance in the activation and inhibition of inflammatory cells or pathways (Arakelyan et al., 2017). One potential regulatory mechanism that controls immune activation, and thus can become dysregulated, is the imbalance of kinases and phosphatases. Functionally, phosphatases and kinases have an inverse relationship: kinases are considered to activate signaling, and phosphatases are thought to inhibit signaling (Protein Phosphatases and Kinases | NEB, n.d.). Therefore, to study the pathogenic signaling mechanisms in SLE this technical report investigates phosphatases and kinases. Specifically, the report aims to identify groups of genes whose expression allows for meaningful separation between patients with SLE and control. To accomplish this goal, the technical project set out three aims: design gene sets of phosphatases and kinases, analyze patterns in the expression of gene sets, and evaluate the ability of gene sets to discriminate between SLE and control. The project used whole blood microarray data from three large data sets. The methods used to complete these aims include literature analysis, gene set variation analysis (GSVA), co-expression, binary classification machine learning (ML), and classification and regression trees (CART). Through this careful approach, this project was able to identify gene sets that differentiate between SLE and control and thus provide insight to the pathogenesis of SLE. The second piece of this portfolio observes another developing field of genomics: genetic testing. The research paper specifically investigates the role of preimplantation genetic testing (PGT) which is a technology sometimes used in in vitro fertilization (IVF) (Klimczak et al., 2021). IVF is an alternative to sex as a method used to impregnate a woman. In IVF the embryo is fertilized outside of the body. In the IVF process, multiple embryos are fertilized but only one is eventually implanted into the womb. PGT is a method for conducting genetic tests on the fertilized embryo and is used to determine which embryo should be implanted for development. PGT is a controversial technology as it provides doctors, parents, and legislators the opportunity to decide which embryo is chosen. Internationally PGT is regulated to varying degrees (Ginoza & Isasi, 2020). This research paper investigates the regulation of two countries with opposite approaches to regulation: Switzerland and the United States. In order to understand the sociotechnical system related to PGT, this paper takes a holistic approach by investigating policy, history, culture, and ethics related to PGT. Progress in genomic research has led to the development of many new technologies. These technologies have allowed for great discoveries but also led to widespread concern about possible gross misuse. This portfolio investigates two of these new technologies using one to attempt to understand a disease, and another to understand the sociotechnical system surrounding the technology itself. Together this work allows one to thoroughly understand the current applications of genomic technology.

It is widely known that root morphology has different response mechanisms at different phosphorus (P) levels. However, the effects of P application times on wheat (Triticum aestivum L.) root morphology and, therefore, on growth and nutrient uptake are still unclear. In this study, we investigated the feedback from the physiological indexes of wheat on different P application times after planting. Compared with the P application at planting, the shoot biomass with delayed P application for 21 days (d) increased by 11.8% (p < 0.05). Compared with the P application at planting, the shoot P uptake with delayed P application for 14–21 days increased by 38.4–71.2%, while the shoot N uptake and K uptake with delayed P application for 21 days increased by 16.0% and 14.1% (p < 0.05). Compared with the P application at planting, P use efficiency, N use efficiency, and K use efficiency with delayed P application for 21 days increased by 16.4%, 12.4%, and 12.4%. Delayed P application for 14–21 days promoted wheat shoot growth, nutrient (P, N, and K) uptake, and their use efficiencies. This is particularly important for optimizing the P fertilizer input and nutrient management for wheat growth.

Plant adaptations to low soil phosphorus (P) availability have been intensively studied in Brassica sp. in an attempt to identify the mechanisms involved in P uptake and utilization. The present pot experiment was conducted to evaluate the relationships between plant shoot and root growth, P uptake and use efficiency parameters, and P fractions and enzyme activity, in two species grown in three soil types. The aim of this study was to determine whether adaptation mechanisms are soil-dependent. Two kale species were grown in soils typical for coastal Croatia (terra rossa, rendzina, and fluvisol) with low P availability. Plants grown in fluvisol had the highest shoot biomass and accumulated most P, whereas plants developed the longest roots in terra rossa. Phosphatase activity differed among soils. P use efficiency differed among soils and species. Genotype IJK 17 showed better adaptation to low P availability, which was related to better uptake efficiency. In general, soils differed in inorganic and organic P fractions in rhizosphere soil, but no difference between genotypes was found. The activities of alkaline phosphatase and phosphodiesterase were negatively correlated with most organic P fractions, suggesting their function in the mineralization of soil organic P. Kale species activate different mechanisms of P uptake and utilization when grown in contrasting soil types, suggesting that specific responses to the soil type were more important than the genotypic difference.

A mesiodens is a supernumerary tooth located in the midline of the premaxilla. To investigate the genetic cause of mesiodens, clinical and radiographic examination were performed on 23 family members of a two-generation Hmong family. Whole exome sequencing (WES) or Sanger sequencing were performed in 22 family members and two unrelated Thai patients with mesiodens. WES in the Hmong family revealed a missense mutation (c.1807G>A;p.Glu603Lys) in PTPN23 in seven affected members and six unaffected members. The mode of inheritance was autosomal dominance with incomplete penetrance (53.84%). Two additional mutations in PTPN23, c.2248C>G;p.Pro750Ala and c.3298C>T;p.Arg1100Cys were identified in two unrelated patients with mesiodens. PTPN23 is a regulator of endosomal trafficking functioning to move activated membrane receptors, such as EGFR, from the endosomal sorting complex towards the ESCRT-III complex for multivesicular body biogenesis, lysosomal degradation, and subsequent downregulation of receptor signaling. Immunohistochemical study and RNAscope on developing mouse embryos showed broad expression of PTPN23 in oral tissues, while immunofluorescence showed that EGFR was specifically concentrated in the midline epithelium. Importantly, PTPN23 mutant protein was shown to have reduced phosphatase activity. In conclusion, mesiodens were associated with genetic variants in PTPN23, suggesting that mesiodens may form due to defects in endosomal trafficking, leading to disrupted midline signaling.

Protein phosphatases play important roles in the regulation of various cellular processes in eukaryotes. The ascomycete Colletotrichum gloeosporioides is a causal agent of anthracnose disease on some important crops and trees. In this study, CgPPZ1, a protein phosphate gene and a homolog of yeast PPZ1, was identified in C. gloeosporioides. Targeted gene deletion showed that CgPpz1 was important for vegetative growth and asexual development, conidial germination, and plant infection. Cytological examinations revealed that CgPpz1 was localized to the cytoplasm. The ΔCgppz1 mutant was hypersensitive to osmotic stresses, cell wall stressors, and oxidative stressors. Taken together, our results indicated that CgPpz1 plays an important role in the fungal development and virulence of C. gloeosporioides and the multiple stress responses generated.

South China has been experiencing very high rate of acid deposition and severe soil acidification in recent decades, which has been proposed to exacerbate the regional ecosystem phosphorus (P) limitation. We conducted a 10-year field experiment of simulated acid deposition to examine how acidification impacts seasonal changes of different soil P fractions in a tropical forest with highly acidic soils in south China. As expected, acid addition significantly increased occluded P pool but reduced the other more labile P pools in the dry season. In the wet season, however, acid addition did not change microbial P, soluble P and labile organic P pools. Acid addition significantly increased exchangeable Al3+ and Fe3+ and the activation of Fe oxides in both seasons. Different from the decline of microbial abundance in the dry season, acid addition increased ectomycorrhizal fungi and its ratio to arbuscular mycorrhiza fungi in the wet season, which significantly stimulated phosphomonoesterase activities and likely promoted the dissolution of occluded P. Our results suggest that, even in already highly acidic soils, the acidification-induced P limitation could be alleviated by stimulating ectomycorrhizal fungi and phosphomonoesterase activities. The differential responses and microbial controls of seasonal soil P transformation revealed here should be implemented into ecosystem biogeochemical model for predicting plant productivity under future acid deposition scenarios.

In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.

Background: How signals from activated angiotensin type-2 receptors (AT 2 R) mediate inhibition of sodium ion (Na + ) reabsorption in renal proximal tubule cells is currently unknown. Protein phosphatases including PP2A (protein phosphatase 2A) have been implicated in AT 2 R signaling in tissues other than kidney. We investigated whether inhibition of protein phosphatase PP2A reduced AT 2 R-mediated natriuresis and evaluated changes in PP2A activity and localization after renal AT 2 R activation in normal 4- and 10-week-old control Wistar-Kyoto rats and 4-week-old prehypertensive and 10-week-old hypertensive spontaneously hypertensive rats. Methods and Results: In Wistar-Kyoto rats, direct renal interstitial administration of selective AT 2 R nonpeptide agonist Compound-21 (C-21) increased renal interstitial cyclic GMP (cGMP) levels, urine Na + excretion, and simultaneously increased PP2A activity ≈2-fold in homogenates of renal cortical tubules. The cyclic GMP and natriuretic responses were abolished by concurrent renal interstitial administration of protein phosphatase inhibitor calyculin A. In renal proximal tubule cells in response to C-21, PP2A subunits A, B55α and C, but not B56γ, were recruited to apical plasma membranes together with AT 2 Rs. Calyculin A treatment abolished C-21-induced translocation of both AT 2 R and PP2A regulatory subunit B55α to apical plasma membranes. Immunoprecipitation of AT 2 R solubilized from renal cortical homogenates demonstrated physical association of AT 2 R with PP2A A, B55α, and C but not B56γ subunits. In contrast, in spontaneously hypertensive rats, administration of C-21 did not alter urine Na + excretion or PP2A activity and failed to translocate AT 2 Rs and PP2A subunits to apical plasma membranes. Conclusions: In renal proximal tubule cells of Wistar-Kyoto rats, PP2A is activated and PP2A subunits AB55αC are recruited to C-21-activated AT 2 Rs during induction of natriuresis. This response is defective in prehypertensive and hypertensive spontaneously hypertensive rats, presenting a potential novel therapeutic target for treating renal Na + retention and hypertension.

Cellular function is based on protein–protein interactions. A large proportion of these interactions involves the binding of short linear motifs (SLiMs) by folded globular domains. These interactions are regulated by post-translational modifications, such as phosphorylation, that create and break motif binding sites or tune the affinity of the interactions. In addition, motif-based interactions are involved in targeting serine/threonine kinases and phosphatases to their substrate and contribute to the specificity of the enzymatic actions regulating which sites are phosphorylated. Here, we review how SLiM-based interactions assist in determining the specificity of serine/threonine kinases and phosphatases, and how phosphorylation, in turn, affects motif-based interactions. We provide examples of SLiM-based interactions that are turned on/off, or are tuned by serine/threonine phosphorylation and exemplify how this affects SLiM-based protein complex formation.

Exposure to extended periods of darkness is a common source of abiotic stress that significantly affects plant growth and development. To understand how Nicotiana benthamiana responds to dark stress, the proteomes and metabolomes of leaves treated with darkness were studied. In total, 5763 proteins and 165 primary metabolites were identified following dark treatment. Additionally, the expression of autophagy‐related gene (ATG) proteins was transiently upregulated. Weighted gene coexpression network analysis (WGCNA) was utilized to find the protein modules associated with the response to dark stress. A total of four coexpression modules were obtained. The results indicated that heat‐shock protein (HSP70), SnRK1‐interacting protein 1, 2A phosphatase‐associated protein of 46 kDa (Tap46), and glutamate dehydrogenase (GDH) might play crucial roles in N. benthamiana’s response to dark stress. Furthermore, a protein–protein interaction (PPI) network was constructed and top‐degreed proteins were predicted to identify potential key factors in the response to dark stress. These proteins include isopropylmalate isomerase (IPMI), eukaryotic elongation factor 5A (ELF5A), and ribosomal protein 5A (RPS5A). Finally, metabolic analysis suggested that some amino acids and sugars were involved in the dark‐responsive pathways. Thus, these results provide a new avenue for understanding the defensive mechanism against dark stress at the protein and metabolic levels in N. benthamiana., Exposure to prolonged periods of darkness can affect plant growth and development. In this study, the response of Nicotiana benthamiana to dark stress was investigated by analyzing changes to the proteome and metabolome across 13 time points. A number of hub proteins were filtered through weighted gene coexpression network analysis to identify potential key proteins involved in the dark stress response.

Understanding the mechanisms underlying the responses to inorganic phosphate (Pi) deficiency in alfalfa will help enhance Pi acquisition efficiency and the sustainable use of phosphorous resources. Integrated global metabolomic and transcriptomic analyses of mid-vegetative alfalfa seedlings under 12-day Pi deficiency were conducted. Limited seedling growth were found, including 13.24%, 16.85% and 33.36% decreases in height, root length and photosynthesis, and a 24.10% increase in root-to-shoot ratio on day 12. A total of 322 and 448 differentially abundant metabolites and 1199 and 1061 differentially expressed genes were identified in roots and shoots. Increased (>3.68-fold) inorganic phosphate transporter 1;4 and SPX proteins levels in the roots (>2.15-fold) and shoots (>2.50-fold) were related to Pi absorption and translocation. The levels of phospholipids and Pi-binding carbohydrates and nucleosides were decreased, while those of phosphatases and pyrophosphatases in whole seedlings were induced under reduced Pi. In addition, nitrogen assimilation was affected by inhibiting high-affinity nitrate transporters (NRT2.1 and NRT3.1), and nitrate reductase. Increased delphinidin-3-glucoside might contribute to the gray-green leaves induced by Pi limitation. Stress-induced MYB, WRKY and ERF transcription factors were identified. The responses of alfalfa to Pi deficiency were summarized as local systemic signaling pathways, including root growth, stress-related responses consisting of enzymatic and nonenzymatic systems, and hormone signaling and systemic signaling pathways including Pi recycling and Pi sensing in the whole plant, as well as Pi recovery, and nitrate and metal absorption in the roots. This study provides important information on the molecular mechanism of the response to Pi deficiency in alfalfa.

Reversible phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) is essential for gene expression control. How altering the phosphorylation of the CTD contributes to gene expression in mammalian systems remains poorly understood. Methods: Primary mouse embryonic fibroblasts, hepatocytes, and embryonic stem cells were isolated from conditional Ssu72f/f mice. To knockout the mouse Ssu72 gene, we infected the cells with adenoviruses of incorporated luciferase and Cre recombinase, respectively. RNA sequencing, ChIP sequencing, ChIP assay, immunoblot analyses, qRT-PCR assay, and immunostaining were performed to gain insights into the functional mechanisms of Ssu72 loss in Pol II dynamics. Results: Using primary cells isolated from Ssu72 conditional knockout and transgenic mice, we found that mammalian Ssu72-mediated transcriptional elongation rather than polyadenylation or RNA processing contributed to the transcriptional regulation of various genes. Depletion of Ssu72 resulted in aberrant Pol II pausing and elongation defects. Reduced transcriptional elongation efficiency tended to preferentially affect expression levels of actively transcribed genes in a tissue-specific manner. Furthermore, Ssu72 CTD phosphatase seemed to regulate the phosphorylation levels of CTD Ser2 and Thr4 through accurate modulation of P-TEFb activity and recruitment. Conclusions: Our findings demonstrate that mammalian Ssu72 contributes to the transcription of tissue-specific actively transcribed gene expression by regulating reciprocal phosphorylation of Pol II CTD.

Abscisic acid (ABA) plays an important role in seed germination, stomatal closure, and seedling growth inhibition in plants. Among downstream genes whose expression levels are regulated by AFA1 (Arabidopsis F-box Protein Hypersensitive to ABA 1), one gene, AtHAD1 upregulated by ABA was selected from Arabidopsis. AtHAD1 was induced by drought and salt stresses as well as by ABA and was found in dry seeds. Its loss-of-function mutants exhibited increased ABA-sensitivity in germination, seedling growth, and stomatal closure. In addition, the mutants displayed a lower water loss rate and higher survival rate under drought stress than the wild-type plants, indicating that a loss of AtHAD1 leads to enhanced drought tolerance. These results show that AtHAD1 has an inhibitory role in ABA response and ABA-mediated drought tolerance. The expression levels of several ABA-responsive genes in athad1 were higher than those in the wild-type under the ABA treatment, suggesting that AtHAD1, as a negative regulator in ABA response, could be associated with the downregulation of the ABA-responsive genes. The phosphatase assay showed that AtHAD1 exhibits phosphatase activity. Monitoring of the subcellular localization of GFP-fused AtHAD1 proteins indicated that AtHAD1 exists in the nucleus and cytoplasm. Overall, this study shows that Arabidopsis HAD1 as an intracellular phosphatase negatively functions in the ABA-mediated cellular responses. This research could serve as a research basis to understand the functional link between ABA signaling and the regulation process of the cellular phosphate level.

Cyclin M (CNNM) and its prokaryotic ortholog CorC belong to a family of proteins that function as Mg2+-extruding transporters by stimulating Na+/Mg2+ exchange, and thereby control intracellular Mg2+ levels. The Mg2+-extruding function of CNNM is inhibited by the direct binding of an oncogenic protein, phosphatase of regenerating liver (PRL), and this inhibition is responsible for the PRL-driven malignant progression of cancers. Studies with mouse strains deficient for the CNNM gene family revealed the importance of CNNM4 and CNNM2 in maintaining organismal Mg2+ homeostasis by participating in intestinal Mg2+ absorption and renal reabsorption, respectively. Moreover, CNNM proteins are involved in various diseases, and gene mutations in CNNM2 and CNNM4 cause dominant familial hypomagnesemia and Jalili syndrome, respectively. Genome wide association studies have also revealed the importance of CNNM2 in multiple major diseases, such as hypertension and schizophrenia. Collectively, the molecular and biological characterizations of CNNM/CorC show that they are an intriguing therapeutic target; the current status of drug development targeting these proteins is also discussed.

Nitric oxide (NO) production in the tumor microenvironment is a common element in cancer. S-nitrosylation, the post-translational modification of cysteines by NO, is emerging as a key transduction mechanism sustaining tumorigenesis. However, most oncoproteins that are regulated by S-nitrosylation are still unknown. Here we show that S-nitrosoglutathione reductase (GSNOR), the enzyme that deactivates S-nitrosylation, is hypo-expressed in several human malignancies. Using multiple tumor models, we demonstrate that GSNOR deficiency induces S-nitrosylation of focal adhesion kinase 1 (FAK1) at C658. This event enhances FAK1 autophosphorylation and sustains tumorigenicity by providing cancer cells with the ability to survive in suspension (evade anoikis). In line with these results, GSNOR-deficient tumor models are highly susceptible to treatment with FAK1 inhibitors. Altogether, our findings advance our understanding of the oncogenic role of S-nitrosylation, define GSNOR as a tumor suppressor, and point to GSNOR hypo-expression as a therapeutically exploitable vulnerability in cancer. ispartof: CELL REPORTS vol:42 issue:1 ispartof: location:United States status: published

Phosphorylation of beta-amyloid peptide (Aβ) at the Ser8 residue affects its neurotoxicity, metal-dependent oligomerisation, amyloidogenicity, and other pathogenic properties. Phosphorylated Aβ (pS8-Aβ) was detected in vivo in AD model mice and in the brains of patients with AD. However, the pS8-Aβ production and the regulation of its levels have not been previously studied in detail. In this paper, immunochemical methods together with radioactive labelling were used to study the Aβ phosphorylation by intracellular and surface protein kinases of HEK293 cells and brain endothelial cells (bEnd.3). It was found that HEK293 robustly phosphorylated Aβ, likely with contribution from casein kinase 2 (CK2), whereas in bEnd.3, the activity of Aβ phosphorylation was relatively low. Further, the study showed that both HEK293 and bEnd.3 could dephosphorylate pS8-Aβ, mainly due to the activity of protein phosphatases PP1 and PP2A. The Aβ dephosphorylation efficiency in bEnd.3 was three times higher than in HEK293, which correlated with the reduced abundance of pS8-Aβ in vascular amyloid deposits of patients with AD compared to senile plaques. These data suggest an important role of CK2, PP1, and PP2A as regulators of Aβ phosphorylation, and point to the involvement of the blood–brain barrier in the control of Aβ modification levels.

Murray-Darling Freshwater Research CentreMDFRC item.Continuous centrifugation and tangential flow ultrafiltration were used to size-fractionate and concentrate suspended particles from various turbid rivers of south-eastern Australia. These preparative steps produced highly concentrated samples, but the high load of suspended solids (up to 3.28 gl-1) did not interfere with the determination of phosphatase activity with an enzyme assay using p-nitrophenyl phosphate as the substrate. Phosphomonoesterase activity was an order of magnitude greater than phosphodiesterase activity; acid, neutral and alkaline phosphatases were detected. Most neutral (pH 7.5) and alkaline (pH 9.0) phosphatase activity was in the 0.2 - 1 mum size fraction: the next most active fractions were usually the 1-25 mum and the 100,000 daltons - 0.2 mum classes. Activity associated with free enzymes (i.e., that in the 10,000 - 100,000 daltons fraction) was usually low. Similar profiles were obtained with p-nitrophenyl phosphate and methylumbelliferyl phosphate substrates. Across the various size fractions within each river-water sample, phosphatase activity was usually more highly correlated with organic-matter content than with dry-matter content. Phosphatase activity was poorly related to chlorophyll-a contents. Phosphatase-producing bacteria were detected in all particulate fractions. The high activity in the 0.2 - 1 mum size fraction of turbid rivers might, therefore, be a function of either phosphatases associated with planktonic bacteria, or phosphatases adsorbed to the organic coating of suspended inorganic particles. In the coarser fractions, the additional contribution of attached bacteria cannot be excluded.

Phosphatase and tensin homologue (PTEN) and SH2-containing inositol 5′-phosphatase 2 (SHIP2) are structurally and functionally similar. They both consist of a phosphatase (Ptase) domain and an adjacent C2 domain, and both proteins dephosphorylate phosphoinositol-tri(3,4,5)phosphate, PI(3,4,5)P3; PTEN at the 3-phophate and SHIP2 at the 5-phosphate. Therefore, they play pivotal roles in the PI3K/Akt pathway. Here, we investigate the role of the C2 domain in membrane interactions of PTEN and SHIP2, using molecular dynamics simulations and free energy calculations. It is generally accepted that for PTEN, the C2 domain interacts strongly with anionic lipids and therefore significantly contributes to membrane recruitment. In contrast, for the C2 domain in SHIP2, we previously found much weaker binding affinity for anionic membranes. Our simulations confirm the membrane anchor role of the C2 domain in PTEN, as well as its necessity for the Ptase domain in gaining its productive membrane-binding conformation. In contrast, we identified that the C2 domain in SHIP2 undertakes neither of these roles, which are generally proposed for C2 domains. Our data support a model in which the main role of the C2 domain in SHIP2 is to introduce allosteric interdomain changes that enhance catalytic activity of the Ptase domain. Phosphatase and tensin homologue (PTEN) and SH2-containing inositol 5′-phosphatase 2 (SHIP2) are structurally and functionally similar. They both consist of a phosphatase (Ptase) domain and an adjacent C2 domain, and both proteins dephosphorylate phosphoinositol-tri(3,4,5)phosphate, PI(3,4,5)P3; PTEN at the 3-phophate and SHIP2 at the 5-phosphate. Therefore, they play pivotal roles in the PI3K/Akt pathway. Here, we investigate the role of the C2 domain in membrane interactions of PTEN and SHIP2, using molecular dynamics simulations and free energy calculations. It is generally accepted that for PTEN, the C2 domain interacts strongly with anionic lipids and therefore significantly contributes to membrane recruitment. In contrast, for the C2 domain in SHIP2, we previously found much weaker binding affinity for anionic membranes. Our simulations confirm the membrane anchor role of the C2 domain in PTEN, as well as its necessity for the Ptase domain in gaining its productive membrane-binding conformation. In contrast, we identified that the C2 domain in SHIP2 undertakes neither of these roles, which are generally proposed for C2 domains. Our data support a model in which the main role of the C2 domain in SHIP2 is to introduce allosteric interdomain changes that enhance catalytic activity of the Ptase domain.

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

Platelet-derived growth factors (PDGF) constitute a family of five functional dimers that bind to two structurally related tyrosine kinase receptors i.e. PDGF receptor α and β (PDGFRα and PDGFRβ, respectively), controlling cell growth, proliferation, and migration in cells of mesenchymal origin. However, the aberrant activation of PDGF-induced intracellular signalling pathways is a frequent event in cancer. Therefore, the aim of this thesis has been to discover novel molecular mechanisms of modulation of PDGFRβ signalling. Since mitogen-activated protein (MAP) kinases are activated in PDGF signalling and their spatiotemporal activity is defined by a balance in phosphorylation and dephosphorylation events, in paper I we focused on dual-specificity MAPK phosphatases (MPKs or DUSPs). We found MKP2/DUSP4 to be induced in response to PDGF-BB stimulation. We then demonstrated that the expression of MKP2/DUSP4 was dependent on ERK1/2 activation and on the STAT3/p53 signalling. Endocytosis of RTKs is another mechanism that serves for signal attenuation and termination and this process can be regulated by ubiquitination or deubiquitination of cell-surface receptors. In paper II, we have identified that ubiquitin specific proteases USP4 and USP17 act as deubiquitinases (DUBs) for PDGFRβ. Both deubiquitinases impacted the timing of PDGFRβ trafficking and prolonged STAT3 activation. Consequently, high transcriptional activity of STAT3 led to the increased expression of STAT3-inducible genes c-MYC, CSF1, JUNB and CDKN1A. USP4 deletion attenuated cell proliferation in response to PDGF-BB stimulation. The family of Cbl E3 ligases is essential for ubiquitination of PDGFRβ upon ligand stimulation, followed by the receptor internalization from the cell surface and downregulation of signalling. In paper III, we have identified a new E3 ligase, i.e. tripartite motif-containing protein TRIM21, that deubiquitinates PDGFRβ and regulates its basal levels and its availability on the cell surface in a PDGF-BB independent manner. In paper IV, we described a regulatory role of the endoribonuclease Ras GTPase-activating protein-binding protein 1 (G3BP1) in PDGF signalling. G3BP1 was identified as a PDGFRβ interacting protein which also interacts with BAF155, a core component of SWI/SNF chromatin remodelling complex. G3BP1 depletion upregulated c-FOS, c-MYC and c-JUN mRNA and negatively affected STAT3 and ERK1/2 mRNA and protein levels, stalling cell proliferation. Collectively, we present new mechanisms that regulate PDGF signalling by controlling either PDGFRβ protein levels, availability on the cell surface, subcellular trafficking or activation of downstream signalling affecting regulation of cell proliferation.

Globally, forests are impacted by atmospheric nitrogen (N) deposition, affecting their structure and functioning above and below ground. All trees form mutualistic root symbioses with mycorrhizal fungi. Of the two kinds of mycorrhizal symbioses of trees, the ectomycorrhizal (EcM) symbiosis is much more sensitive to N enrichment than the arbuscular mycorrhizal (AM) symbiosis. Due to increasing N deposition, significant declines in the richness and abundance of EcM fungal species and shifts in community composition and functional traits have been recorded. Under increasing N deposition, ectomycorrhizal forests usually show enhanced foliar mass fractions of N, reduced foliar mass fractions of phosphorus (P), and, consequently, an increasing imbalance in the foliar N:P stoichiometry, ultimately impacting tree performance. The question has been raised of whether, under conditions of high N deposition, EcM trees can select EcM fungi that are both tolerant to high N availability and efficient in the acquisition of P, which could to some extent mitigate the negative impact of N deposition on nutrient balances. Here we evaluate the literature for mechanisms through which certain EcM fungi could increase P acquisition under increased N loading. We find very little evidence that under N enrichment, EcM fungi that have on average higher P efficiency might be selected and thereby prevent or delay tree N:P imbalances. However, methodological issues in some of these studies make it imperative to treat this conclusion with caution. Considering the importance of avoiding tree N:P disbalances under N enrichment and the need to restore EcM forests that have suffered from long-term excess N loading, further research into this question is urgently required.

Introduction: Organic phosphorus (Po) compounds constitute an important pool in soil P cycling, but their decomposition dynamics are poorly understood. Further, it has never been directly tested whether low molecular weight Po compounds are taken up by soil microbes in an intact form, which reduces the dependence of their P acquisition on extracellular phosphatases. Methods: We investigated the short-term fate (24 h) of five 33P-labelled Po compounds (teichoic acids, phospholipids, DNA, RNA and soluble organophosphates) and 33P-labelled inorganic P (Pi) in two soils. Results: We found indications that soil microbial breakdown of phosphodiesters was limited by the depolymerization step, and that direct microbial uptake of Po occurred to a substantial extent. Discussion: We postulate a trade-off between direct Po uptake and complete extracellular Po mineralization. These findings have profound consequences for our understanding of microbial P cycling in soils., Frontiers in Soil Science, 3, ISSN:2673-8619

Bacteria that exhibit polar growth, i.e. build their peptidoglycan cell walls in restricted zones at cell poles, often show large morphological diversity and plasticity. However, their mechanisms for regulation of cell shape and cell wall assembly are poorly understood. The Gram-positive Streptomyces bacteria, like other Actinobacteria, depend on the essential coiled coil protein DivIVA for establishment of cell polarity and direction of polar growth. Streptomycetes grow as filamentous hyphae that exhibit tip extension. New hyphal tips are generated by lateral branching. Cell shape is largely determined by the control of cell wall growth at these hyphal tips. The Ser/Thr protein kinase AfsK is involved in controlling polar growth and directly phosphorylates DivIVA. Here, we identify a protein phosphatase in Streptomyces coelicolor, SppA, that dephosphorylates DivIVA in vivo and in vitro and affects growth and cell shape. An sppA mutant shows reduced rate of hyphal tip extension, altered hyphal branching patterns, and exhibits frequent spontaneous hyphal growth arrests, all contributing to the unusually dense mycelial structure and slow growth rate that characterize sppA mutants. These phenotypes are largely suppressed in an afsK sppA double mutant, showing that AfsK and SppA partially affect the same regulatory pathway and share target proteins that are involved control of polar growth in S. coelicolor. Strains with a non-phosphorylatable mutant DivIVA were constructed and confirm that the effect of afsK on hyphal branching during normal growth is mediated by DivIVA phosphorylation. However, the phenotypic effects of sppA deletion are independent of DivIVA phosphorylation and must be mediated via other substrates. Altogether, this study identifies a PPP-family protein phosphatase directly involved in the control of polar growth and cell shape determination in S. coelicolor and underscore the importance of eukaryotic-type Ser/Thr phosphorylation in regulation of growth and cell envelope biogenesis in Actinobacteria.Author summaryGram-positive bacteria often use eukaryotic-like Ser/Thr protein phosphorylation in the regulation of central processes related to growth and cell cycle progression. Members of the Actinobacteria, including pathogenic mycobacteria and antibiotic-producing streptomycetes, exhibit a distinctive mode of polar growth, with cell wall synthesis being restricted to zones at cell poles and directed by the essential cell polarity determinant DivIVA. Previously, we have shown that Streptomyces coelicolor modulates its machinery for polar growth and cell shape determination via the Ser/Thr kinase AfsK, which phosphorylates DivIVA. Here, we identify a phosphoprotein phosphatase that targets DivIVA and reverses the AfsK-mediated phosphorylation. We show that this phosphatase has strong effects on polar growth and cell shape, including hyphal branching and the rate of tip extension. The effects are to a large extent dependent on the kinase AfsK but do not require DivIVA phosphorylation as such. The results identify a new regulator of polar growth in streptomycetes, and highlight the importance of Ser/Thr phosphorylation in regulation of actinobacterial growth and cell shape determination.

Secreted acid phosphatases (APases) dephosphorylate extracellular organic phosphate compounds to supply inorganic phosphate (Pi) to maintain cellular functions. Here, we show that APases are necessary to maintain a normal replicative lifespan in Saccharomyces cerevisiae. Deletion of all four APase genes shortened the lifespan in yeast strains on synthetic media (irrespective of the concentrations of Pi in the media), but it did not affect the intracellular ortho- and polyphosphate levels. Deletion of IPK1, which encodes inositol-pentakisphosphate 2-kinase, restored the lifespan in APase-null mutants, and IPK1 overexpression shortened the lifespan in wild-type strains. Overexpression of inositol hexakisphosphate (IP6 ) and heptakisphosphate kinases, KCS1 and VIP1, recovered the lifespan in APase-null mutants. Thus, yeast APases modulate the replicative lifespan, probably through dephosphorylation of intracellular IP6 .

PPM1A (magnesium-dependent phosphatase 1 A, also known as PP2Cα) is a member of the Ser/Thr protein phosphatase family. Protein phosphatases catalyze the removal of phosphate groups from proteins via hydrolysis, thus opposing the role of protein kinases. The PP2C family is generally considered a negative regulator in the eukaryotic stress response pathway. PPM1A can bind and dephosphorylate various proteins and is therefore involved in the regulation of a wide range of physiological processes. It plays a crucial role in transcriptional regulation, cell proliferation, and apoptosis and has been suggested to be closely related to the occurrence and development of cancers of the lung, bladder, and breast, amongst others. Moreover, it is closely related to certain autoimmune diseases and neurodegenerative diseases. In this review, we provide an insight into currently available knowledge of PPM1A, including its structure, biological function, involvement in signaling pathways, and association with diseases. Lastly, we discuss whether PPM1A could be targeted for therapy of certain human conditions.

Graphical abstract, Introduction Previously, we have reported a withanolide-type steroid, named tubocapsenolide A (TA), which shows potent anti-proliferative activity in several cancer cell lines. However, its inhibitory effect on the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway and therapeutic potential on osteosarcoma have not been reported. Objectives In the present study, we aimed to investigate the effect and molecular mechanism of TA in osteosarcoma. Methods The biological functions of TA in U2OS cells were investigated using colony formation, 5-ethynyl-20-deoxyuridine (EDU) staining, and cell cycle/apoptosis assays. The interaction between TA and Src homology 2 phosphatase 2 (SHP-2) was detected by enzyme activity and validated by target-identification methods such as drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and biolayer interferometry (BLI). The in vivo anti-tumor efficacy of TA was analyzed in the xenograft tumor model. Western blotting analysis was performed to detect the protein expression levels. Results TA exhibited antitumor activity against osteosarcoma both in vitro and in vivo by regulating the JAK/STAT3 signaling pathway. Mechanically, TA interacted with SHP-2 directly and activated its phosphatase activity. Importantly, protein tyrosine phosphatase (PTP) inhibitor, SHP-2 inhibitor, and SHP-2 siRNA could reverse the inhibitory effect of TA on the JAK/STAT3 signaling pathway and restored the TA-induced cell death. Conclusion TA activated the phosphatase activity of SHP-2, which resulted in the inhibition of the JAK/STAT3 pathway and contributed to the antitumor efficacy of TA. Collectively, these findings suggested that TA could serve as a novel therapeutic agent for the treatment of osteosarcoma.

We have previously reported an important role of PR55α, a regulatory subunit of PP2A Ser/Thr phosphatase, in the support of critical oncogenic pathways required for oncogenesis and the malignant phenotype of pancreatic cancer. The studies in this report reveal a novel mechanism by which the p53 tumor suppressor inhibits the protein-stability of PR55α via FBXL20, a p53-target gene that serves as a substrate recognition component of the SCF (Skp1_Cullin1_F-box) E3 ubiquitin ligase complex that promotes proteasomal degradation of its targeted proteins. Our studies show that inactivation of p53 by siRNA-knockdown, gene-deletion, HPV-E6-mediated degradation, or expression of the loss-of-function mutant p53R175H results in increased PR55α protein stability, which is accompanied by reduced protein expression of FBXL20 and decreased ubiquitination of PR55α. Subsequent studies demonstrate that knockdown of FBXL20 by siRNA mimics p53 deficiency, reducing PR55α ubiquitination and increasing PR55α protein stability. Functional tests indicate that ectopic p53R175H or PR55α expression results in an increase of c-Myc protein stability with concomitant dephosphorylation of c-Myc-T58, which is a PR55α substrate, whose phosphorylation otherwise promotes c-Myc degradation. A significant increase in anchorage-independent proliferation is also observed in normal human pancreatic cells expressing p53R175H or, to a greater extent, overexpressing PR55α. Consistent with the common loss of p53 function in pancreatic cancer, FBXL20 mRNA expression is significantly lower in pancreatic cancer tissues compared to pancreatic normal tissues and low FBXL20 levels correlate with poor patient survival. Collectively, these studies delineate a novel mechanism by which the p53/FBXL20 axis negatively regulates PR55α protein stability.

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28- days iPTH (80µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1-/- mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in femora of WT mice following iPTH administration but remained unchanged in femora of Phospho1-/- mice. After 28-days of iPTH administration the anabolic response in femora of WT was greater than that noted in Phospho1-/- mice. Specifically, cortical and trabecular BV/TV, as well as cortical thickness were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1-/- mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1-/- mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.Significance Statement: Intermittent administration of PTH (iPTH) is recognised to increase osteoblast number and promote bone formation. A critical component of this anabolic response is the mineralisation of the osteoid matrix but the effects iPTH on the expression of mineralisation regulating enzymes is unclear. This study’s principal finding was that iPTH administration increased the expression of Phospho1, Alpl, Enpp1 and Smpd3 in vivo which was consistent with a bone anabolic PTH regimen. The data also disclosed that in the absence of PHOSPHO1, the iPTH anabolic response was dampened, suggesting that amplified Phospho1 expression is a prerequisite for a full iPTH mediated bone anabolic response.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a dual lipid and protein phosphatase. Multiple mechanisms contributing to the regulation of PTEN levels have been identified thus far, including post-translational modifications, epigenetic mechanisms, and transcriptional mechanisms. In the present study, we identified Ubiquitin specific peptidase 22 (USP22) as a novel deubiquitination-modifying enzyme of PTEN. Furthermore, by inducing deubiquitination and inhibiting the degradation of PTEN, USP22 could induce Cyclin dependent kinase inhibitor 1A (CDKN1A, also symbolled as p21) expression in pancreatic cancer. Besides, MDM2 proto-oncogene (MDM2) inhibitor enhanced the anti-pancreatic cancer effects of USP22 overexpression. In addition to its regulation of MDM2-Tumor protein p53 (p53) signaling, we found that PTEN could induce p21 expression by interacting with Ankyrin repeat and KH domain containing 1 (ANKHD1) and inhibiting ANKHD1 binding to the p21 promoter. Taken together, our results indicate that ANKHD1 and MDM2 might be novel therapeutic targets in pancreatic cancer.

Protein phosphorylation, which regulates many critical aspects of cell biology, is dynamically governed by kinases and phosphatases. Many diseases are associated with dysregulated hyperphosphorylation of critical proteins, such as retinoblastoma protein in cancer. Although kinase inhibitors have been widely applied in the clinic, growing evidence of off-target effects and increasing drug resistance prompts the need to develop a new generation of drugs. Here, we propose a proof-of-concept study of phosphorylation targeting chimeras (PhosTACs). Similar to PROTACs in their ability to induce ternary complexes, PhosTACs focus on recruiting a Ser/Thr phosphatase to a phosphosubstrate to mediate its dephosphorylation. However, distinct from PROTACs, PhosTACs can uniquely provide target gain-of-function opportunities to manipulate protein activity. In this study, we applied a chemical biology approach to evaluate the feasibility of PhosTACs by recruiting the scaffold and catalytic subunits of the PP2A holoenzyme to protein substrates such as PDCD4 and FOXO3a for targeted protein dephosphorylation. For FOXO3a, this dephosphorylation resulted in the transcriptional activation of a FOXO3a-responsive reporter gene.

This work explored the mechanism of augmented stress-induced vascular reactivity of senescent murine femoral arteries (FAs). Mechanical and pharmacological reactivity of young (12-25 weeks, y-FA) and senescent (>104 weeks, s-FAs) femoral arteries was measured by wire myography. Expression and protein phosphorylation of selected regulatory proteins were studied by western blotting. Expression ratio of the Exon24 in/out splice isoforms of the regulatory subunit of myosin phosphatase, MYPT1 (MYPT1-Exon24 in/out), was determined by polymerase chain reaction (PCR). While the resting length-tension relationship showed no alteration, the stretch-induced-tone increased to 8.3 ± 0.9 mN in s-FA versus only 4.6 ± 0.3 mN in y-FAs. Under basal conditions, phosphorylation of the regulatory light chain of myosin at S19 was 19.2 ± 5.8% in y-FA versus 49.2 ± 12.6% in s-FA. Inhibition of endogenous NO release raised tone additionally to 10.4 ± 1.2 mN in s-FA, whereas this treatment had a negligible effect in y-FAs (4.8 ± 0.3 mN). In s-FAs, reactivity to NO donor was augmented (pD2 = -4.5 ± 0.3 in y-FA vs. -5.2 ± 0.1 in senescent). Accordingly, in s-FAs, MYPT1-Exon24-out-mRNA, which is responsible for expression of the more sensitive to protein-kinase G, leucine-zipper-positive MYPT1 isoform, was increased. The present work provides evidence that senescent murine s-FA undergoes vascular remodelling associated with increases in stretch-activated contractility and sensitivity to NO/cGMP/PKG system.

The modulatory effect of miR-21 on the proliferation of melanoma cells through stimulation of PTEN (Phosphatase and tensin homologue deleted on chromosome 10) expression was investigated in the current study. PTEN, as a tumor suppressor, is expressed in low levels in melanoma tissues and cell lines. Nevertheless, miR-21 can stimulate cancer development and suppress cell apoptosis. Overexpression of PTEN substantially impaired the proliferation of miR-21-treated melanoma cells. In addition, miR-21 and PTEN were observed to exhibit a combinatorial effect, whereas miR-21 could negatively regulate the expression of PTEN. In conclusion, these findings demonstrate that miR-21 affects melanoma development by targeting PTEN, establishing a new strategy for treating malignant melanoma. Furthermore, in this study, microparticles and nanoparticles were employed as carriers to construct, through the self-assembly method, nanocapsules carrying miR-21 in order to develop an efficient nanocapsule delivery system of miR-21 against melanoma cells.

Protein phosphatase Z1 (Ppz1) has been shown to take part in important physiological functions in fungi including a contribution to virulence of Candida albicans. Although its involvement in the oxidative stress response has also been documented, the exact mechanism of action of its protective effect against oxidative damage remains unknown. By developing a pipeline to analyze the biophysical properties of the cell membrane in fungi, we demonstrate that the plasma membrane of Ppz1-KO Candida albicans displays increased sensitivity to tert-butyl-hydroperoxide-induced oxidative damage. In particular, the response to the oxidizing agent, characterized by increased lipid peroxidation, reduced lipid order, and inhibited lateral mobility of plasma membrane components, is significantly more pronounced in the Ppz1-KO C. albicans strain than in the wild-type counterpart. Remarkably, membrane constituents became almost completely immobile in the phosphatase deletion mutant exposed to oxidative stress. Furthermore, moderately elevated membrane lipid peroxidation accompanied by the aforementioned changes in the biophysical characteristics of the plasma membrane are already detectable in untreated Ppz1-KO cells indicating latent membrane damage even in the absence of oxidative stress. In conclusion, the hypersensitivity of cells lacking Ppz1 to oxidative damage establishes that potential Ppz1 inhibitors may synergize with oxidizing agents in prospective anti-fungal combination therapies.

Digital bioassays have attracted extensive attention in biomedical applications due to their ultrahigh sensitivity. However, traditional digital bioassays require numerous microchambers such as droplets or microwells, which restricts their application scope. Herein, we propose a microchamber-free flow cytometric method for the digital quantification of T4 polynucleotide kinase phosphatase (T4 PNKP) based on an unprecedented phenomenon that each T4 PNKP molecule-catalyzed reaction can be spatially self-confined on a single microbead, which ultimately enables the one-target-to-one-fluorescence-positive microbead digital signal transduction. The digital signal-readout mode can clearly detect T4 PNKP concentrations as low as 1.28 × 10-10 U/μL, making it most sensitive method to date. Significantly, T4 PNKP can be specifically distinguished from other phosphatases and nucleases in complex samples by digitally counting the fluorescence-positive microbeads, which cannot be realized by traditional bulk measurement-based methods. Taking advantage of the novel space-confined enzymatic feature of T4 PNKP, this digital mechanism can use T4 PNKP as the enzyme label to fabricate digital sensing systems toward various biomolecules such as digital enzyme-linked immunosorbent assay (ELISA). Therefore, this work not only enlarges the toolbox for high-sensitivity biomolecule detection but also opens new gates to fabricate next-generation digital assays.

Many human diseases, including cancer and neurological abnormalities, are linked to deficiencies of phosphatase and tensin homolog deleted on chromosome ten (PTEN), a dual phosphatase that dephosphorylates both lipids and proteins. PTEN functions in multiple intracellular locations, including the plasma membrane and nucleus. Therefore, a critical challenge to understand the pathogenesis of PTEN-associated diseases is to determine the specific role of PTEN at different locations. Toward this goal, the current study generated a mouse line in which lysine 13, which is critical for the nuclear localization of PTEN, is changed to arginine in the lipid-binding domain using the CRISPR-Ca9 gene-editing system. We found that PTENK13R mice show a strong decrease in the localization of PTEN in the nucleus without affecting the protein stability, phosphatase activity, and phosphorylation in the C-terminal tail region. PTENK13R mice are viable but produce smaller neurons and develop microcephaly. These data demonstrate that PTENK13R mice provide a useful animal model to study the role of PTEN in the nucleus in vivo.

Mitogen-activated protein kinases (MPKs) play roles as critical signal components in the environmental stress responses and developmental processes in plants. Calcium ion (Ca2+) is one of the most essential ubiquitous intracellular second messengers involved in many signal transduction pathways in plants. It was previously known that MPKs are activated by the increasing Ca2+ concentration. However, the mechanism of how Ca2+ activates MPKs is not elucidated yet. In this study, we revealed that Ca2+ could activate MPK signaling pathway via inhibiting the activity of a dual-specificity protein phosphatase1 (DsPTP1) by Ca2+/calmodulin (CaM). We showed that DsPTP1 directly interacts with MPK6 in vitro and in vivo. DsPTP1 was able to inactivate the active MPK6 by dephosphorylation. Interestingly, the DsPTP1-mediated dephosphorylation of MPK6 was strongly inhibited by Ca2+/CaM. Moreover, this inhibition was caused by the binding of CaM to the calmodulin-binding domain II (CaMBDII) of DsPTP1. This study implies that Ca2+/CaM is involved in the activation of MPKs through the inhibition of DsPTP1.

How plants balance growth and stress adaptation is a long-standing topic in plant biology. Abscisic acid (ABA) induces the expression of the stress-responsive Asparagine Rich Protein (NRP), which promotes the vacuolar degradation of PP6 phosphatase FyPP3, releasing ABI5 transcription factor to initiate transcription. Whether NRP is required for growth remains unknown. We generated an nrp1 nrp2 double mutant, which had a dwarf phenotype that can be rescued by inhibiting auxin transport. Insufficient auxin in the transition zone and over-accumulation of auxin at the root tip was responsible for the short elongation zone and short-root phenotype of nrp1 nrp2. The auxin efflux carrier PIN2 over-accumulated in nrp1 nrp2 and became de-polarized at the plasma membrane, leading to slower root basipetal auxin transport. Knock-out of PIN2 suppressed the dwarf phenotype of nrp1 nrp2. Furthermore, ABA can induce NRP-dependent vacuolar degradation of PIN2 to inhibit primary root elongation. FyPP3 also is required for NRP-mediated PIN2 turnover. In summary, in growth condition, NRP promotes PIN2 vacuolar degradation to help maintain PIN2 protein concentration and polarity, facilitating the establishment of the elongation zone and primary root elongation. When stressed, ABA employs this pathway to inhibit root elongation for stress adaptation.

Brassinosteroids (BRs) play essential roles in regulating plant growth and development, however, gaps still remain in our understanding of the BR signaling network. We previously cloned a grain length quantitative trait locus qGL3, encoding a rice (Oryza sativa L.) protein phosphatase with Kelch-like repeat domain (OsPPKL1), that negatively regulates grain length and BR signaling. To further explore the BR signaling network, we performed phosphoproteomic analysis to screen qGL3-regulated downstream components. We selected a 14-3-3 protein OsGF14b from the phosphoproteomic data for further analysis. qGL3 promoted the phosphorylation of OsGF14b and induced the interaction intensity between OsGF14b and OsBZR1. In addition, phosphorylation of OsGF14b played an important role in regulating nucleocytoplasmic shuttling of OsBZR1. The serine acids (Ser258Ser259) residues of OsGF14b play an essential role in BR-mediated responses and plant development. Genetic and molecular analyses indicated that OsGF14b functions as a negative regulator in BR signaling and represses the transcriptional activation activity of OsBZR1. Collectively, these results demonstrate that qGL3 induces the phosphorylation of OsGF14b, which modulates nucleocytoplasmic shuttling and transcriptional activation activity of OsBZR1, to eventually negatively regulate BR signaling and grain length in rice.

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal in vivo pharmacokinetic (PK) profile. Hypothesis-driven scaffold optimization led us to a series of pyrazolopyrazines with excellent PK properties across species but a narrow human Ether-a-go-go-Related Gene (hERG) window. Subsequent optimization of properties led to the discovery of the pyrimidinone series, in which multiple members possessed excellent potency, optimal in vivo PK across species, and no off-target activities including no hERG liability up to 100 μM. Importantly, compound 30 (IACS-15414) potently suppressed the mitogen-activated protein kinase (MAPK) pathway signaling and tumor growth in RTK-activated and KRASmut xenograft models in vivo.

Staphylococcus aureus, a gram-positive pathogen, invades cells mainly in an integrin-dependent manner. As the activity or conformation of several integrin-associated proteins can be regulated by phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2 ), we investigated the roles of PI-4,5-P2 and PI-4,5-P2 -producing enzymes in cellular invasion by S. aureus. PI-4,5-P2 accumulated upon contact of S. aureus with the host cell and targeting of an active PI-4,5-P2 phosphatase to the plasma membrane reduced bacterial invasion. Knockdown of individual phosphatidylinositol-4-phosphate 5-kinases revealed that phosphatidylinositol-4-phosphate 5-kinase γ (PIP5KIγ) plays an important role in bacterial internalization. Specific ablation of the talin and FAK binding motif in PIP5KIγ90 reduced bacterial invasion, which could be rescued by re-expression of an active, but not inactive PIP5KIγ90. Furthermore, PIP5KIγ90-deficient cells showed normal basal PI-4,5-P2 levels in the plasma membrane but reduced accumulation of PI-4,5-P2 and talin at sites of S. aureus attachment, and overall lower levels of FAK phosphorylation. These results highlight the importance of local synthesis of PI-4,5-P2 by a focal adhesion-associated lipid kinase for integrin-mediated internalization of S. aureus. published

Background information Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B-type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools. GIT1 gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasing GIT1 expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization. Results We find that the α-arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI-treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs in aly1∆ aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α-arrestin Aly1 by the protein phosphatase calcineurin improves steady-state and substrate-induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α-arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [3 H]-inositol-labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a-arrestins in this regulation. Conclusions The α-arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance. Significance To our knowledge, this is the first example of α-arrestin regulation of phosphatidyliniositol-3-phosphate levels. In future studies it will be exciting to determine if other α-arrestins similarly alter PI and PIPs to change the cellular landscape.

Toxic cyanobacteria blooms have been reported in freshwater sources worldwide and may lead to aquatic biota toxin accumulation and trophic chain transfer, resulting in ecological and public health concerns. To assess cyanobacteria effects on microcystin uptake and accumulation and on phosphatase, acethylcholinesterase (AChE) and carboxylesterase (CarbE) enzymatic activities, an in vivo experiment was carried out employing the golden mussel Limnoperna fortunei. These mussels were exposed to a Microcystis aeruginosa NPLJ-4 strain (NPLJ-4) for 48 hours at different cell densities. Subsequently, algal cell counts were carried out and enzymatic activities were assayed. All three enzymes (Phosphatase, AChE and CarbE) were inhibited at the end of the exposure experiment. Mussels exposed to higher in vivo M. aeruginosa densities exhibited microcystin uptake and accumulation. In vitro assays were also carried out, exposing soluble L. fortunei enzyme fractions to M. aeruginosa extracts containing microcystin, and phosphatase inhibition was observed, whereas acetylcholinesterase and carboxylesterase were not inhibited. The results indicate that metabolites other than mycrocystin probably caused the observed in vivo esterase inhibitions, requiring further investigations.

Ras-related GTP binding (Rag) GTPases are required to activate mechanistic target of rapamycin complex 1 (mTORC1), which plays a central role in cell growth and metabolism and is considered as one of the most important oncogenic pathways. Therefore, Rag GTPases have been speculated to play a pro-cancer role via mTOR induction. However, aside from stimulation of mTOR signaling, firm links connecting Rag GTPase activity and their downstream effectors with cancer progression, remain largely unreported. In this study, we reported a novel link between RagB/C and a known oncoprotein phosphatase of regenerating liver-3 (PRL-3) by screening 22 pairs of tumors and their adjacent normal tissues from gastric, liver and lung cancers, and validating our findings in cancer cell lines with ectopic RagB/C expression. RagB/C was found to enhance PRL-3 stability by modulating two major cellular protein degradation pathways: lysosomal-autophagy and ubiquitin-proteasome system (UPS). Functionally, we identified the correlation between RagB/C expression with poor clinical outcomes in breast or colon cancer patients who also showed low PRL-3 mRNA expression from data retrieved from TCGA datasets, highlighting the potential relevance of Rag GTPase and PRL-3 mRNA in combination as a prognostic clinical biomarker.

Introduction: This study aims to evaluate the expression of PTEN on the development of cisplatin resistance in breast cancer by TGF-β1 signal pathway in order to investigate the prognostic value of PTEN and TGF-β1 for breast cancer. Material and Methods: In this study, HE staining, Immunohistochemical staining, Immunofluorescence and Western blotting were used to detect the expression of PTEN and TGF-β1 in breast cancer MCF-7 cells to explain clearly the relationship between PTEN and TGF-β1 on TGF-β1 treated MCF-7 Cells. Results: The experiment results showed the expression of PTEN and TGF-β1 in Human breast cancer increased obviously compared with paracancerous tissues (P < 0.05). The expression of PTEN and TGF-β1 was closely correlated with tumor size, distant metastasis, pathological stage and progesterone receptor status, but not with age and lymph node status. At the same time, the expression of PTEN and TGF-β1 in cisplatin-treated MCF-7 Cells reduced as compared with untreated breast cancer cells (P < 0.05). In addition, the expression of PTEN increased by TGF-β1 inducer treated Human Breast Cancer Cells. However, the expression of PTEN in TGF-β1 inhibitor treated Human Breast Cancer Cells was lower than in untreated breast cancer cells (P < 0.05). Conclusions: PTEN and TGF-β1 played an important role in Human Breast cancer. In addition, the expression of PTEN could be regulated by TGF-β1 in Cisplatin and TGF-β1 treated Human Breast Cancer Cells.

Nine new highly oxygenated 3,5-dimethylorsellinic acid-derived meroterpenoids, talaromynoids A-I (1-9), were isolated from the marine-derived fungus Talaromyces purpureogenus SCSIO 41517. Their structures including absolute configurations were elucidated by HRMS, NMR, single-crystal X-ray diffraction analysis, and electronic circular dichroism calculations. Compounds 1 and 7-9 possessed unprecedented 5/7/6/5/6/6, 6/7/6/6/6/5, 6/7/6/5/6/5/4, and 7/6/5/6/5/4 polycyclic systems, respectively. Biologically, compound 5 showed selective inhibitory activity against phosphatase CDC25B with an IC50 value of 13 μM. Moreover, 7-9 and 12 exhibited the activity of reducing triglyceride in 3T3-L1 adipocytes in a dosage-dependent manner.