Protein Phosphatase Inhibitor Research Articles

Alpha-mangostin dephosphorylates ERM to induce adhesion and decrease surface stiffness in KG-1 cells.

Surface stiffness is a unique indicator of various cellular states and events and needs to be tightly controlled. α-Mangostin, a natural compound with numerous bioactivities, reduces the mechanical stiffness of various cells; however, the mechanism by which it affects the actin cytoskeleton remains unclear. We aimed to elucidate the mechanism underlying α-mangostin activity on the surface stiffness of leukocytes. We treated spherical non-adherent myelomonocytic KG-1 cells with α-mangostin; it clearly reduced their surface stiffness and disrupted their microvilli. The α-mangostin-induced reduction in surface stiffness was inhibited by calyculin A, a protein phosphatase inhibitor. α-Mangostin also induced KG-1 cell adhesion to a fibronectin-coated surface. In KG-1 cells, a decrease in surface stiffness and the induction of cell adhesion are largely attributed to the dephosphorylation of ezrin/radixin/moesin proteins (ERMs); α-mangostin reduced the levels of phosphorylated ERMs. It further increased protein kinase C (PKC) activity. α-Mangostin-induced KG-1 cell adhesion and cell surface softness were inhibited by the PKC inhibitor GF109203X. The results of the present study suggest that α-mangostin decreases stiffness and induces adhesion of KG-1 cells via PKC activation and ERM dephosphorylation.

Renal AT2 Receptors Mediate Natriuresis via Protein Phosphatase PP2A.

How signals from activated angiotensin type-2 receptors (AT2R) 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 AT2R signaling in tissues other than kidney. We investigated whether inhibition of protein phosphatase PP2A reduced AT2R-mediated natriuresis and evaluated changes in PP2A activity and localization after renal AT2R 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. In Wistar-Kyoto rats, direct renal interstitial administration of selective AT2R 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 AT2Rs. Calyculin A treatment abolished C-21-induced translocation of both AT2R and PP2A regulatory subunit B55α to apical plasma membranes. Immunoprecipitation of AT2R solubilized from renal cortical homogenates demonstrated physical association of AT2R 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 AT2Rs and PP2A subunits to apical plasma membranes. In renal proximal tubule cells of Wistar-Kyoto rats, PP2A is activated and PP2A subunits AB55αC are recruited to C-21-activated AT2Rs 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.

Convergent Protein Phosphatase Inhibitor Design for PTP1B and TCPTP: Exchangeable Vanadium Coordination Complexes on Graphene Quantum Dots

AbstractDevelopment of potent and specific inhibitors of protein tyrosine phosphatase 1B (PTP1B) with desired drug‐like properties is still a challenge. Based on the crystal structures of PTP1B transition state analog consisting of a vanadate peptide, a novel approach is proposed to design PTP1B inhibitors, in which the tyrosyl vanadate ester of a PTP1B peptide mimic (PL1) is stably integrated on the membrane permeable graphene quantum dots (GQDs). The vanadate complexes (GQD‐PL1‐VV) prepared exhibit high potency (Ki = 6 ± 1 × 10−9 m) and selectivity (selectivity index SI >200 for PTP1B versus the T‐cell protein tyrosine phosphatase, TCPTP) in solution and in HepG2 cells. Oral administration of GQD‐PL1‐VV in db/db model mice shows selective PTP1B inhibition in liver and fat tissues and exhibits improved anti‐diabetic activity compared to bis(maltolato)oxovanadium(IV). Moreover, exchange of PL1 to a TCPTP‐specific ligand (PL2) results in potent TCPTP inhibition (Ki = 59 ± 12 × 10−9 m) as expected with SI ≈23 versus PTP1B. Overall, the present results provide a paradigm shift and a general design of phosphatase inhibitors consisting of GQDs, a complexing targeting ligand and vanadium (V) for selective regulation of PTP1B both in vitro and vivo.

Investigation of the Occurrence of Cyanotoxins in Lake Karaoun (Lebanon) by Mass Spectrometry, Bioassays and Molecular Methods.

Lake Karaoun is the largest artificial lake in Lebanon and serves multiple purposes. Recently, intensive cyanobacterial blooms have been reported in the lake, raising safety and aesthetic concerns related to the presence of cyanotoxins and cyanobacterial taste and odor (T&O) compounds, respectively. Here, we communicate for the first time results from a recent investigation by LC-MS/MS covering multiple cyanotoxins (microcystins (MCs), anatoxin-a, cylindrospermopsin, nodularin) in water and fish collected between 2019 and 2020. Eleven MCs were identified reaching concentrations of 211 and 199 μg/L for MC-LR and MC-YR, respectively. Cylindrospermopsin, anatoxin-a and nodularin were not detected. The determination of the total MCs was also carried out by ELISA and Protein Phosphatase Inhibition Assay yielding comparable results. Molecular detection of cyanobacteria (16S rRNA) and biosynthetic genes of toxins were carried out by qPCR. Untargeted screening analysis by GC-MS showed the presence of T&O compounds, such as β-cyclocitral, β-ionone, nonanal and dimethylsulfides that contribute to unpleasant odors in water. The determination of volatile organic compounds (VOCs) showed the presence of anthropogenic pollutants, mostly dichloromethane and toluene. The findings are important to develop future monitoring schemes in order to assess the risks from cyanobacterial blooms with regard to the lake’s ecosystem and its uses.

The phosphatase/kinase balance affects phytochrome A and its native pools, phyA' and phyA″, in etiolated maize roots: evidence from the induction of phyA' destruction by a protein phosphatase inhibitor sodium fluoride.

Phytochrome A (phyA) comprises two native types, phyA' and phyA″, with distinct spectroscopic, photochemical, and functional properties, differing at the N-terminal extension, probably, by the state of phosphorylation. To find out if and how protein phosphatases (PP) affect the state of the phyA species in planta, we studied the effect of the non-specific phosphatase inhibitor NaF on etiolated maize seedlings with the use of low-temperature fluorescence spectroscopy and photochemistry. In roots, phosphatase inhibition facilitated photoreceptor destruction in its labile phyA' form and shifted the phyA'/phyA″ ratio towards the more stable phyA″. The effect of NaF was not observed in stems. It was similar, though less pronounced, in comparison to the effects of the serine/threonine PP inhibitors, okadaic and cantharidic acids (OA and CA), which likewise facilitate the destruction of phyA' in etiolated maize stems, not, however, in roots (Sineshchekov et al., Photochem. Photobiol 89:83-96, 2013). The phyA'/phyA″ balance thus depends on the kinase/phosphatase equilibrium in the root cells. The relatively low effect of NaF on phyA in roots, together with the lack of the effect of OA and CA in them, may imply that the mechanism controlling the phyA'/phyA″ balance in roots can be different from that in shoots.

Sodium cantharidate induces Apoptosis in breast cancer cells by regulating energy metabolism via the protein phosphatase 5-p53 axis

Breast cancer is the leading cause of cancer-related death in women worldwide, and despite multiple chemotherapeutic approaches, effective treatment strategies for advanced metastatic breast cancer are still lacking. Metabolic reprogramming is essential for tumor cell growth and propagation, and most cancers, including breast cancer, are accompanied by abnormalities in energy metabolism. Here, we confirmed that sodium cantharidate inhibited cell viability using the Cell Counting Kit-8, clonogenic assay, and Transwell assay. The cell cycle and apoptosis assays indicated that sodium cantharidate induced apoptosis and cell cycle arrest in breast cancer cells. Additionally, proteomic assays, western blots, and metabolic assays revealed that sodium cantharidate converted the metabolic phenotype of breast cancer cells from glycolysis to oxidative phosphorylation. Furthermore, bioinformatics analysis identified possible roles for p53 with respect to the effects of sodium cantharidate on breast cancer cells. Western blot, docking, and phosphatase assays revealed that the regulation of p53 activity by sodium cantharidate was related to its inhibition of protein phosphatase 5 activity. Moreover, sodium cantharidate significantly inhibited tumor growth in tumor-bearing nude mice. In summary, our study provides evidence for the use of sodium cantharidate as an effective and new therapeutic candidate for the treatment of human breast cancer in clinical trials.

Cellular oxidative stress stimulated by microcystin: review

Introduction: Cyanobacteria are organisms capable of producing a high number of bioactive molecules, known as cyanotoxins. Among the cyanotoxins, microcystins stand out, compounds with hepatotoxic potential. Studies claim that the most common and most toxic isoform among microcystins is microcystin-LR. One of the most frequently detected properties of microcystins is their ability to generate cellular oxidative stress. Thus, the present study is a bibliographic research about the biochemical mechanism of free radical generation caused by Microcystin LR. Methodology: for the preparation of this review, a survey was carried out in the national and international literature. The inclusion criteria for the construction of this work were original and review articles that addressed the ability of microcystin LR to generate oxidative damage. Results: Once they enter the body, microcystins accumulate in the liver, so that toxicity is associated with specific inhibition of protein phosphatase 1 and 2A (PP1 and PP2A), leading to disruption of cell integrity. Studies prove that MCs produce oxidative stress in vitro and in vivo and that they can act as tumor promoters. Conclusion: there is a possible relationship between cellular oxidative stress caused by microcystin. Thus, cyanobacterial blooms represent a threat to the health of several animals, including man, however, further studies on the topic addressed are needed.

Dephosphorylation of annexin A2 by protein phosphatase 1 regulates endothelial cell barrier.

Annexin A2 (ANXA2) is a multifunctional protein expressed in nearly all human tissues and cell types, playing a role in various signaling pathways. It is subjected to phosphorylation, but no specific protein phosphatase has been identified in its posttranslational regulation yet. Using pull-down assay followed by liquid chromatography-mass spectrometry analysis we found that ANXA2 interacts with TIMAP (TGF-beta-inhibited membrane-associated protein) in pulmonary artery endothelial cells. TIMAP is highly expressed in endothelial cells, where it acts as a regulatory and targeting subunit of protein phosphatase 1 (PP1). TIMAP plays an important role in the regulation of the endothelial barrier maintenance through the dephosphorylation of its several substrate proteins. In the present work, phosphorylation of Ser25 side chain in ANXA2 by protein kinase C (PKC) was shown both in vivo and in vitro. Phosphorylation level of ANXA2 at Ser25 increased greatly by inhibition of PP1 and by depletion of its regulatory subunit, TIMAP, implying a role of this PP1 holoenzyme in the dephosphorylation of ANXA2. Immunofluorescence staining and subcellular fractionations revealed a diffuse subcellular localization for the endogenous ANXA2, but phospho-Ser25 ANXA2 was mainly detected in the membrane. ANXA2 depletion lowered the basal endothelial barrier and inhibited cell migration, but had no significant effect on cell proliferation or viability. ANXA2 depleted cells failed to respond to PMA treatment, indicating an intimately involvement of phospho-ANXA2 in PKC signaling. Moreover, phosphorylation of ANXA2 disrupted its interaction with S100A10 suggesting a phosphorylation dependent multiple regulatory role of ANXA2 in endothelial cells. Our results demonstrate the pivotal role of PKC-ANXA2-PP1 pathway in endothelial cell signaling, especially in barrier function and cell migration.

Repeated exposure to microcystin-leucine-arginine potentiates excitotoxicity induced by a low dose of kainate

Microcystin-leucine-arginine (MLCR) is a cyanobacterial toxin, and has been demonstrated to cause neurotoxicity. In addition, MCLR has been identified as an inhibitor of protein phosphatase (PP)1 and PP2A, which are known to regulate the phosphorylation of various molecules related to synaptic excitability. Thus, in the present study, we examined whether MCLR exposure affects seizures induced by a low dose of kainic acid (KA; 0.05 μg, i.c.v.) administration. KA-induced seizure occurrence and seizure score significantly increased after repeated exposure to MCLR (2.5 or 5.0 μg/kg, i.p., once a day for 10 days), but not after acute MCLR exposure (2.5 or 5.0 μg/kg, i.p., 2 h and 30 min prior to KA administration), and hippocampal neuronal loss was consistently facilitated by repeated exposure to MCLR. In addition, repeated MCLR significantly elevated the membrane expression of kainate receptor GluK2 subunits, p-pan-protein kinase C (PKC), and p-extracellular signal-related kinase (ERK) at 1 h after KA. However, KA-induced membrane expression of Ca2+/calmodulin-dependent kinase II (CaMKII) was significantly reduced by repeated MCLR exposure. Consistent with the enhanced seizures and neurodegeneration, MCLR exposure significantly potentiated KA-induced oxidative stress and microglial activation, which was accompanied by increased expression of p-ERK and p-PKCδ in the hippocampus. The combined results suggest that repeated MCLR exposure potentiates KA-induced excitotoxicity in the hippocampus by increasing membrane GluK2 expression and enhancing oxidative stress and neuroinflammation through the modulation of p-CaMKII, p-PKC, and p-ERK.

Microcystin Toxicokinetics, Molecular Toxicology, and Pathophysiology in Preclinical Rodent Models and Humans.

Microcystins are ubiquitous toxins produced by photoautotrophic cyanobacteria. Human exposures to microcystins occur through the consumption of contaminated drinking water, fish and shellfish, vegetables, and algal dietary supplements and through recreational activities. Microcystin-leucine-arginine (MCLR) is the prototypical microcystin because it is reported to be the most common and toxic variant and is the only microcystin with an established tolerable daily intake of 0.04 µg/kg. Microcystin toxicokinetics is characterized by low intestinal absorption, rapid and specific distribution to the liver, moderate metabolism to glutathione and cysteinyl conjugates, and low urinary and fecal excretion. Molecular toxicology involves covalent binding to and inhibition of protein phosphatases, oxidative stress, cell death (autophagy, apoptosis, necrosis), and cytoskeleton disruption. These molecular and cellular effects are interconnected and are commonly observed together. The main target organs for microcystin toxicity are the intestine, liver, and kidney. Preclinical data indicate microcystins may also have nervous, pulmonary, cardiac, and reproductive system toxicities. Recent evidence suggests that exposure to other hepatotoxic insults could potentiate microcystin toxicity and increase the risk for chronic diseases. This review summarizes the current knowledge for microcystin toxicokinetics, molecular toxicology, and pathophysiology in preclinical rodent models and humans. More research is needed to better understand human toxicokinetics and how multifactorial exposures contribute to disease pathogenesis and progression.

Charge‐Reversal Polymer Nano‐modulators for Photodynamic Immunotherapy of Cancer

AbstractNanomedicine can regulate the balance between cytotoxic T lymphocytes (CTLs) and suppressive regulatory T lymphocytes (Tregs), which however has been rarely exploited for cancer immunotherapy. We report a charge‐reversal polymer nano‐modulator (SPDMCN) activated by tumor microenvironment (TME) for photodynamic immunotherapy of cancer. SPDMCN is constructed by conjugating an immunomodulator (demethylcantharidin, DMC) to the side chains of a photodynamic polymer via an acid‐liable linker. The negative charge of SPDMCN ensures its high stability in blood circulation and ideal tumor accumulation; exposure to acidic TME reverses its surface charge to positive, enhancing tumor penetration and locally releasing DMC. Upon near‐infrared photoirradiation, SPDMCN generates singlet oxygen to ablate tumors and promote maturation of dendritic cells. Released DMC inhibits protein phosphatase 2 (PP2A) activity and decreases Tregs differentiation. Such combinational action induces a sharp increase in CTL/Treg ratio in TME and effectively inhibits both primary and distant tumors in living mice.

Charge-Reversal Polymer Nano-modulators for Photodynamic Immunotherapy of Cancer.

Nanomedicine can regulate the balance between cytotoxic T lymphocytes (CTLs) and suppressive regulatory T lymphocytes (Tregs), which however has been rarely exploited for cancer immunotherapy. We report a charge-reversal polymer nano-modulator (SPDMC N) activated by tumor microenvironment (TME) for photodynamic immunotherapy of cancer. SPDMC N is constructed by conjugating an immunomodulator (demethylcantharidin, DMC) to the side chains of a photodynamic polymer via an acid-liable linker. The negative charge of SPDMC N ensures its high stability in blood circulation and ideal tumor accumulation; exposure to acidic TME reverses its surface charge to positive, enhancing tumor penetration and locally releasing DMC. Upon near-infrared photoirradiation, SPDMC N generates singlet oxygen to ablate tumors and promote maturation of dendritic cells. Released DMC inhibits protein phosphatase 2 (PP2A) activity and decreases Tregs differentiation. Such combinational action induces a sharp increase in CTL/Treg ratio in TME and effectively inhibits both primary and distant tumors in living mice.

Investigation of structural, electronical and in vitro cytotoxic activity properties of some heterocyclic compounds

A series of heterocyclic compounds (15) were synthesized, characterized and tested towards two human cancer cell lines for learning their in vitro antiproliferative activities. Compound 3 demonstrated the most promising activity in breast cancer cell line with a half maximal inhibitory concentration (IC50) value of 23.73 μM compared to other compounds (1, 2, 4, 5). Cytotoxic activity studies revealed that compounds 24 did not have antiproliferative activity towards liver cancer cell line. Computational methods were used to determine various quantum chemical parameters in order to identify correlations with the measured biological activity, which can assist in the molecular modeling of new heterocyclic systems. The biological activities of heterocyclic molecules against cancer cell proteins that are the crystal structure of the BRCT repeat region from the breast cancer-associated protein, ID: 1JNX, crystal structure of VEGFR kinase (liver cancer) protein, ID: 3WZE, and crystal structure of an allosteric Eya2 phosphatase inhibitor (lung cancer) protein, ID: 5ZMA, were compared. Finally, ADME/T analysis was performed for heterocyclic molecules and their future possibilities as a drug were investigated.

Protein Phosphatase 1 Regulates Human Cytomegalovirus Protein Translation by Restraining AMPK Signaling

Human cytomegalovirus (HCMV) carries the human protein phosphatase 1 (PP1) and other human proteins important for protein translation in its tegument layer for a rapid supply upon infection. However, the biological relevance behind PP1 incorporation and its role during infection is unclear. Additionally, PP1 is a difficult molecular target due to its promiscuity and similarities between the catalytic domain of multiple phosphatases. In this study, we circumvented these shortcomings by using 1E7-03, a small molecule protein–protein interaction inhibitor, as a molecular tool of noncatalytic PP1 inhibition. 1E7-03 treatment of human fibroblasts severely impaired HCMV replication and viral protein translation. More specifically, PP1 inhibition led to the deregulation of metabolic signaling pathways starting at very early time points post-infection. This effect was at least partly mediated by the prevention of AMP-activated protein kinase dephosphorylation, leading to elongation factor 2 hyperphosphorylation and reduced translation rates. These findings reveal an important mechanism of PP1 for lytic HCMV infection.

Salubrinal Mediated Fetal Hemoglobin Induction through the eIF2α-ATF4 Signaling Pathway

Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the adult β-globin gene affecting ~100,000 people in the United States and millions worldwide. The clinical symptoms of SCD include chronic hemolytic anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. An effective therapeutic intervention for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents to ameliorate clinical symptoms. Hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD, however, it is not effective in all people. Moreover, a critical barrier to the development of additional effective HbF inducers is the limited knowledge of mechanisms involved in γ-globin gene regulation. The goal of this project is to determine if Salubrinal (SAL) can be used as a therapeutic HbF inducer in SCD. Salubrinal is a selective inhibitor of protein phosphatase 1 leading to increased levels of p-eIF2α (phosphorylated eukaryotic initiation factor 2α) and a block in protein synthesis. It is known that ATF4 (activating transcription factor 4) is a downstream target of p-eIF2α under oxidant stress conditions. Previously, we identified a Gγ-globin cAMP response element (G-CRE) that binds ATF2, a known binding partner of ATF4 (Sangerman et al. Blood 2006). Furthermore, ENCODE in silico analysis showed predicted ATF4 binding sites at -822Gγ and in the second intron of the β-globin gene. Therefore, studies were conducted to determine if ATF4 is involved in the mechanism of HbF induction by SAL.Initial experiments were conducted with day 8 erythroid progenitors generated from human CD34+ stem cells isolated from healthy donors. Cells were treated with SAL (5 µM and 12 µM), HU (100 μM), and 1% DMSO (vehicle control) for 48 h; cell viability remained >90% for all drug conditions. SAL produced a 2.2-fold increase in γ-globin mRNA and a similar increase in HbF levels by western blot analysis; flow cytometry revealed a 2.1-fold increase (p<0.05) in F-cells (HbF positive cells). Subsequently, similar studies were completed in sickle erythroid progenitors generated from peripheral blood mononuclear cells where SAL (18 μM) increased γ-globin mRNA 3.2-fold and F-cells 2.5-fold (p<0.05). To gain insights into mechanisms of HbF induction, K562 cells were treated with SAL (12 µM and 18µM), Hemin (50 μM), and 1% DMSO for 48 h. Western blot analysis showed a parallel increase of HbF levels, p-eIF2α and p-ATF4 mediated by SAL. To evaluate whether SAL protects against oxidative stress, K562 cells were treated for 48 h and then 10 µM dichlorofluorescin diacetate was added to measure reactive oxygen species (ROS). SAL decreased ROS by 50% compared to DMSO controls; in contrast hemin known to mediate oxidative stress, produced a 3.7-fold increase in ROS levels.To determine if SAL activates HbF expression in vivo ,the Townes knock-in SCD mice were treated with SAL (1mg/kg) and 0.5% DMSO (vehicle control), 5 days a week for 2 weeks; Dectiabine (1.25mg/kg), a known HbF inducer, was given once a week for 2 weeks. Blood was drawn at week 0 (baseline) and week 2 after completion of treatment. The SAL treatment was well tolerated and did not cause a significant change in the complete blood count and differential or reticulocyte count. Flow cytometry analysis of peripheral blood showed that SAL produced a 4.5-fold increase in F-cells by 2 weeks of treatment compared to the DMSO treated mice (p<0.05). At the dose of Decitabine given, there was not a significant change in F-cells.In summary, our in vitro data supports HbF induction by SAL involves p-eIF2α mediated activation of ATF4. The interaction of ATF4 in the G-CRE and/or other predicted binding sites to activate γ-globin expression will be investigated. To support clinical development, studies in the SCD mouse model support the ability of SAL to induce HbF in vivo . Additional studies are underway to determine if longer treatment periods will be more effective. Defining the mechanism of HbF induction by SAL has the potential to impact treatment for SCD and other β-hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Efforts to Generate a Male Contraception Just by Prevention of Sperm Motility Acquirement in Epididymis Without Influencing the Physiology of Spermatogenesis: Yet Still a Little Far Goal

In view of the world population escalating at an alarming rate anticipated population will reach 9.8 billion by 2050. In contrast to female contraceptive measures male contraceptive methods are relatively negligible. Maximum researchers have concentrated on generation of male contraceptive methods by trying to modulate hormones but had to abandon this strategy as the pharmaceutical companies were not happy in view of marked adverse actions. Maximum nonhormonal strategies in research concentrate on spermatogenesis repression or reversible physical barriers. Sperm motility represents one of the maximum significant markers of semen quality as it is necessary for sperm motion appropriately in the female reproductive tract for it to arrive intricately towards the egg as well as fertilize it. The group of Vijayraghavan S, et al. have been concentrating on generation of a male contraceptive by utilization of targeting sperm motility at epididymal level by extensively studying the regulation of flaggelar protein motion by various subunits like Protein Phosphatase 1 (PP1), Protein Phosphatase Inhibitor-2 (PPPIR2), its phosphorylation by Glycogen Synthase Kinase-3 (GSK3), A kinase anchor protein 4(AKAP4). Recently they attempted to generate a bioportide that could simulate binding of the anchor protein AKAP4 that had been earlier revealed as a PPγ2 interactor in human sperm,that are implicated in sperm motility .Unfortunately that did not abolish sperm motility completely .This seemed to be surprising as PP1/ PPPIR2crosstalk seems to be central in regulation of sperm motility. Thus Silva et al., pointed that evaluation of the probable part of PPPIR7 as well as PPPIR11 in the manipulation of sperm motility in men might be the aim of future studies that needs to be continued to get more insight in this field.

Protein phosphatase 1: life‐course regulation by SDS22 and Inhibitor‐3

Protein phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a sizable fraction of protein Ser/Thr dephosphorylation events. It is tightly regulated in space and time through association with a wide array of regulatory interactors of protein phosphatase one (RIPPOs). Suppressor-of-Dis2-number 2 (SDS22) and Inhibitor-3 (I3), which form a ternary complex with PP1, are the first two evolved and most widely expressed RIPPOs. Their deletion causes mitotic-arrest phenotypes and is lethal in some organisms. The role of SDS22 and I3 in PP1 regulation has been a mystery for decades as they were independently identified as both activators and inhibitors of PP1. This conundrum has largely been solved by recent reports showing that SDS22 and I3 control multiple steps of the life course of PP1. Indeed, they contribute to (a) the stabilization and activation of newly translated PP1, (b) the translocation of PP1 to the nucleus, and (c) the storage of PP1 as a reserve for holoenzyme assembly. Preliminary evidence suggests that SDS22 and I3 may also function as scavengers of released or aged PP1 for re-use in holoenzyme assembly or proteolytical degradation, respectively. Hence, SDS22 and I3 are emerging as master regulators of the life course of PP1.

MAP4K4 expression in cardiomyocytes: multiple isoforms, multiple phosphorylations and interactions with striatins.

The Ser/Thr kinase MAP4K4, like other GCKIV kinases, has N-terminal kinase and C-terminal citron homology (CNH) domains. MAP4K4 can activate c-Jun N-terminal kinases (JNKs), and studies in the heart suggest it links oxidative stress to JNKs and heart failure. In other systems, MAP4K4 is regulated in striatin-interacting phosphatase and kinase (STRIPAK) complexes, in which one of three striatins tethers PP2A adjacent to a kinase to keep it dephosphorylated and inactive. Our aim was to understand how MAP4K4 is regulated in cardiomyocytes. The rat MAP4K4 gene was not properly defined. We identified the first coding exon of the rat gene using 5′-RACE, we cloned the full-length sequence and confirmed alternative-splicing of MAP4K4 in rat cardiomyocytes. We identified an additional α-helix C-terminal to the kinase domain important for kinase activity. In further studies, FLAG-MAP4K4 was expressed in HEK293 cells or cardiomyocytes. The Ser/Thr protein phosphatase inhibitor calyculin A (CalA) induced MAP4K4 hyperphosphorylation, with phosphorylation of the activation loop and extensive phosphorylation of the linker between the kinase and CNH domains. This required kinase activity. MAP4K4 associated with myosin in untreated cardiomyocytes, and this was lost with CalA-treatment. FLAG-MAP4K4 associated with all three striatins in cardiomyocytes, indicative of regulation within STRIPAK complexes and consistent with activation by CalA. Computational analysis suggested the interaction was direct and mediated via coiled-coil domains. Surprisingly, FLAG-MAP4K4 inhibited JNK activation by H2O2 in cardiomyocytes and increased myofibrillar organisation. Our data identify MAP4K4 as a STRIPAK-regulated kinase in cardiomyocytes, and suggest it regulates the cytoskeleton rather than activates JNKs.

Blocking Protein Phosphatase 1 [PP1] Prevents Loss of Tether Elasticity in Anaphase Crane-Fly Spermatocytes.

In normal anaphase cells, telomeres of each separating chromosome pair are connected to each other by tethers. Tethers are elastic at the start of anaphase: arm fragments cut from anaphase chromosomes in early anaphase move across the equator to the oppositely-moving chromosome, telomere moving toward telomere. Tethers become inelastic later in anaphase as the tethers become longer: arm fragments no longer move to their partners. When early anaphase cells are treated with Calyculin A (CalA), an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), at the end of anaphase chromosomes move backward from the poles, with telomeres moving toward partner telomeres. Experiments described herein show that in cells treated with CalA, backwards movements are stopped in a variety of ways, by cutting the tethers of backwards moving chromosomes, by severing arms of backwards moving chromosomes, by severing arms before the chromosomes reach the poles, and by cutting the telomere toward which a chromosome is moving backwards. Measurements of arm-fragment velocities show that CalA prevents tethers from becoming inelastic as they lengthen. Since treatment with CalA causes tethers to remain elastic throughout anaphase and since inhibitors of PP2A do not cause the backwards movements, PP1 activity during anaphase causes the tethers to become inelastic.

Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization

Phospholipase A1 (PLA1) hydrolyzes the fatty acids of glycerophospholipids, which are structural components of the cellular membrane. Genetic mutations in DDHD1, an intracellular PLA1, result in hereditary spastic paraplegia (HSP) in humans. However, the regulation of DDHD1 activity has not yet been elucidated in detail. In the present study, we examined the phosphorylation of DDHD1 and identified the responsible protein kinases. We performed MALDI-TOF MS/MS analysis and Phos-tag SDS-PAGE in alanine-substitution mutants in HEK293 cells and revealed multiple phosphorylation sites in human DDHD1, primarily Ser8, Ser11, Ser723, and Ser727. The treatment of cells with a protein phosphatase inhibitor induced the hyperphosphorylation of DDHD1, suggesting that multisite phosphorylation occurred not only at these major, but also at minor sites. Site-specific kinase-substrate prediction algorithms and in vitro kinase analyses indicated that cyclin-dependent kinase CDK1/cyclin A2 phosphorylated Ser8, Ser11, and Ser727 in DDHD1 with a preference for Ser11 and that CDK5/p35 also phosphorylated Ser11 and Ser727 with a preference for Ser11. In addition, casein kinase CK2α1 was found to phosphorylate Ser104, although this was not a major phosphorylation site in cultivated HEK293 cells. The evaluation of the effects of phosphorylation revealed that the phosphorylation mimic mutants S11/727E exhibit only 20% reduction in PLA1 activity. However, the phosphorylation mimics were mainly localized to focal adhesions, whereas the phosphorylation-resistant mutants S11/727A were not. This suggested that phosphorylation alters the subcellular localization of DDHD1 without greatly affecting its PLA1 activity.