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Reversible protein phosphorylation regulates various cellular mechanisms in eukaryotes by altering the conformation, activity, localization, and stability of substrate proteins. In Arabidopsis thaliana root meristems, histone post-translational modifications are crucial for proper cell division, and they are also involved in oxidative stress signaling. To investigate the link between reactive oxygen species (ROS) and mitosis, we treated various Arabidopsis genotypes, including wild-types and mutants showing dysfunctional PP2A, with the ROS-inducing herbicide diquat (DQ). Studying the c3c4 double catalytic subunit mutant and fass regulatory subunit mutants of PP2A provided insights into phosphorylation-dependent mitotic processes. DQ treatment reduced mitotic activity in all genotypes and caused early mitotic arrest in PP2A mutants, likely due to oxidative stress-induced damage to essential mitotic processes. DQ had a minimal effect on reversible histone H3 phosphorylation in wild-type plants but significantly decreased phospho-histone H3 levels in PP2A mutants. Following drug treatment, the phosphatase activity decreased only in the stronger phenotype mutant plants (fass-5 and c3c4). Our findings demonstrate that (i) the studied PP2A loss-of-function mutants are more sensitive to increased intracellular ROS and (ii) DQ has indirect altering effects of mitotic activities and histone H3 phosphorylation. All these findings underscore the importance of PP2A in stress responses. [ABSTRACT FROM AUTHOR]

Recently, significant advances have been achieved in gene editing of plant protein kinases and phosphatases using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. Progress in computer science and programming could significantly improve CRISPR/Cas9 gene-editing predictions and subsequent wet-laboratory experiments on protein kinases and phosphatases. Gene-edited protein kinases and phosphatases could considerably improve desirable crop traits and stress tolerance. Protein phosphorylation, the most common and essential post-translational modification, belongs to crucial regulatory mechanisms in plants, affecting their metabolism, intracellular transport, cytoarchitecture, cell division, growth, development, and interactions with the environment. Protein kinases and phosphatases, two important families of enzymes optimally regulating phosphorylation, have now become important targets for gene editing in crops. We review progress on gene-edited protein kinases and phosphatases in crops using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). We also provide guidance for computational prediction of alterations and/or changes in function, activity, and binding of protein kinases and phosphatases as consequences of CRISPR/Cas9-based gene editing with its possible application in modern crop molecular breeding towards sustainable agriculture. [ABSTRACT FROM AUTHOR]

Protein phosphatases are primarily responsible for dephosphorylation modification within signal transduction pathways. Phosphatase of regenerating liver-3 (PRL-3) is a dual-specific phosphatase implicated in cancer pathogenesis. Understanding PRL-3's intricate functions and developing targeted therapies is crucial for advancing cancer treatment. This review highlights its regulatory mechanisms, expression patterns, and multifaceted roles in cancer progression. PRL-3's involvement in proliferation, migration, invasion, metastasis, angiogenesis, and drug resistance is discussed. Regulatory mechanisms encompass transcriptional control, alternative splicing, and post-translational modifications. PRL-3 exhibits selective expressions in specific cancer types, making it a potential target for therapy. Despite advances in small molecule inhibitors, further research is needed for clinical application. PRL-3-zumab, a humanized antibody, shows promise in preclinical studies and clinical trials. Our review summarizes the current understanding of the cancer-related cellular function of PRL-3, its prognostic value, and the research progress of therapeutic inhibitors. [ABSTRACT FROM AUTHOR]

Self-organized and excitable signaling activities play important roles in a wide range of cellular functions in eukaryotic and prokaryotic cells. Cells require signaling networks to communicate amongst themselves, but also for response to environmental cues. Such signals involve complex spatial and temporal loops that may propagate as oscillations or waves. When Dictyostelium become starved for nutrients, cells within a localized space begin to secrete cAMP. Starved cells also become chemotactic to cAMP. cAMP signals propagate as outwardly moving waves that oscillate at ~6 min intervals, which creates a focused territorial region for centralized cell aggregation. Proximal cells move inwardly toward the cAMP source and relay cAMP outwardly to recruit additional cells. To ensure directed inward movement and outward cAMP relay, cells go through adapted and de-adapted states for both cAMP synthesis/degradation and for directional cell movement. Although many immediate components that regulate cAMP signaling (including receptors, G proteins, an adenylyl cyclase, phosphodiesterases, and protein kinases) are known, others are only inferred. Here, using biochemical experiments coupled with gene inactivation studies, we model an integrated large, multi-component kinetic pathway involving activation, inactivation (adaptation), re-activation (resensitization), feed-forward, and feed-back controls to generate developmental cAMP oscillations. [ABSTRACT FROM AUTHOR]

Reversible protein phosphorylation regulates various cellular mechanisms in eukaryotes by altering the conformation, activity, localization, and stability of substrate proteins. In Arabidopsis thaliana root meristems, histone post-translational modifications are crucial for proper cell division, and they are also involved in oxidative stress signaling. To investigate the link between reactive oxygen species (ROS) and mitosis, we treated various Arabidopsis genotypes, including wild-types and mutants showing dysfunctional PP2A, with the ROS-inducing herbicide diquat (DQ). Studying the c3c4 double catalytic subunit mutant and fass regulatory subunit mutants of PP2A provided insights into phosphorylation-dependent mitotic processes. DQ treatment reduced mitotic activity in all genotypes and caused early mitotic arrest in PP2A mutants, likely due to oxidative stress-induced damage to essential mitotic processes. DQ had a minimal effect on reversible histone H3 phosphorylation in wild-type plants but significantly decreased phospho-histone H3 levels in PP2A mutants. Following drug treatment, the phosphatase activity decreased only in the stronger phenotype mutant plants (fass-5 and c3c4). Our findings demonstrate that (i) the studied PP2A loss-of-function mutants are more sensitive to increased intracellular ROS and (ii) DQ has indirect altering effects of mitotic activities and histone H3 phosphorylation. All these findings underscore the importance of PP2A in stress responses.

Tumor angiogenesis, the formation of new blood vessels to support tumor growth and metastasis, is a complex process regulated by a multitude of signaling pathways. Dysregulation of signaling pathways involving protein kinases has been extensively studied, but the role of protein phosphatases in angiogenesis within the tumor microenvironment remains less explored. However, among angiogenic pathways, protein phosphatases play critical roles in modulating signaling cascades. This review provides a comprehensive overview of the involvement of protein phosphatases in tumor angiogenesis, highlighting their diverse functions and mechanisms of action. Protein phosphatases are key regulators of cellular signaling pathways by catalyzing the dephosphorylation of proteins, thereby modulating their activity and function. This review aims to assess the activity of the protein tyrosine phosphatases and serine/threonine phosphatases. These phosphatases exert their effects on angiogenic signaling pathways through various mechanisms, including direct dephosphorylation of angiogenic receptors and downstream signaling molecules. Moreover, protein phosphatases also crosstalk with other signaling pathways involved in angiogenesis, further emphasizing their significance in regulating tumor vascularization, including endothelial cell survival, sprouting, and vessel maturation. In conclusion, this review underscores the pivotal role of protein phosphatases in tumor angiogenesis and accentuate their potential as therapeutic targets for anti-angiogenic therapy in cancer.

Self-organized and excitable signaling activities play important roles in a wide range of cellular functions in eukaryotic and prokaryotic cells. Cells require signaling networks to communicate amongst themselves, but also for response to environmental cues. Such signals involve complex spatial and temporal loops that may propagate as oscillations or waves. When Dictyostelium become starved for nutrients, cells within a localized space begin to secrete cAMP. Starved cells also become chemotactic to cAMP. cAMP signals propagate as outwardly moving waves that oscillate at ∼6 min intervals, which creates a focused territorial region for centralized cell aggregation. Proximal cells move inwardly toward the cAMP source and relay cAMP outwardly to recruit additional cells. To ensure directed inward movement and outward cAMP relay, cells go through adapted and de-adapted states for both cAMP synthesis/degradation and for directional cell movement. Although many immediate components that regulate cAMP signaling (including receptors, G proteins, an adenylyl cyclase, phosphodiesterases, and protein kinases) are known, others are only inferred. Here, using biochemical experiments coupled with gene inactivation studies, we model an integrated large, multi-component kinetic pathway involving activation, inactivation (adaptation), re-activation (re-sensitization), feed-forward, and feed-back controls to generate developmental cAMP oscillations.

Background: Protein phosphatases (PP2Cs) are the main classes of protein phosphatases in plants, having pivotal functions in different plant stages and abiotic stresses. The PP2C genes are suggested to have critical roles in barley by exposure to heat and cold treatments. Objectives: We surveyed HvPP2C genes from the barley genome in the present study. Phylogenetic analysis, functional annotation, synteny analysis, chromosomal distribution, prediction of cis-elements, and gene expression of HvPP2Cs under abiotic stresses were studied. Methods: In this study, HvPP2Cs of barley were surveyed using bioinformatics tools, and HvPP2C gene expression profiles under cold and heat stresses in 3 tissues (root, leaf, and stem) were analyzed. Results: In this study, 61 HvPP2C genes were detected from barley, and a phylogenetic tree was divided into 13 subfamilies. The results of chromosomal distribution showed that the HvPP2C genes were located on 7 chromosomes. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis of HvPP2Cs showed that they were largely expressed in different tissues (root, stem, and leaf) in the Azaran and Jolge barley cultivars. In Azaran, the HvPP2C gene expression increased in response to cold stress, whereas, in the Jolge cultivar, the HvPP2C gene expression increased in response to heat stress. The analysis of synteny revealed that HvPP2C24b with HvPP2C24a, HvPP2C24 with HvPP2C24a and HvPP2C24b; HvPP2C5 with HvPP2C5a, and HvPP2C41 with HvPP2C41a were paralogous. Conclusions: Results revealed a broad understanding of the HvPP2C gene family in barley, which can be valuable for the functional description of HvPP2Cs in plant response to abiotic stresses. [ABSTRACT FROM AUTHOR]

Protein phosphatases are primarily responsible for dephosphorylation modification within signal transduction pathways. Phosphatase of regenerating liver-3 (PRL-3) is a dual-specific phosphatase implicated in cancer pathogenesis. Understanding PRL-3’s intricate functions and developing targeted therapies is crucial for advancing cancer treatment. This review highlights its regulatory mechanisms, expression patterns, and multifaceted roles in cancer progression. PRL-3’s involvement in proliferation, migration, invasion, metastasis, angiogenesis, and drug resistance is discussed. Regulatory mechanisms encompass transcriptional control, alternative splicing, and post-translational modifications. PRL-3 exhibits selective expressions in specific cancer types, making it a potential target for therapy. Despite advances in small molecule inhibitors, further research is needed for clinical application. PRL-3-zumab, a humanized antibody, shows promise in preclinical studies and clinical trials. Our review summarizes the current understanding of the cancer-related cellular function of PRL-3, its prognostic value, and the research progress of therapeutic inhibitors.

Protein phosphorylation is a universal mechanism regulating a wide range of cellular responses across all domains of life. The antagonistic activities of kinases and phosphatases can orchestrate the life cycle of an organism. The availability of bacterial genome sequences, particularly Bacillus species, followed by proteomics and functional studies have aided in the identification of putative protein kinases and protein phosphatases, and their downstream substrates. Several studies have established the role of phosphorylation in different physiological states of Bacillus species as they pass through various life stages such as sporulation, germination, and biofilm formation. The most common phosphorylation sites in Bacillus proteins are histidine, aspartate, tyrosine, serine, threonine, and arginine residues. Protein phosphorylation can alter protein activity, structural conformation, and protein–protein interactions, ultimately affecting the downstream pathways. In this review, we summarize the knowledge available in the field of Bacillus signaling, with a focus on the role of protein phosphorylation in its physiological processes. [ABSTRACT FROM AUTHOR]

Optimizing physical training regimens to increase muscle aerobic capacity requires an understanding of the internal processes that occur during exercise that initiate subsequent adaptation. During exercise, muscle cells undergo a series of metabolic events that trigger downstream signaling pathways and induce the expression of many genes in working muscle fibers. There are a number of studies that show the dependence of changes in the activity of AMP-activated protein kinase (AMPK), one of the mediators of cellular signaling pathways, on the duration and intensity of single exercises. The activity of various AMPK isoforms can change in different directions, increasing for some isoforms and decreasing for others, depending on the intensity and duration of the load. This review summarizes research data on changes in the activity of AMPK, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and other components of the signaling pathways in skeletal muscles during exercise. Based on these data, we hypothesize that the observed changes in AMPK activity may be largely related to metabolic and signaling transients rather than exercise intensity per se. Probably, the main events associated with these transients occur at the beginning of the exercise in a time window of about 1–10 min. We hypothesize that these transients may be partly due to putative trigger-like kinase/protein phosphatase interactions regulated by feedback loops. In addition, numerous dynamically changing factors, such as [Ca2+], metabolite concentration, and reactive oxygen and nitrogen species (RONS), can shift the switching thresholds and change the states of these triggers, thereby affecting the activity of kinases (in particular, AMPK and CaMKII) and phosphatases. The review considers the putative molecular mechanisms underlying trigger-like interactions. The proposed hypothesis allows for a reinterpretation of the experimental data available in the literature as well as the generation of ideas to optimize future training regimens. [ABSTRACT FROM AUTHOR]

Among the most common muscular dystrophies in adults is Myotonic Dystrophy type 1 (DM1), an autosomal dominant disorder characterized by myotonia, muscle wasting and weakness, and multisystemic dysfunctions. This disorder is caused by an abnormal expansion of the CTG triplet at the DMPK gene that, when transcribed to expanded mRNA, can lead to RNA toxic gain of function, alternative splicing impairments, and dysfunction of different signaling pathways, many regulated by protein phosphorylation. In order to deeply characterize the protein phosphorylation alterations in DM1, a systematic review was conducted through PubMed and Web of Science databases. From a total of 962 articles screened, 41 were included for qualitative analysis, where we retrieved information about total and phosphorylated levels of protein kinases, protein phosphatases, and phosphoproteins in DM1 human samples and animal and cell models. Twenty-nine kinases, 3 phosphatases, and 17 phosphoproteins were reported altered in DM1. Signaling pathways that regulate cell functions such as glucose metabolism, cell cycle, myogenesis, and apoptosis were impaired, as seen by significant alterations to pathways such as AKT/mTOR, MEK/ERK, PKC/CUGBP1, AMPK, and others in DM1 samples. This explains the complexity of DM1 and its different manifestations and symptoms, such as increased insulin resistance and cancer risk. Further studies can be done to complement and explore in detail specific pathways and how their regulation is altered in DM1, to find what key phosphorylation alterations are responsible for these manifestations, and ultimately to find therapeutic targets for future treatments. [ABSTRACT FROM AUTHOR]

Microcystins (MCs) in irrigation water could inhibit crop growth and yield. Protein phosphatases (PPs) play an important role in regulating physiological mechanisms in plants to adapt abiotic stresses. To clarify the adaptation mechanism in plants to MCs stress, we compared PPs in rice and cucumber leaves by analyzing PPs total activity, protein phosphatase-2A (PP2A) activity and expression, as well as related growth and gas exchange parameters. After 7-day exposure of MCs (5 ~ 100 µg/L) and 7-day recovery without MCs, rice showed higher tolerance to MCs by analyzing dry weight and gas exchange parameters. Both crops may regulate PPs activity to adapt MCs stress by increasing the expression of genes encoding PPs. Among them, PP2A activity in two crops showed more sensitivity to MCs than total PPs activity. In addition, the higher expressions of PP2A catalytic and regulatory subunits and lower decrease PP2A activity were observed in rice leaves compared to cucumber. All results suggest that the expression levels of PP2A subunits could play a role in maintaining the activity of PP2A to regulating plant tolerance to MCs stress. [ABSTRACT FROM AUTHOR]

Protein kinases and phosphatases catalyze the phosphorylation and dephosphorylation of their protein substrates, respectively, and these are important mechanisms in cellular signal transduction. The rice blast fungus Magnaporthe oryzae possesses 6 protein phosphatases of type 2C class, including MoPtc1, 2, 5, 6, 7 and 8. However, only very little is known about the roles of these phosphatases in filamentous fungi. Here in, we deployed genetics and molecular biology techniques to identify, characterize and establish the roles of MoPtc5 and MoPtc7 in M. oryzae development and pathogenicity. We found that during pathogen-host interaction, MoPTC7 is differentially expressed. Double deletion of MoPTC7 and MoPTC5 suppressed the fungal vegetative growth, altered its cell wall integrity and reduced its virulence. The two genes were found indispensable for stress tolerance in the phytopathogen. We also demonstrated that disruption of any of the two genes highly affected appressorium turgor generation and Mps1 and Osm1 phosphorylation levels. Lastly, we demonstrated that both MoPtc5 and MoPtc7 are localized to mitochondria of different cellular compartments in the blast fungus. Taken together, our study revealed synergistic coordination of M. oryzae development and pathogenesis by the type 2C protein phosphatases. [ABSTRACT FROM AUTHOR]

Sodium orthovanadate (Na3VO4) is an inhibitor of phosphatases that acts as a phosphate analog and is being developed as an anti-diabetes drug. Phosphatases play important roles in inflammatory signal pathways by modulating the removal of phosphate moieties of key signaling proteins. However, the role of protein phosphatases on the inflammatory response has not been fully established. In this study, we investigated how phosphatases can control the inflammatory response using Na3VO4 in LPS-stimulated RAW264.7 cells and explored the molecular mechanisms by NO assay, mRNA analysis, immunoblotting analysis, kinase assay, luciferase reporter gene assay, and mutation strategy. Na3VO4 decreased the release of nitric oxide (NO) and suppressed the expression of pro-inflammatory genes at the transcriptional level, without cytotoxicity. The translocation of nuclear factor (NF)-κB subunits into the nucleus and the level of p-IκBα were reduced by Na3VO4, as was IKKβ activity. Na3VO4 inhibited NF-κB-Luc activity under AKT1/2 and IKKβ overexpression. However, the inhibitory effect of Na3VO4 against NF-κB-Luc was not observed in the group overexpressing both AKT2 and IKKβ-M10, a mutant in which the 10 serine residues in the autophosphorylated region of the C-terminal were replaced with alanine. Na3VO4 directly decreased the activity of protein phosphatase 1α (PP1α) and protein phosphatase 2 A (PP2A) by 95%. Phosphatase inhibition by Na3VO4 also selectively suppressed AKT-IKKβ signaling by directly blocking the phosphatase activity of PP1 and PP2A, consequently down-regulating NF-κB and inflammatory gene expression. Therefore, these results suggest that vanadium compounds including Na3VO4 can be developed as anti-inflammatory drugs.

Exercise induces cardioprotection against myocardial infarction, despite obesity, by restoring pro-survival pathways and increasing resistance of mitochondrial permeability transition pore (mPTP) opening at reperfusion. Among the mechanisms involved in the inactivation of these pathways, oxysterols appear interesting. Thus, we investigated the influence of regular exercise on the reperfusion injury salvage kinase (RISK) pathway, oxysterols, and mitochondria, in the absence of ischemia-reperfusion. We also studied 7β-hydroxycholesterol (7βOH) concentration (mass spectrometry) in human lean and obese subjects. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise. Exercise significantly increased Akt phosphorylation, whereas 7βOH concentration was reduced. Moreover, exercise induced the translocation of PKCε from the cytosol to mitochondria. However, exercise did not affect the calcium concentration required to open mPTP in the mitochondria, neither in WT nor in ob/ob animals. Finally, human plasma 7βOH concentration was consistent with observations made in mice. In conclusion, regular exercise enhanced the RISK pathway by increasing kinase phosphorylation and PKCε translocation and decreasing 7βOH concentration. This activation needs the combination with stress conditions, i.e., ischemia-reperfusion, in order to inhibit mPTP opening at the onset of reperfusion. The human findings suggest 7βOH as a candidate marker for evaluating cardiovascular risk factors in obesity. [ABSTRACT FROM AUTHOR]

Plain English summary Transcription factor CREB plays an important role in the development of pulmonary hypertension (PH). However, paradoxical roles have been reported in the pathogenesis of PH, and the regulatory mechanisms of CREB activation in pulmonary artery smooth muscle cells (PASMCs) proliferation remained unknown. In this study, we showed that CRE-containing genes were overrepresented among the differentially expressed genes in experimental PH, which resulted from the sustained activation of CREB pathway. The sustained activation of CREB pathway may be associated with the activation of multiple protein kinases that positively regulate CREB and down-regulation of numerous phosphatases involved in CREB dephosphorylation. Additionally, we found that the proliferation of PAMSCs was dependent on the CREB-mediated transcriptional activity in experimental PH. Moreover, the raised intracellular labile zinc possibly from ZIP12 may be associated with reduced protein phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Collectively, we found CREB-mediated transcriptional activity in the proliferation of PASMCs in PH, which may be associated with multiple protein kinases and/or reduced phosphatases and elevated intracellular zinc. This study may reveal a critical role of zinc-mediated activation of CREB pathway in the proliferation of PASMCs, thus providing a more comprehensive understanding of CREB pathway in the pathogenesis of PH.

Optimizing physical training regimens to increase muscle aerobic capacity requires an understanding of the internal processes that occur during exercise that initiate subsequent adaptation. During exercise, muscle cells undergo a series of metabolic events that trigger downstream signaling pathways and induce the expression of many genes in working muscle fibers. There are a number of studies that show the dependence of changes in the activity of AMP-activated protein kinase (AMPK), one of the mediators of cellular signaling pathways, on the duration and intensity of single exercises. The activity of various AMPK isoforms can change in different directions, increasing for some isoforms and decreasing for others, depending on the intensity and duration of the load. This review summarizes research data on changes in the activity of AMPK, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and other components of the signaling pathways in skeletal muscles during exercise. Based on these data, we hypothesize that the observed changes in AMPK activity may be largely related to metabolic and signaling transients rather than exercise intensity per se. Probably, the main events associated with these transients occur at the beginning of the exercise in a time window of about 1–10 min. We hypothesize that these transients may be partly due to putative trigger-like kinase/protein phosphatase interactions regulated by feedback loops. In addition, numerous dynamically changing factors, such as [Ca2+], metabolite concentration, and reactive oxygen and nitrogen species (RONS), can shift the switching thresholds and change the states of these triggers, thereby affecting the activity of kinases (in particular, AMPK and CaMKII) and phosphatases. The review considers the putative molecular mechanisms underlying trigger-like interactions. The proposed hypothesis allows for a reinterpretation of the experimental data available in the literature as well as the generation of ideas to optimize future training regimens.

The cell wall provides a crucial barrier to stress imposed by the external environment. In the rice blast fungus Magnaporthe oryzae, this stress response is mediated by the cell wall integrity (CWI) pathway, consisting of a well‐characterized protein phosphorylation cascade. However, other regulators that maintain CWI phosphorylation homeostasis, such as protein phosphatases (PPases), remain unclear. Here, we identified two PPases, MoPtc1 and MoPtc2, that function as negative regulators of the CWI pathway. MoPtc1 and MoPtc2 interact with MoMkk1, one of the key components of the CWI pathway, and are crucial for the vegetative growth, conidial formation, and virulence of M. oryzae. We also demonstrate that both MoPtc1 and MoPtc2 dephosphorylate MoMkk1 in vivo and in vitro, and that CWI stress leads to enhanced interaction between MoPtc1 and MoMkk1. CWI stress abolishes the interaction between MoPtc2 and MoMkk1, providing a means of deactivation for CWI signalling. Our studies reveal that CWI signalling in M. oryzae is a highly coordinated regulatory mechanism vital for stress response and pathogenicity. [ABSTRACT FROM AUTHOR]

We recently reported pheochromocytoma 12 (PC12) cells and rats subjected to intermittent hypoxia (IH), a hallmark manifestation of obstructive sleep apnea (OSA), exhibit reduced histone deacetylase activity and HDAC5 protein. Our study further suggested that posttranslational modifications rather than transcriptional mechanism(s) mediate IH-induced HDAC5 degradation. These observations prompted our current study to investigate the mechanism(s) underlying HDAC5 degradation by IH in PC12 cell cultures. IH-induced HDAC5 degradation was blocked by an antioxidant, and reactive oxygen species (ROS) mimetics decreased HDAC5 protein, suggesting that ROS mediates HDAC5 degradation by IH. NADPH oxidases (NOX) 2 and 4 were identified as sources of ROS that mediate the effects of IH. HDAC5 degradation during IH was associated with dephosphorylation of HDAC5 at serine259, and this response was blocked by a NOX inhibitor, suggesting that ROS-dependent dephosphorylation mediates HDAC5 degradation. IH-induced dephosphorylation of HDCA5 was inhibited by calyculin A, an inhibitor of protein phosphatase (PP)-1 and -2, or by the overexpression of nuclear inhibitor of PP1 (NIPP1). HDAC5 dephosphorylation by IH lead to augmented hypoxia-inducible factor (HIF)-1α protein and an increase in its transcriptional activity. These data suggest that PP1-dependent dephosphorylation of S259 destabilizes HDAC5 protein in response to IH, resulting in HIF-1α stabilization and transcriptional activity. Our findings highlight hither to unexplored role of protein phosphatases, especially PP1 in regulating HDAC5 protein, which is an upstream activator of HIF-1 signaling by IH. [ABSTRACT FROM AUTHOR]

Seed germination is critical for early plantlet development and is tightly controlled by environmental factors. Nevertheless, the signaling networks underlying germination control remain elusive. In this study, the remodeling of Arabidopsis seed phosphoproteome during imbibition was investigated using stable isotope dimethyl labeling and nanoLC-MS/MS analysis. Freshly harvested seeds were imbibed under dark or constant light to restrict or promote germination, respectively. For each light regime, phosphoproteins were extracted and identified from dry and imbibed (6 h, 16 h, and 24 h) seeds. A large repertoire of 10,244 phosphopeptides from 2546 phosphoproteins, including 110 protein kinases and key regulators of seed germination such as Delay Of Germination 1 (DOG1), was established. Most phosphoproteins were only identified in dry seeds. Early imbibition led to a similar massive downregulation in dormant and non-dormant seeds. After 24 h, 411 phosphoproteins were specifically identified in non-dormant seeds. Gene ontology analyses revealed their involvement in RNA and protein metabolism, transport, and signaling. In addition, 489 phosphopeptides were quantified, and 234 exhibited up or downregulation during imbibition. Interaction networks and motif analyses revealed their association with potential signaling modules involved in germination control. Our study provides evidence of a major role of phosphosignaling in the regulation of Arabidopsis seed germination. [ABSTRACT FROM AUTHOR]

Zinc is essential for human growth and development. As a trace nutrient, zinc plays important roles in numerous signal transduction pathways involved in distinct physiologic or pathologic processes. Protein phosphorylation is a posttranslational modification which regulates protein activity, degradation, and interaction with other molecules. Protein kinases (PKs) and phosphatases (PPs), with their effects of adding phosphate to or removing phosphate from certain substrates, are master regulators in controlling the phosphorylation of proteins. In this review, we summarize the disturbance of zinc homeostasis and role of zinc disturbance in regulating protein kinases and protein phosphatases in neurodegenerative diseases, with the focus of that in Alzheimer's disease, providing a new perspective for understanding the mechanisms of these neurologic diseases. [ABSTRACT FROM AUTHOR]

The fact that overexpression of the yeast Ser/Thr protein phosphatase Ppz1 induces a dramatic halt in cell proliferation was known long ago, but only work in the last few years has provided insight into the molecular basis for this toxicity. Overexpression of Ppz1 causes abundant changes in gene expression and modifies the phosphorylation state of more than 150 proteins, including key signaling protein kinases such as Hog1 or Snf1. Diverse cellular processes are altered: halt in translation, failure to properly adapt to low glucose supply, acidification of the cytosol, or depletion of intracellular potassium content are a few examples. Therefore, the toxicity derived from an excess of Ppz1 appears to be multifactorial, the characteristic cell growth blockage thus arising from the combination of various altered processes. Notably, overexpression of the Ppz1 regulatory subunit Hal3 fully counteracts the toxic effects of the phosphatase, and this process involves intracellular relocation of the phosphatase to internal membranes. [ABSTRACT FROM AUTHOR]

Parasites belonging to the Apicomplexa phylum still represent a major public health and world-wide socioeconomic burden that is greatly amplified by the spread of resistances against known therapeutic drugs. Therefore, it is essential to provide the scientific and medical communities with innovative strategies specifically targeting these organisms. In this review, we present an overview of the diversity of the phosphatome as well as the variety of functions that phosphatases display throughout the Apicomplexan parasites' life cycles. We also discuss how this diversity could be used for the design of innovative and specific new drugs/therapeutic strategies. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammation undermines multiple forms of neuroplasticity. Although inflammation and its influence on plasticity in multiple neural systems has been extensively studied, its effects on plasticity of neural networks controlling vital life functions, such as breathing, are less understood. In this study, we investigated the signaling mechanisms whereby lipopolysaccharide (LPS)-induced systemic inflammation impairs plasticity within the phrenic motor system—a major spinal respiratory motor pool that drives contractions of the diaphragm muscle. Here, we tested the hypotheses that lipopolysaccharide-induced systemic inflammation (1) blocks phrenic motor plasticity by a mechanism that requires cervical spinal okadaic acid-sensitive serine/threonine protein phosphatase (PP) 1/2A activity and (2) prevents phosphorylation/activation of extracellular signal-regulated kinase 1/2 mitogen activated protein kinase (ERK1/2 MAPK)—a key enzyme necessary for the expression of phrenic motor plasticity. Methods To study phrenic motor plasticity, we utilized a well-characterized model for spinal respiratory plasticity called phrenic long-term facilitation (pLTF). pLTF is characterized by a long-lasting, progressive enhancement of inspiratory phrenic nerve motor drive following exposures to moderate acute intermittent hypoxia (mAIH). In anesthetized, vagotomized and mechanically ventilated adult Sprague Dawley rats, we examined the effect of inhibiting cervical spinal serine/threonine PP 1/2A activity on pLTF expression in sham-vehicle and LPS-treated rats. Using immunofluorescence optical density analysis, we compared mAIH-induced phosphorylation/activation of ERK 1/2 MAPK with and without LPS-induced inflammation in identified phrenic motor neurons. Results We confirmed that mAIH-induced pLTF is abolished 24 h following low-dose systemic LPS (100 μg/kg, i.p.). Cervical spinal delivery of the PP 1/2A inhibitor, okadaic acid, restored pLTF in LPS-treated rats. LPS also prevented mAIH-induced enhancement in phrenic motor neuron ERK1/2 MAPK phosphorylation. Thus, a likely target for the relevant okadaic acid-sensitive protein phosphatases is ERK1/2 MAPK or its upstream activators. Conclusions This study increases our understanding of fundamental mechanisms whereby inflammation disrupts neuroplasticity in a critical population of motor neurons necessary for breathing, and highlights key roles for serine/threonine protein phosphatases and ERK1/2 MAPK kinase in the plasticity of mammalian spinal respiratory motor circuits.

Salt‐inducible kinase 2 (SIK2) is an important regulator in various intracellular signaling pathways related to apoptosis, tumorigenesis and metastasis. However, the involvement of SIK2 in gastric tumorigenesis and the functional linkage with gastric cancer (GC) progression remain to be defined. Here, we report that SIK2 was significantly downregulated in human GC tissues, and reduced SIK2 expression was associated with poor prognosis of patients. Overexpression of SIK2 suppressed the migration and invasion of GC cells, whereas knockdown of SIK2 enhanced cell migratory and invasive capability as well as metastatic potential. These changes in the malignant phenotype resulted from the ability of SIK2 to suppress epithelial–mesenchymal transition via inhibition of AKT/GSK3β/β‐catenin signaling. The inhibitory effect of SIK2 on AKT/GSK3β/β‐catenin signaling was mediated primarily through inactivation of AKT, due to its enhanced dephosphorylation by the upregulated protein phosphatases PHLPP2 and PP2A. The upregulation of PHLPP2 and PP2A was attributable to SIK2 phosphorylation and activation of mTORC1, which inhibited autophagic degradation of these two phosphatases. These results suggest that SIK2 acts as a tumor suppressor in GC and may serve as a novel prognostic biomarker and therapeutic target for this tumor.

Abstract Cell division is orchestrated by the phosphorylation and dephosphorylation of thousands of proteins. These post-translational modifications underlie the molecular cascades converging to the activation of the universal mitotic kinase, Cdk1, and entry into cell division. They also govern the structural events that sustain the mechanics of cell division. While the role of protein kinases in mitosis has been well documented by decades of investigations, little was known regarding the control of protein phosphatases until the recent years. However, the regulation of phosphatase activities is as essential as kinases in controlling the activation of Cdk1 to enter M-phase. The regulation and the function of phosphatases result from post-translational modifications but also from the combinatorial association between conserved catalytic subunits and regulatory subunits that drive their substrate specificity, their cellular localization and their activity. It now appears that sequential dephosphorylations orchestrated by a network of phosphatase activities trigger Cdk1 activation and then order the structural events necessary for the timely execution of cell division. This review discusses a series of recent works describing the important roles played by protein phosphatases for the proper regulation of meiotic division. Many breakthroughs in the field of cell cycle research came from studies on oocyte meiotic divisions. Indeed, the meiotic division shares most of the molecular regulators with mitosis. The natural arrests of oocytes in G2 and in M-phase, the giant size of these cells, the variety of model species allowing either biochemical or imaging as well as genetics approaches explain why the process of meiosis has served as an historical model to decipher signalling pathways involved in the G2-to-M transition. The review especially highlights how the phosphatase PP2A-B55δ critically orchestrates the timing of meiosis resumption in amphibian oocytes. By opposing the kinase PKA, PP2A-B55δ controls the release of the G2 arrest through the dephosphorylation of their substrate, Arpp19. Few hours later, the inhibition of PP2A-B55δ by Arpp19 releases its opposing kinase, Cdk1, and triggers M-phase. In coordination with a variety of phosphatases and kinases, the PP2A-B55δ/Arpp19 duo therefore emerges as the key effector of the G2-to-M transition.

Among the most common muscular dystrophies in adults is Myotonic Dystrophy type 1 (DM1), an autosomal dominant disorder characterized by myotonia, muscle wasting and weakness, and multisystemic dysfunctions. This disorder is caused by an abnormal expansion of the CTG triplet at the DMPK gene that, when transcribed to expanded mRNA, can lead to RNA toxic gain of function, alternative splicing impairments, and dysfunction of different signaling pathways, many regulated by protein phosphorylation. In order to deeply characterize the protein phosphorylation alterations in DM1, a systematic review was conducted through PubMed and Web of Science databases. From a total of 962 articles screened, 41 were included for qualitative analysis, where we retrieved information about total and phosphorylated levels of protein kinases, protein phosphatases, and phosphoproteins in DM1 human samples and animal and cell models. Twenty-nine kinases, 3 phosphatases, and 17 phosphoproteins were reported altered in DM1. Signaling pathways that regulate cell functions such as glucose metabolism, cell cycle, myogenesis, and apoptosis were impaired, as seen by significant alterations to pathways such as AKT/mTOR, MEK/ERK, PKC/CUGBP1, AMPK, and others in DM1 samples. This explains the complexity of DM1 and its different manifestations and symptoms, such as increased insulin resistance and cancer risk. Further studies can be done to complement and explore in detail specific pathways and how their regulation is altered in DM1, to find what key phosphorylation alterations are responsible for these manifestations, and ultimately to find therapeutic targets for future treatments.

Protein kinases and phosphatases catalyze the phosphorylation and dephosphorylation of their protein substrates, respectively, and these are important mechanisms in cellular signal transduction. The rice blast fungus Magnaporthe oryzae possesses 6 protein phosphatases of type 2C class, including MoPtc1, 2, 5, 6, 7 and 8. However, only very little is known about the roles of these phosphatases in filamentous fungi. Here in, we deployed genetics and molecular biology techniques to identify, characterize and establish the roles of MoPtc5 and MoPtc7 in M. oryzae development and pathogenicity. We found that during pathogen-host interaction, MoPTC7 is differentially expressed. Double deletion of MoPTC7 and MoPTC5 suppressed the fungal vegetative growth, altered its cell wall integrity and reduced its virulence. The two genes were found indispensable for stress tolerance in the phytopathogen. We also demonstrated that disruption of any of the two genes highly affected appressorium turgor generation and Mps1 and Osm1 phosphorylation levels. Lastly, we demonstrated that both MoPtc5 and MoPtc7 are localized to mitochondria of different cellular compartments in the blast fungus. Taken together, our study revealed synergistic coordination of M. oryzae development and pathogenesis by the type 2C protein phosphatases.

Tumor angiogenesis, the formation of new blood vessels to support tumor growth and metastasis, is a complex process regulated by a multitude of signaling pathways. Dysregulation of signaling pathways involving protein kinases has been extensively studied, but the role of protein phosphatases in angiogenesis within the tumor microenvironment remains less explored. However, among angiogenic pathways, protein phosphatases play critical roles in modulating signaling cascades. This review provides a comprehensive overview of the involvement of protein phosphatases in tumor angiogenesis, highlighting their diverse functions and mechanisms of action. Protein phosphatases are key regulators of cellular signaling pathways by catalyzing the dephosphorylation of proteins, thereby modulating their activity and function. This review aims to assess the activity of the protein tyrosine phosphatases and serine/threonine phosphatases. These phosphatases exert their effects on angiogenic signaling pathways through various mechanisms, including direct dephosphorylation of angiogenic receptors and downstream signaling molecules. Moreover, protein phosphatases also crosstalk with other signaling pathways involved in angiogenesis, further emphasizing their significance in regulating tumor vascularization, including endothelial cell survival, sprouting, and vessel maturation. In conclusion, this review underscores the pivotal role of protein phosphatases in tumor angiogenesis and accentuate their potential as therapeutic targets for anti-angiogenic therapy in cancer.

Background: Transcription factor CREB is involved in the development of pulmonary hypertension (PH). However, little is known about the role and regulatory signaling of CREB in PH. Methods: A series of techniques, including bioinformatics methods, western blot, cell proliferation and luciferase reporter assay were used to perform a comprehensive analysis of the role and regulation of CREB in proliferation of pulmonary artery smooth muscle cells (PASMCs) in PH. Results: Using bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total- and phosphorylated-CREB in PASMCs isolated from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation analysis showed increased PASMCs proliferation in MCT-induced PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Conclusions: CREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which was associated with multiple protein kinases and/or reduced CREB phosphatases and raised intracellular zinc. Thus, this study may provide a novel insight into the CREB pathway in the pathogenesis of PH. B6vttFt3ef-LGEoxvha7Ny Video abstract Plain English summary: Transcription factor CREB plays an important role in the development of pulmonary hypertension (PH). However, paradoxical roles have been reported in the pathogenesis of PH, and the regulatory mechanisms of CREB activation in pulmonary artery smooth muscle cells (PASMCs) proliferation remained unknown. In this study, we showed that CRE-containing genes were overrepresented among the differentially expressed genes in experimental PH, which resulted from the sustained activation of CREB pathway. The sustained activation of CREB pathway may be associated with the activation of multiple protein kinases that positively regulate CREB and down-regulation of numerous phosphatases involved in CREB dephosphorylation. Additionally, we found that the proliferation of PAMSCs was dependent on the CREB-mediated transcriptional activity in experimental PH. Moreover, the raised intracellular labile zinc possibly from ZIP12 may be associated with reduced protein phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Collectively, we found CREB-mediated transcriptional activity in the proliferation of PASMCs in PH, which may be associated with multiple protein kinases and/or reduced phosphatases and elevated intracellular zinc. This study may reveal a critical role of zinc-mediated activation of CREB pathway in the proliferation of PASMCs, thus providing a more comprehensive understanding of CREB pathway in the pathogenesis of PH. [ABSTRACT FROM AUTHOR]

Exercise induces cardioprotection against myocardial infarction, despite obesity, by restoring pro-survival pathways and increasing resistance of mitochondrial permeability transition pore (mPTP) opening at reperfusion. Among the mechanisms involved in the inactivation of these pathways, oxysterols appear interesting. Thus, we investigated the influence of regular exercise on the reperfusion injury salvage kinase (RISK) pathway, oxysterols, and mitochondria, in the absence of ischemia-reperfusion. We also studied 7β-hydroxycholesterol (7βOH) concentration (mass spectrometry) in human lean and obese subjects. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise. Exercise significantly increased Akt phosphorylation, whereas 7βOH concentration was reduced. Moreover, exercise induced the translocation of PKCε from the cytosol to mitochondria. However, exercise did not affect the calcium concentration required to open mPTP in the mitochondria, neither in WT nor in ob/ob animals. Finally, human plasma 7βOH concentration was consistent with observations made in mice. In conclusion, regular exercise enhanced the RISK pathway by increasing kinase phosphorylation and PKCε translocation and decreasing 7βOH concentration. This activation needs the combination with stress conditions, i.e., ischemia-reperfusion, in order to inhibit mPTP opening at the onset of reperfusion. The human findings suggest 7βOH as a candidate marker for evaluating cardiovascular risk factors in obesity.

Reversible phospho-dephosphorylation of proteins is a major mechanism for the control of cellular functions. By large, Ser and Thr are the most frequently residues phosphorylated in eukaryotes. Removal of phosphate from these amino acids is catalyzed by a large family of well-conserved enzymes, collectively called Ser/Thr protein phosphatases. The activity of these enzymes has an enormous impact on cellular functioning. In this work we present the members of this family in S. cerevisiae and other fungal species, and review the most recent findings concerning their regulation and the roles they play in the most diverse aspects of cell biology.

Zinc is essential for human growth and development. As a trace nutrient, zinc plays important roles in numerous signal transduction pathways involved in distinct physiologic or pathologic processes. Protein phosphorylation is a posttranslational modification which regulates protein activity, degradation, and interaction with other molecules. Protein kinases (PKs) and phosphatases (PPs), with their effects of adding phosphate to or removing phosphate from certain substrates, are master regulators in controlling the phosphorylation of proteins. In this review, we summarize the disturbance of zinc homeostasis and role of zinc disturbance in regulating protein kinases and protein phosphatases in neurodegenerative diseases, with the focus of that in Alzheimer’s disease, providing a new perspective for understanding the mechanisms of these neurologic diseases.

Summary: Flux through the RAF-MEK-ERK protein kinase cascade is shaped by phosphatases acting on the core components of the pathway. Despite being an established drug target and a hub for crosstalk regulation, little is known about dephosphorylation of MEK, the central kinase within the cascade. Here, we identify PPP6C, a phosphatase frequently mutated or downregulated in melanoma, as a major MEK phosphatase in cells exhibiting oncogenic ERK pathway activation. Recruitment of MEK to PPP6C occurs through an interaction with its associated regulatory subunits. Loss of PPP6C causes hyperphosphorylation of MEK at activating and crosstalk phosphorylation sites, promoting signaling through the ERK pathway and decreasing sensitivity to MEK inhibitors. Recurrent melanoma-associated PPP6C mutations cause MEK hyperphosphorylation, suggesting that they promote disease at least in part by activating the core oncogenic pathway driving melanoma. Collectively, our studies identify a key negative regulator of ERK signaling that may influence susceptibility to targeted cancer therapies.

Introduction: Protein phosphatases (PP) and kinases are known to regulate the cell cycle dynamics. Although kinases have been studied extensively, most of the phosphatases are still unexplored. Therefore, the present study aimed to investigate the association of an isoform of PP1 family protein phosphatases 1 gamma 2 (PP1γ2) in the regulation of cervical cancer HeLa cell proliferation. Material and methods: Expression of PP1γ2 transcript and protein was assessed in the cervical cancer cell line of HeLa cells through RT-PCR and western blotting. Flow cytometry was employed to confirm its expression quantitatively, and Immuno-fluorescence was done to evaluate the distribution of PP1γ2 in the dividing mononuclear and Taxol-induced multipolar HeLa cells. PP1γ2-specific siRNA-mediated silencing was done to understand downstream pathways. The effect of the hypoxic tumour microenvironment on PP1γ2 expression was also evaluated. Results: RT-PCR and western blotting confirmed the expression of PP1γ2 in HeLa cells, and flow cytometry analysis established intracellular expression of PP1γ2. Immunofluorescence is localized PP1γ2 in the nucleus of mononuclear cells during interphase, whereas it is transiently redistributed to spindle poles throughout the cell division and localized back to the nucleus after complete karyokinesis. Taxol-induced multipolar HeLa cells also showed a temporal redistribution of PP1γ2 on the spindle poles. Hypoxic conditions upregulated PP1γ2 expression, but downregulated PP1γ2 levels through siRNA increased GSK3β phosphorylation. Conclusions: Collectively, data suggests that PP1γ2 is modulated during HeLa cell division and regulates GSK3β phosphorylation, which may regulate downstream signalling of cell division. [ABSTRACT FROM AUTHOR]

Several Candida species, the best known example of which is Candida albicans , are opportunistic human pathogens that are responsible for frequent nosocomial infections. A worrisome aspect of the currently available treatments of candidemia is the steady development of resistance to antifungals among these potentially life threatening fungi. Under these circumstances the search for novel drug targets is a well justified research direction. We propose that the principles of signal transduction therapy by targeting protein phosphatases can be adopted as these enzymes carry out important physiological functions in Candida. We demonstrate that C. tropicalis, C. albicans, C. dubliniensis , and S. cerevisiae exhibit the largest repertoire of protein phosphatases among the investigated fungi. Together with other opportunistic pathogen Candida species and the members of the Saccharomycetales order, they expanded their phosphatome by repeated gene duplications. We noted that evolution generated a set of fungus specific phosphatases which can be targeted without the danger of causing undesirable side effects in the human body. Based on the conflicting criteria of effectiveness and selectivity, we identified and characterized 7 phosphatases that are potent virulence determinants and may be utilized as potential antifungal drug targets. Image 1 • The phosphatases of 11 opportunistic pathogen Candida species were surveyed. • From 66 to 79 conserved phosphatases per species were identified. • Frequent gene duplications expanded the phosphatome of these pathogens. • 7 novel phosphatases were pinpointed as putative drug targets. [ABSTRACT FROM AUTHOR]

The fact that overexpression of the yeast Ser/Thr protein phosphatase Ppz1 induces a dramatic halt in cell proliferation was known long ago, but only work in the last few years has provided insight into the molecular basis for this toxicity. Overexpression of Ppz1 causes abundant changes in gene expression and modifies the phosphorylation state of more than 150 proteins, including key signaling protein kinases such as Hog1 or Snf1. Diverse cellular processes are altered: halt in translation, failure to properly adapt to low glucose supply, acidification of the cytosol, or depletion of intracellular potassium content are a few examples. Therefore, the toxicity derived from an excess of Ppz1 appears to be multifactorial, the characteristic cell growth blockage thus arising from the combination of various altered processes. Notably, overexpression of the Ppz1 regulatory subunit Hal3 fully counteracts the toxic effects of the phosphatase, and this process involves intracellular relocation of the phosphatase to internal membranes.

Parasites belonging to the Apicomplexa phylum still represent a major public health and world-wide socioeconomic burden that is greatly amplified by the spread of resistances against known therapeutic drugs. Therefore, it is essential to provide the scientific and medical communities with innovative strategies specifically targeting these organisms. In this review, we present an overview of the diversity of the phosphatome as well as the variety of functions that phosphatases display throughout the Apicomplexan parasites’ life cycles. We also discuss how this diversity could be used for the design of innovative and specific new drugs/therapeutic strategies.