Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

The Effect of PGAM5 on Regulating Mitochondrial Dysfunction in Ischemic Stroke.

BACKGROUND: Ischemic stroke is an acute cerebrovascular disease with high mortality rates and poor prognoses. The influence of ischemic stroke includes a heavy economic burden to patients and society, making the exploration of new therapeutic targets for preventing and treating ischemic stroke urgent. This study aimed to explore the effect of phosphoglycerate mutase family member 5 (PGAM5) on oxidative stress and mitochondrial dysfunction in ischemic stroke. METHODS: The model of ischemic neuronal brain injury was established through culturing purchased human neuroblastoma cells (SH-SY5Y) by oxygen-glucose deprivation/reoxygenation (OGD/R). There were six experimental groups, including the OGD/R model group (SH-cells of OGD/R model), OE-NC group (cells of OGD/R model transfected with scramble cDNA), OE-PGAM5 group (cells of OGD/R model transfected with full-length sequence of PGAM5), si-NC group (cells of OGD/R model transfected with negative control small interference (si)RNA), si-PGAM5 group (cells of OGD/R model transfected with siRNA for PGAM5 knockdown), and a control group (cells cultured normally). Cell counting kit-8 (CCK-8) and flow cytometry were used to determine the activity and apoptosis of cells. Subsequently, the effects of PGAM5 expression on oxidative stress and mitochondrial dysfunction were analyzed. Mitochondrial morphology was observed by transmission electron microscopy (TEM), and mitochondrial membrane potential (MMP) was determined by JC-1 fluorescent probe. The levels of reactive oxygen species (ROS) were measured by flow cytometry, and levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured by enzyme-linked immunosorbent assay (ELISA) assay. The expression of light chain (LC)3-II/I and autophagy-related gene 5 (ATG5) proteins were measured, and the regulation of PGAM5 expression on PTEN-induced putative protein kinase 1 (PINK1)/Parkin pathway was also explored. RESULTS: PGAM5 overexpression in OGD/R cells decreased the cell viability (p < 0.001) while increasing cell apoptosis (p < 0.01) compared to the OGD/R group. Inhibition of PGAM5 expression reversed the decreased cell viability (p < 0.001) and the increased cell apoptosis (p < 0.01). The JC-1 fluorescence showed that OGD/R treatment reduced mitochondrial membrane potential (p < 0.001) and TEM showed an obvious increase in phagosomes. In addition, OGD/R treatment enhanced oxidative stress (increased ROS, p < 0.01; increased MDA, p < 0.001; decreased SOD, p < 0.001), which could be further enhanced by overexpression of PGAM5 (ROS, p < 0.001; MDA, p < 0.001; SOD, p < 0.001) while reversed by the inhibition of PGAM5 (ROS, p < 0.01; MDA, p < 0.001; SOD, p < 0.001). The OGD/R-activated PINK1/Parkin pathway was inhibited by the knockdown of PGAM5 (p < 0.01) but promoted by the overexpression of PGAM5 (p < 0.05). CONCLUSIONS: PGAM5 stimulates oxidative stress and impairs mitochondrial function in ischemic stroke, and regulates the PINK1/Parkin signaling pathway. Therefore, PGAM5 is likely to be a target for the therapy of ischemic stroke.

[KEAP1/PGAM5/AIFM1 mediated oxeiptosis pathway in TDCIPP-induced reduction of TM4 cell viability].

OBJECTIVE: To investigate the toxic effects and potential mechanisms of tri(1, 3-dichloro-2-propyl) phosphate(TDCIPP) exposure on the mouse testicular supporting cell line(TM4 cells). METHODS: TM4 cells were treated with different concentrations of TDCIPP(0, 12.5, 25 and 50 µmol/L), or 50 µmol/L TDCIPP combined with antioxidant N-acetylcysteine(NAC) for 24 h. Cell viability was assessed using the CCK8 assay, intracellular ROS levels were detected using the DCFH-DA probe, and the protein levels of oxeiptosis-related proteins, such as KEAP1, PGAM5, AIFM1 and phosphorylated AIFM1(p-AIFM1), were detected using Western blot. RESULTS: TDCIPP dose-dependently reduced TM4 cell viability(P<0.05). ROS levels in TM4 cells treated with 12.5, 25 and 50 µmol/L TDCIPP were 9.44±1.42, 17.25±1.81 and 18.38±2.66, respectively, significantly higher than the control group's 5.08±0.90(P<0.05). ROS levels in the 5 mmol/L NAC+50 µmol/L TDCIPP group were 14.70±0.50, significantly lower than the corresponding TDCIPP group's 26.44±0.73(P<0.05). The activity of TM4 cells in KEAP1siRNA+TDCIPP group and PGAM5siRNA+TDCIPP group were 77.00±1.73 and 76.67±1.53, respectively, significantly higher than TDCIPP group 68.67±1.53(P<0.05). The relative expression of KEAP1 protein in TM4 cells treated with 25 and 50 µmol/L TDCIPP were 0.77±0.04 and 0.82±0.02, respectively, significantly higher than the control group's 0.57±0.01(P<0.05). The relative expression of PGAM5 protein in TDCIPP-treated TM4 cells were 1.17±0.04, 1.38±0.03 and 1.41±0.03, respectively, significantly higher than the control group's 0.81±0.02(P<0.05). The relative expression of AIFM1 protein were 0.42±0.01, 0.63±0.01 and 0.68±0.02, respectively, significantly higher than the control group's 0.34±0.02(P<0.05). The relative expression of p-AIFM1 protein were 1.73±0.02, 1.52±0.02 and 0.73±0.01, respectively, significantly lower than the control group's 2.25±0.02(P<0.05). In the 5 mmol/L NAC+50 µmol/L TDCIPP group, the relative expression of KEAP1, PGAM5 and AIFM1 proteins in TM4 cells were 0.61±0.01, 0.58±0.01 and 0.48±0.03, respectively, significantly lower than the TDCIPP group's 0.86±0.12(P<0.05), 0.74±0.02(P<0.05) and 0.92±0.01(P<0.05). The relative expression of p-AIFM1 protein in the 5 mmol/L NAC+50 µmol/L TDCIPP group was 0.45±0.11, significantly higher than the TDCIPP group's 0.23±0.01(P<0.05). CONCLUSION: The reduction of TM4 cell viability induced by TDCIPP may be related to ROS-mediated regulation of the KEAP1/PGAM5/AIFM1 pathway, leading to oxeiptosis.

CXADR promote epithelial-mesenchymal transition in endometriosis by modulating AKT/GSK-3ß signaling.

Endometriosis is a benign high prevalent disease exhibiting malignant features. However, the underlying pathogenesis and key molecules of endometriosis remain unclear. By integrating and analysis of existing expression profile datasets, we identified coxsackie and adenovirus receptor (CXADR), as a novel key gene in endometriosis. Based on the results of immunohistochemistry (IHC), we confirmed significant down-regulation of CXADR in ectopic endometrial tissues obtained from women with endometriosis compared with healthy controls. Further in vitro investigation indicated that CXADR regulated the stability and function of the phosphatases and AKT inhibitors PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2) and PTEN (phosphatase and tensin homolog). Loss of CXADR led to phosphorylation of AKT and glycogen synthase kinase-3ß (GSK-3ß), which resulted in stabilization of an epithelial-mesenchymal transition (EMT) factor, SNAIL1 (snail family transcriptional repressor 1). Therefore, EMT processs was induced, and the proliferation, migration and invasion of Ishikawa cells were enhanced. Over-expression of CXADR showed opposite effects. These findings suggest a previously undefined role of AKT/GSK-3ß signaling axis in regulating EMT and reveal the involvement of a CXADR-induced EMT, in pathogenic progression of endometriosis.

Protein phosphatase 6 regulates trametinib sensitivity, a mitogen-activated protein kinase kinase (MEK) inhibitor, by regulating MEK1/2-ERK1/2 signaling in canine melanoma cells.

Melanoma is a highly aggressive and metastatic cancer occurring in both humans and dogs. Canine melanoma accounts for a significant proportion of neoplastic diseases in dogs, and despite standard treatments, overall survival rates remain low. Protein phosphatase 6 (PP6), an evolutionarily conserved serine/threonine protein phosphatase, regulates various biological processes. Additionally, the loss of PP6 function reportedly leads to the development of melanoma in humans. However, there are no reports regarding the role of PP6 in canine cancer cells. We, therefore, conducted a study investigating the role of PP6 in canine melanoma by using four canine melanoma cell lines: CMec1, CMM, KMeC and LMeC. PP6 knockdown increased phosphorylation levels of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) but not Akt. Furthermore, PP6 knockdown decreased sensitivity to trametinib, a MEK inhibitor, but did not alter sensitivity to Akt inhibitor. These findings suggest that PP6 may function as a tumor suppressor in canine melanoma and modulate the response to trametinib treatment. Understanding the role of PP6 in canine melanoma could lead to the development of more effective treatment strategies for this aggressive disease.

Medial prefrontal cortical PPM1F alters depression-related behaviors by modifying p300 activity via the AMPK signaling pathway.

AIMS: Protein phosphatase Mg2+/Mn2+-dependent 1F (PPM1F) is a serine/threonine phosphatase, and its dysfunction in depression in the hippocampal dentate gyrus has been previously identified. Nevertheless, its role in depression of another critical emotion-controlling brain region, the medial prefrontal cortex (mPFC), remains unclear. We explored the functional relevance of PPM1F in the pathogenesis of depression. METHODS: The gene expression levels and colocalization of PPM1F in the mPFC of depressed mice were measured by real-time PCR, western blot and immunohistochemistry. An adeno-associated virus strategy was applied to determine the impact of knockdown or overexpression of PPM1F in the excitatory neurons on depression-related behaviors under basal and stress conditions in both male and female mice. The neuronal excitability, expression of p300 and AMPK phosphorylation levels in the mPFC after knockdown of PPM1F were measured by electrophysiological recordings, real-time PCR and western blot. The depression-related behavior induced by PPM1F knockdown after AMPKα2 knockout or the antidepressant activity of PPM1F overexpression after inhibiting acetylation activity of p300 was evaluated. RESULTS: Our results indicate that the expression levels of PPM1F were largely decreased in the mPFC of mice exposed to chronic unpredictable stress (CUS). Behavioral alterations relevant to depression emerged with short hairpin RNA (shRNA)-mediated genetic knockdown of PPM1F in the mPFC, while overexpression of PPM1F produced antidepressant activity and ameliorated behavioral responses to stress in CUS-exposed mice. Molecularly, PPM1F knockdown decreased the excitability of pyramidal neurons in the mPFC, and restoring this low excitability decreased the depression-related behaviors induced by PPM1F knockdown. PPM1F knockdown reduced the expression of CREB-binding protein (CBP)/E1A-associated protein (p300), a histone acetyltransferase (HAT), and induced hyperphosphorylation of AMPK, resulting in microglial activation and upregulation of proinflammatory cytokines. Conditional knockout of AMPK revealed an antidepressant phenotype, which can also block depression-related behaviors induced by PPM1F knockdown. Furthermore, inhibiting the acetylase activity of p300 abolished the beneficial effects of PPM1F elevation on CUS-induced depressive behaviors. CONCLUSION: Our findings demonstrate that PPM1F in the mPFC modulates depression-related behavioral responses by regulating the function of p300 via the AMPK signaling pathway.

The Mitogen-Activated Protein Kinase Slt2 Promotes Asymmetric Cell Cycle Arrest and Reduces TORC1-Sch9 Signaling in Yeast Lacking the Protein Phosphatase Ptc1.

Mitogen-activated protein kinase (MAPK) pathways regulate essential processes in eukaryotes. However, since uncontrolled activation of these cascades has deleterious effects, precise negative regulation of signaling flow through them, mainly executed by protein phosphatases, is crucial. Previous studies showed that the absence of Ptc1 protein phosphatase results in the upregulation of the MAPK of the cell wall integrity (CWI) pathway, Slt2, and numerous functional defects in Saccharomyces cerevisiae, including a failure to undergo cell separation under heat stress. In this study, we demonstrate that multibudded ptc1Δ cells also exhibit impaired mitochondrial inheritance and that excessive Slt2 kinase activity is responsible for their growth deficiency and daughter-specific G1 cell cycle arrest, as well as other physiological alterations, namely, mitochondrial hyperpolarization and reactive oxygen species (ROS) accumulation. We bring to light the fact that sustained Slt2 kinase activity inhibits signaling through the Sch9 branch of the TORC1 pathway in ptc1Δ cells, leading to increased autophagy. After cytokinesis, septin rings asymmetrically disassembled in ptc1Δ multibudded cells, abnormally remaining at the daughter cell side and eventually relocalizing at the daughter cell periphery, where they occasionally colocalized with the autophagic protein Atg9. Finally, we show that the inability of ptc1Δ cells to undergo cell separation is not due to a failure in the regulation of Ace2 and morphogenesis (RAM) pathway, since the transcription factor Ace2 correctly enters the daughter cell nuclei. However, the Ace2-regulated endochitinase Cts1 did not localize to the septum, preventing the proper degradation of this structure. IMPORTANCE This study provides further evidence that the cell cycle is regulated by complex signaling networks whose purpose is to guarantee a robust response to environmental threats. Using the S. cerevisiae eukaryotic model, we show that, under the stress conditions that activate the CWI MAPK pathway, the absence of the protein phosphatase Ptc1 renders Slt2 hyperactive, leading to numerous physiological alterations, including perturbed mitochondrial inheritance, oxidative stress, changes in septin dynamics, increased autophagy, TORC1-Sch9 inhibition, and ultimately cell cycle arrest and the failure of daughter cells to separate, likely due to the absence of key degradative enzymes at the septum. These results imply novel roles for the CWI pathway and unravel new cell cycle-regulatory controls that operate beyond the RAM pathway, arresting buds in G1 without compromising further division rounds in the mother cell.

MARCH1 silencing suppresses growth of oral squamous cell carcinoma through regulation of PHLPP2

PurposeOral squamous cell carcinoma (OSCC) is the most frequent type of oral cancer and is associated with high mortality. Membrane-associated ring-CH type finger 1 (MARCH1) is an E3 ubiquitin ligase with roles in immune regulation and cancer development. Whether MARCH1 has a specific role in OSCC, and if so through what mechanism, has not been explored.MethodsImmunohistochemistry was performed to examine MARCH1 expression in OSCC clinical samples and adjacent paracancerous tissues. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot were conducted to determine mRNA expression and protein levels, respectively. Knockdown and overexpression experiments were carried out to evaluate the effects of MARCH1 on proliferation and apoptosis. To test protein–protein interaction, co-immunoprecipitation assay was performed. Finally, tumor cell grafting was utilized to test the function of MARCH in vivo.ResultsHigh MARCH1 expression in OSCC clinical samples correlated with poor patient prognosis. Functionally, MARCH1 knockdown in OSCC cells suppressed proliferation and promoted apoptosis, while MARCH1 overexpression displayed the opposite effects. We identified PH Domain And Leucine Rich Repeat Protein Phosphatase (PHLPP) 2 as an important target of MARCH1. Mechanistically, MARCH1 interacted with PHLPP2 and promoted PHLPP2 ubiquitination. Lastly, MARCH1 knockdown suppressed OSCC tumorigenicity in vivo and increased PHLPP2 protein level.

Identification of potential therapeutic and diagnostic characteristics of Alzheimer disease by targeting the miR-132-3p/FOXO3a-PPM1F axis in APP/PS1 mice.

Alzheimer disease (AD) is a neurodegenerative disorder, which is characterized by progressive impairment of memory and cognition. Early diagnosis and treatment of AD has become a leading topic of research. In this study, we explored the effects of the miR-132-3p/FOXO3a-PPM1F axis on the onset of AD for possible early diagnosis and therapy. We found that miR-132-3p levels in the hippocampus and blood were drastically decreased in APP/PS1 mice from 9 months of age, and bi-directional manipulation of miR-132-3p levels induced magnified effects on learning memory behaviors, and manifestation of AD-related pathological characteristics and inflammatory cytokines in APP/PS1 mice of relevant ages. The hippocampal PPM1F expression levels were significantly elevated in APP/PS1 mice from 3 months of age, which was correlated with miR-132-3p levels at different ages. Overexpression of PPM1F remarkably accelerated the progression of learning memory deficits and associated pathological factors in APP/PS1 mice. Further, we showed that miR-132-3p modulated the expression of PPM1F via FOXO3a in HT22 cells. Finally, using peripheral blood samples of human study participants, we found that the miR-132-3p and PPM1F expression levels in patients with AD were also altered with prominent correlations. In conclusion, miR-132-3p indirectly regulates PPM1F expression by targeting FOXO3a, which could play an extensive role in contributing to the establishment of early diagnosis, treatment, and pathogenesis of AD.

Ruscogenin Attenuates Lipopolysaccharide-Induced Septic Vascular Endothelial Dysfunction by Modulating the miR-146a-5p/NRP2/SSH1 Axis.

Introduction: Endothelial dysfunction (ED) is associated with the progression of sepsis. Ruscogenin (RUS) has shown considerable efficacy in treating ED and sepsis. In the current study, the effects of RUS on sepsis-induced ED were assessed, and the mechanism was explored by focusing on the interactions of RUS with miRs. Methods: Sepsis was induced in mice and in human umbilical vein endothelial cells (HUVECs) using LPS method. Expression profile of miRs responding to sepsis was determined. Symptoms associated with sepsis and ED were examined after treatment with RUS. Changes in mouse survival, arterial structure, systemic inflammation, cell viability, apoptosis, and the miR-146a-5p/NRP2/SSH1 axis were analyzed. Results: Based on the microarray results, miR-146a-5p was selected as the therapeutic target. RUS improved survival rates and arterial structure, suppressed proinflammatory cytokines, down-regulated miR-146a-5p, and up-regulated NPR2 and SSH1 in septic mice. In HUVECs, RUS increased cell viability, suppressed apoptosis, inhibited inflammation, downregulated miR-146a-5p, and increased NRP2 and SSH1 levels. The re-induction of miR-146a-5p-5p impaired the protective effects of RUS on HUVECs. Discussion: Effects of RUS on sepsis-induced impairments in endothelium relied on the suppression of miR-146a-5p.

MARCH1 silencing suppresses growth of oral squamous cell carcinoma through regulation of PHLPP2.

PURPOSE: Oral squamous cell carcinoma (OSCC) is the most frequent type of oral cancer and is associated with high mortality. Membrane-associated ring-CH type finger 1 (MARCH1) is an E3 ubiquitin ligase with roles in immune regulation and cancer development. Whether MARCH1 has a specific role in OSCC, and if so through what mechanism, has not been explored. METHODS: Immunohistochemistry was performed to examine MARCH1 expression in OSCC clinical samples and adjacent paracancerous tissues. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot were conducted to determine mRNA expression and protein levels, respectively. Knockdown and overexpression experiments were carried out to evaluate the effects of MARCH1 on proliferation and apoptosis. To test protein-protein interaction, co-immunoprecipitation assay was performed. Finally, tumor cell grafting was utilized to test the function of MARCH in vivo. RESULTS: High MARCH1 expression in OSCC clinical samples correlated with poor patient prognosis. Functionally, MARCH1 knockdown in OSCC cells suppressed proliferation and promoted apoptosis, while MARCH1 overexpression displayed the opposite effects. We identified PH Domain And Leucine Rich Repeat Protein Phosphatase (PHLPP) 2 as an important target of MARCH1. Mechanistically, MARCH1 interacted with PHLPP2 and promoted PHLPP2 ubiquitination. Lastly, MARCH1 knockdown suppressed OSCC tumorigenicity in vivo and increased PHLPP2 protein level. CONCLUSION: Our study uncovered a function of MARCH1 in OSCC and identified PHLPP2 as an important target of MARCH1 to modulate OSCC cell proliferation and apoptosis.

Fine-Tuning of Platelet Responses by Serine/Threonine Protein Kinases and Phosphatases-Just the Beginning.

Comprehensive proteomic analyses of human and murine platelets established an extraordinary intracellular repertoire of signaling components, which control crucial functions. The spectrum of platelet serine/threonine protein kinases (more than 100) includes the AGC family (protein kinase A, G, C [PKA, PKG, PKC]), the mitogen-activated protein kinases (MAPKs), and others. PKA and PKG have multiple significantly overlapping substrates in human platelets, which possibly affect functions with clear "signaling nodes" of regulation by multiple protein kinases/phosphatases. Signaling nodes are intracellular Ca2+ stores, the contractile system (myosin light chains), and other signaling components such as G-proteins, protein kinases, and protein phosphatases. An example for this fine-tuning is the tyrosine kinase Syk, a crucial component of platelet activation, which is controlled by several serine/threonine and tyrosine protein kinases as well as phosphatases. Other protein kinases including PKA/PKG modulate protein phosphatase 2A, which may be a master regulator of MAPK signaling in human platelets. Protein kinases and in particular MAPKs are targeted by an increasing number of clinically used inhibitors. However, the precise regulation and fine-tuning of these protein kinases and their effects on other signaling components in platelets are only superficially understood-just the beginning. However, promising future approaches are in sight.

Inhibition of Heparanase Expression Results in Suppression of Invasion, Migration and Adhesion Abilities of Bladder Cancer Cells.

Heparan sulfate proteoglycan syndecan-1, CD138, is known to be associated with cell proliferation, adhesion, and migration in malignancies. We previously reported that syndecan-1 (CD138) may contribute to urothelial carcinoma cell survival and progression. We investigated the role of heparanase, an enzyme activated by syndecan-1 in human urothelial carcinoma. Using human urothelial cancer cell lines, MGH-U3 and T24, heparanase expression was reduced with siRNA and RK-682, a heparanase inhibitor, to examine changes in cell proliferation activity, induction of apoptosis, invasion ability of cells, and its relationship to autophagy. A bladder cancer development mouse model was treated with RK-682 and the bladder tissues were examined using immunohistochemical analysis for Ki-67, E-cadherin, LC3, and CD31 expressions. Heparanase inhibition suppressed cellular growth by approximately 40% and induced apoptosis. The heparanase inhibitor decreased cell activity in a concentration-dependent manner and suppressed invasion ability by 40%. Inhibition of heparanase was found to suppress autophagy. In N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer mice, treatment with heparanase inhibitor suppressed the progression of cancer by 40%, compared to controls. Immunohistochemistry analysis showed that heparanase inhibitor suppressed cell growth, and autophagy. In conclusion, heparanase suppresses apoptosis and promotes invasion and autophagy in urothelial cancer.

Centrobin plays a role in the cellular response to DNA damage.

DNA repair proteins have been found to localize to the centrosomes and defects in these proteins cause centrosome abnormality. Centrobin is a centriole-associated protein that is required for centriole duplication and microtubule stability. A recent study revealed that centrobin is a candidate substrate for ATM/ATR kinases. However, whether centrobin is involved in DNA damage response (DDR) remains unexplored. Here we show that centrobin is phosphorylated after UV exposure and that the phosphorylation is detected exclusively in the detergent/DNase I-resistant nuclear matrix. UV-induced phosphorylation of centrobin is largely dependent on ATR activity. Centrobin-depleted cells show impaired DNA damage-induced microtubule stabilization and increased sensitivity to UV radiation. Interestingly, depletion of centrobin leads to defective homologous recombination (HR) repair, which is reversed by expression of wild-type centrobin. Taken together, these results strongly suggest that centrobin plays an important role in DDR.

Is RK-682 a promiscuous enzyme inhibitor? Synthesis and in vitro evaluation of protein tyrosine phosphatase inhibition of racemic RK-682 and analogues.

RK-682 (1) is a natural product known to selectively inhibit protein tyrosine phosphatases (PTPases) and is used commercially as a positive control for phosphatase inhibition in in vitro assays. Protein phosphatases are involved in several human diseases including diabetes, cancer and inflammation, and are considered important targets for pharmaceutical development. Here we report the synthesis of racemic RK-682 (rac-1) and a focused set of compounds, including racemic analogues of 1, dihydropyranones and C-acylated Meldrum's acid derivatives, the later obtained in one synthetic step from commercially available starting material. We further characterized the behavior of some representative compounds in aqueous solution and evaluated their in vitro PTPase binding and inhibition. Our data reveal that rac-1 and some derivatives are able to form large aggregates in solution, in which the aggregation capacity is dependent on the acyl side chain size. However, compound aggregation per se is not able to promote PTPase inhibition. Our data disclose a novel family of PTPase inhibitors (C-acylated Meldrum's acid derivatives) and that rac-1 and derivatives with an exposed latent negatively charged substructure (e.g.: the tetronic acid core of 1) can bind to the PTPase binding site, as well promiscuously to protein surfaces. The combined capacity of compounds to bind to proteins together with their intrinsic capacity to aggregate in solution seems essential to promote enzyme aggregation and thus, its inhibition. We also observed that divalent cations, such as magnesium frequently used in enzyme buffer solutions, can deplete the inhibitory activity of rac-1, thus influencing the enzyme inhibition experiment. Overall, these data help to characterize the mechanism of PTPase inhibition by rac-1 and derivatives, revealing that enzyme inhibition is not solely dependent on compound binding to the PTPase catalytic site as generally accepted in the literature. In addition, our results point to promiscuous mechanisms that influence significantly the in vitro evaluation of enzyme inhibition by rac-1. Therefore, we recommend caution when using natural or synthetic RK-682 (1) as an internal control for evaluating PTPase inhibition and selectivity, since many events can modulate the apparent enzyme inhibition.

Role of cortactin in dynamic actin remodeling events in gonadotrope cells.

GnRH induces marked activation of the actin cytoskeleton in gonadotropes; however, the physiological consequences and cellular mechanisms responsible have yet to be fully elucidated. The current studies focus on the actin scaffolding protein cortactin. Using the gonadotrope-derived αT3-1 cell line, we found that cortactin is phosphorylated at Y(421), S(405), and S(418) in a time-dependent manner in response to the GnRH agonist buserelin (GnRHa). GnRHa induced translocation of cortactin to the leading edge of the plasma membrane where it colocalizes with actin and actin-related protein 3 (Arp3). Incubation of αT3-1 cells with the c-src inhibitor phosphoprotein phosphatase 1, blocked tyrosine phosphorylation of cortactin, reduced cortactin association with Arp3, and blunted actin reorganization in response to GnRHa. Additionally, we used RNA silencing strategies to knock down cortactin in αT3-1 cells. Knockdown of cortactin blocked the ability of αT3-1 cells to generate filopodia, lamellipodia, and membrane ruffles in response to GnRHa. We show that lamellipodia and filopodia are capable of LHß mobilization in primary pituitary culture after GnRHa treatment, and disruption of these structures using jasplakinolide reduces LH secretion. Collectively, our findings suggest that after GnRHa activation, src activity leads to tyrosine phosphorylation of cortactin, which facilitates its association with Arp3 to engage the actin cytoskeleton. The reorganization of actin by cortactin potentially underlies GnRHa-induced secretory events within αT3-1 cells.

Serine/threonine protein phosphatase 5 regulates glucose homeostasis in vivo and apoptosis signalling in mouse pancreatic islets and clonal MIN6 cells.

AIMS/HYPOTHESIS: During the development of type 2 diabetes mellitus, beta cells are often exposed to a high glucose/hyperlipidaemic environment, in which the levels of reactive oxygen species (ROS) are elevated. In turn, ROS can trigger an apoptotic response leading to beta cell death, by activating mitogen-activated protein kinase (MAPK) signalling cascades. Here we test the hypothesis that serine/threonine protein phosphatase 5 (PP5) acts to suppress proapoptotic c-Jun N-terminal kinase (JNK) signalling in beta cells. METHODS: Ppp5c(-/-) and Ppp5c(+/+) mice were subjected to intraperitoneal glucose (IPGTT) or insulin tolerance tests. Pancreatic islets from Ppp5c(-/-) and Ppp5c(+/+) mice or MIN6 cells treated with short-interfering RNA targeting PP5 were exposed to palmitate or H(2)O(2) to activate MAPK signalling. Changes in protein phosphorylation, mRNA expression, apoptosis and insulin secretion were detected by western blot analysis, quantitative RT-PCR or ELISA. RESULTS: Ppp5c(-/-) mice weighed less and exhibited reduced fasting glycaemia and improved glucose tolerance during IPGTT, but retained normal insulin sensitivity and islet volume. Comparison of MAPK signalling in islets from Ppp5c(-/-) mice and MIN6 cells revealed that the lack of PP5 was associated with enhanced H(2)O(2)-induced phosphorylation of JNK and c-Jun. Cells with reduced PP5 also showed enhanced JNK phosphorylation and apoptosis after palmitate treatment. PP5 suppression in MIN6 cells correlated with hypersecretion of insulin in response to glucose. CONCLUSIONS/INTERPRETATION: PP5 deficiency in mice is associated with reduced weight gain, lower fasting glycaemia, and improved glucose tolerance during IPGTT. At a molecular level, PP5 helps suppress apoptosis in beta cells by a mechanism that involves regulation of JNK phosphorylation.

Baculovirus expression system: an alternative for producing catalytically active human PTP-1B.

Protein tyrosine phosphatases (PTPs) play multiple roles in many physiological processes. Over-expression of the PTPs has been shown to be associated with cellular toxicity, which may also lead to the deletion of the respective gene from stable cell clones. We also observed that PTP-1B over-expression in CHO and HEK293 stable cell clones led to cytotoxicity and low revival rates during clone generation and maintenance. To address these issues, bacmid transposition technology was utilized to generate recombinant PTP-1B baculovirus, and Spodoptera frugiperda (Sf9 and Sf21) insect cell lines were infected with the virus. The data obtained on expression and activity of the PTP-1B highlights clear advantage of the recombinant baculovirus-insect cell expression system over the mammalian cell line technique due to increase in enzyme activity, strongly inhibited by phosphatase specific inhibitor RK682. Possible application of the expression system for producing active enzymes in bulk quantity for a new drug discovery is also discussed.

Protein phosphatase magnesium-dependent 1A-mediated inhibition of BMP signaling is independent of Smad dephosphorylation.

Phosphorylation of Smad1/5/8 at carboxyl-terminal serine residues by type I receptors activates downstream bone morphogenetic protein (BMP) signaling. Protein phosphatase magnesium-dependent 1A (PPM1A) has been shown to suppress BMP activity by dephosphorylating phospho-Smads. We report here that PPM1A suppresses BMP signaling via a novel mechanism. PPM1A inhibited a constitutively activated Smad1 mutant lacking BMP receptor phosphorylation sites. PPM1A reduced the protein levels not only of Smad1 but also of Smad5 and Smad8. A proteasome inhibitor blocked the inhibitory effects of PPM1A on Smad1, but the Smurf-binding motif in the Smad1 linker region was not involved in this inhibition. The phosphatase activity of PPM1A is essential for inhibition. Taken together, these findings suggest that through the dephosphorylation of unidentified substrate(s), PPM1A inhibits BMP signaling by decreasing Smad protein levels via the proteasome pathway. Moreover, knockdown of endogenous PPM1A stimulated osteoblastic differentiation, suggesting that PPM1A may physiologically suppress BMP signaling via Smads.

Negative regulation of multifunctional Ca2+/calmodulin-dependent protein kinases: physiological and pharmacological significance of protein phosphatases.

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) play pivotal roles in intracellular Ca2+ signaling pathways. There is growing evidence that CaMKs are involved in the pathogenic mechanisms underlying various human diseases. In this review, we begin by briefly summarizing our knowledge of the involvement of CaMKs in the pathogenesis of various diseases suggested to be caused by the dysfunction/dysregulation or aberrant expression of CaMKs. It is widely known that the activities of CaMKs are strictly regulated by protein phosphorylation/dephosphorylation of specific phosphorylation sites. Since phosphorylation status is balanced by protein kinases and protein phosphatases, the mechanism of dephosphorylation/deactivation of CaMKs, corresponding to their 'switching off', is extremely important, as is the mechanism of phosphorylation/activation corresponding to their 'switching on'. Therefore, we focus on the regulation of multifunctional CaMKs by protein phosphatases. We summarize the current understanding of negative regulation of CaMKs by protein phosphatases. We also discuss the biochemical properties and physiological significance of a protein phosphatase that we designated as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), and those of its homologue CaMKP-N. Pharmacological applications of CaMKP inhibitors are also discussed. These compounds may be useful not only for exploring the physiological functions of CaMKP/CaMKP-N, but also as novel chemotherapies for various diseases.