Putative Protein Phosphatase Research Articles

The Toxoplasma protein phosphatase 6 catalytic subunit (TgPP6C) is essential for cell cycle progression and virulence.

Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.

Giving a signal: how protein phosphorylation helps Bacillus navigate through different life stages.

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.

The Calcium Chloride Responsive Type 2C Protein Phosphatases Play Synergistic Roles in Regulating MAPK Pathways in Magnaporthe oryzae.

Reversible protein phosphorylation is essential in cellular signal transduction. The rice blast fungus Magnaporthe oryzae contains six putative type 2C protein phosphatases, namely MoPtc1, MoPtc2, MoPtc5, MoPtc6, MoPtc7, and MoPtc8. The major functions of MoPtc1 and MoPtc2 have been reported recently. In this communication, we found that MoPtc1 and MoPtc2 were induced by calcium chloride. We also found that the deletion of both MoPtc1 and MoPtc2 resulted in the overstimulation of both the high-osmolarity glycerol (Hog1) and pathogenicity MAP kinase 1 (Pmk1) pathways in M. oryzae. MoPtc1 was recruited directly to Osm1 (the osmotic stress-sensitive mutant) by the adaptor protein MoNbp2 to inactivate the Osm1 during hypoosmotic stress, distinct from the budding yeast. Moreover, we showed that MoPtc1 and MoPtc2 were localized in different cellular compartments in the fungal development. Taken together, we added some new findings of type 2C protein phosphatases MoPtc1 and MoPtc2 functions to the current knowledge on the regulation of MAPK signaling pathways in M. oryzae.

Srg1, a putative protein phosphatase from the HAD-family, is involved in stress adaptation in Candida albicans

BackgroundThe cell stress response plays an important role in the survival of organisms. Studies have revealed that the pathogenic yeast Candida albicans that constantly encounters various environmental insults inside the host has emerged as an ideal system to understand the molecular mechanism (s) of stress response. In this study, we characterize a stress-inducible gene SRG1 which is a Halo Acid Dehalogenase (HAD) family member from C. albicans. MethodsWe used confocal microscopy, site-directed mutagenesis, gene deletion techniques, and tandem-affinity purification and co-immunoprecipitation studies to functionally characterize SRG1. ResultsThe sub-cellular localization of Srg1 is predominantly cytoplasmic and includes punctate mitochondrial staining in the presence of salt. Protein purification studies coupled with LC-MS analysis showed that Srg1 is a phosphoprotein. The Srg1 mutant carrying S47A and S49A mutations failed to migrate to mitochondria in the presence of salt but retained its phosphatase activity. Srg1 migrates to the nucleus in ∆hog1 mutant cells indicating an unorthodox role for HAD family proteins in stress-mediated transcriptional response. Srg1 also interacts with Erg13, a component involved in the mitochondrial membrane lipid biosynthesis pathway. ConclusionsA multistep relay mechanism that includes a positive modulation by the MAP kinase Hog1 and a negative modulation by the global repressor Tup1 controls SRG1 expression. General significanceTaken together, our work contributes towards gaining a functional insight into a class of phosphatases that probably have evolved with novel specificities in the pathogenic yeast C. albicans to counteract stressful conditions.

The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity.

How intracellular organelles acquire their characteristic sizes is a fundamental question in cell biology. Given stereotypical changes in nuclear size in cancer, it is important to understand the mechanisms that control nuclear size in human cells. Using a high-throughput imaging RNAi screen, we identify and mechanistically characterize ELYS, a nucleoporin required for post-mitotic nuclear pore complex (NPC) assembly, as a determinant of nuclear size in mammalian cells. ELYS knockdown results in small nuclei, reduced nuclear lamin B2 localization, lower NPC density, and decreased nuclear import. Increasing nuclear import by importin α overexpression rescues nuclear size and lamin B2 import, while inhibiting importin α/β-mediated nuclear import decreases nuclear size. Conversely, ELYS overexpression increases nuclear size, enriches nuclear lamin B2 at the nuclear periphery, and elevates NPC density and nuclear import. Consistent with these observations, knockdown or inhibition of exportin 1 increases nuclear size. Thus, we identify ELYS as a novel positive effector of mammalian nuclear size and propose that nuclear size is sensitive to NPC density and nuclear import capacity.

Rice qGL3/OsPPKL1 Functions with the GSK3/SHAGGY-Like Kinase OsGSK3 to Modulate Brassinosteroid Signaling.

Brassinosteroids (BRs) are steroid hormones that play essential roles in plant growth and development. We previously cloned qGL3, a major quantitative trait locus regulating grain length in rice (Oryza sativa). The O. sativa japonica var N411 has extra-large grains compared with the O. sativa indica var 9311, and the recessive qgl3 allele from N411 contributes positively to grain length. qGL3 encodes a putative protein phosphatase with Kelch-like repeat domains, an ortholog of Arabidopsis (Arabidopsis thaliana) brassinosteroid-insensitive1 SUPPRESSOR1 (BSU1). BSU1 positively regulates BR signaling, while overexpression of qGL3 induced BR loss-of-function phenotypes. Both qGL3N411 and qGL39311 physically interact with the rice glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase 3 (OsGSK3), an ortholog of Arabidopsis BR INSENSITIVE2 (BIN2). qGL39311 dephosphorylates OsGSK3, but qGL3N411 lacks this activity. Knocking out OsGSK3 enhances BR signaling and induces nuclear localization of O. sativa BRASSINAZOLE RESISTANT1 (OsBZR1). Unlike the dephosphorylation of BIN2 (which leads to protein degradation) in Arabidopsis, qGL3 dephosphorylates and stabilizes OsGSK3 in rice. These results demonstrate that qGL3 suppresses BR signaling by regulating the phosphorylation and stability of OsGSK3, which modulates OsBZR1 phosphorylation and subcellular distribution. Our study clarifies the role of qGL3 in the regulation of grain length and provides insight into BR signaling, including the differences between rice and Arabidopsis.

The CIF1 protein is a master orchestrator of trypanosome cytokinesis that recruits several cytokinesis regulators to the cytokinesis initiation site

To proliferate, the parasitic protozoan Trypanosoma brucei undergoes binary fission in a unidirectional manner along the cell's longitudinal axis from the cell anterior toward the cell posterior. This unusual mode of cell division is controlled by a regulatory pathway composed of two evolutionarily conserved protein kinases, Polo-like kinase and Aurora B kinase, and three trypanosome-specific proteins, CIF1, CIF2, and CIF3, which act in concert at the cytokinesis initiation site located at the distal tip of the newly assembled flagellum attachment zone (FAZ). However, additional regulators that function in this cytokinesis signaling cascade remain to be identified and characterized. Using proximity biotinylation, co-immunofluorescence microscopy, and co-immunoprecipitation, we identified 52 CIF1-associated proteins and validated six CIF1-interacting proteins, including the putative protein phosphatase KPP1, the katanin p80 subunit KAT80, the cleavage furrow-localized proteins KLIF and FRW1, and the FAZ tip-localized proteins FAZ20 and FPRC. Further analyses of the functional interplay between CIF1 and its associated proteins revealed a requirement of CIF1 for localization of a set of CIF1-associated proteins, an interdependence between KPP1 and CIF1, and an essential role of katanin in the completion of cleavage furrow ingression. Together, these results suggest that CIF1 acts as a master regulator of cytokinesis in T. brucei by recruiting a cohort of cytokinesis regulatory proteins to the cytokinesis initiation site.

Flagellum inheritance in Trypanosoma brucei requires a kinetoplastid-specific protein phosphatase

Trypanosoma brucei causes sleeping sickness in humans and nagana in cattle in sub-Saharan Africa and alternates between its mammalian hosts and its insect vector, the tsetse fly. T. brucei uses a flagellum for motility, cell division, and cell-cell communication. Proper positioning and attachment of the newly assembled flagellum rely on the faithful duplication and segregation of flagellum-associated cytoskeletal structures. These processes are regulated by the polo-like kinase homolog TbPLK, whose activity and abundance are under stringent control to ensure spatiotemporally regulated phosphorylation of its substrates. However, it remains unclear whether a protein phosphatase that counteracts TbPLK activity is also involved in this regulation. Here, we report that a putative kinetoplastid-specific protein phosphatase, named KPP1, has essential roles in regulating flagellum positioning and attachment in T. brucei KPP1 localized to multiple flagellum-associated cytoskeletal structures and co-localized with TbPLK in several cytoskeletal structures at different cell-cycle stages. KPP1 depletion abolished basal body segregation, inhibited the duplication of the centrin arm and the hook complex of the bilobe structure, and disrupted the elongation of the flagellum attachment zone, leading to flagellum misplacement and detachment and cytokinesis arrest. Importantly, KPP1-depleted cells lacked dephosphorylation of TbCentrin2, a TbPLK substrate, at late cell-cycle stages. Together, these results suggest that KPP1-mediated protein dephosphorylation regulates the duplication and segregation of flagellum-associated cytoskeletal structures, thereby promoting flagellum positioning and attachment. These findings highlight the requirement of reversible protein phosphorylation, mediated by TbPLK and KPP1, in regulating flagellum inheritance in T. brucei.

Search for Partner Proteins of A. thaliana Immunophilins Involved in the Control of Plant Immunity

The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes (AtCYP19-1/ROC3, AtFKBP65/ROF2, and AtCYP57) in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H) system for protein–protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1) and putative protein phosphatase (At2g30020; АТ2), whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1) and RmlC-like cupin (At5g39120). The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1) and clathrin adaptor complex-related protein (At5g05010). Identified interactors confirm our previous findings that immunophilins ROC3, ROF2, and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.

The Phosphatome of Medicinal and Edible Fungus Wolfiporia cocos.

Wolfiporia cocos is an important medicinal and edible fungus that grows in association with pine trees, and its dried sclerotium has been used as a traditional medicine in China for centuries. However, the commercial production of W. cocos sclerotia is currently limited by shortages in pine wood resources. Since protein phosphatases (PPs) play significant roles in growth, signal transduction, development, metabolism, sexual reproduction, cell cycle, and environmental stress responses in fungi, the phosphatome of W. cocos was analyzed in this study by identifying PP genes, studying transcript profiles and assigning PPs to orthologous groups. Fifty-four putative PP genes were putatively identified in W. cocos genome based on homologous sequences searching using BLASTx program against the Saccharomyces cerevisiae, Fusarium graminearum, and Sclerotinia sclerotiorum databases. Based on known and presumed functions of orthologues of these PP genes found in other fungi, the putative roles of these W. cocos PPs in colonization, hyphal growth, sclerotial formation, secondary metabolism, and stress tolerance to environment were discussed in this study. And the level of transcripts from PP genes in the mycelium and sclerotium stages was also analyzed by qRT-PCR. Our study firstly identified and functional discussed the phosphatome in the medicinal and edible fungus W. cocos. The data from our study contribute to a better understanding of PPs potential roles in various cellar processes of W. cocos, and systematically provide comprehensive and novel insights into W. cocos economically important traits that could be extended to other fungi.

Phosphorylation-mediated Regulatory Networks in Mycelia of Pyricularia oryzae Revealed by Phosphoproteomic Analyses

Protein phosphorylation is known to regulate pathogenesis, mycelial growth, conidiation and stress response in Pyricularia oryzae However, phosphorylation mediated regulatory networks in the fungal pathogen remain largely to be uncovered. In this study, we identified 1621 phosphorylation sites of 799 proteins in mycelia of P. oryzae, including 899 new p-sites of 536 proteins and 47 new p-sites of 31 pathogenicity-related proteins. From the sequences flanking the phosphorylation sites, 19 conserved phosphorylation motifs were identified. Notably, phosphorylation was detected in 7 proteins that function upstream of Pmk1, but not in Pmk1 and its downstream Mst12 and Sfl1 that have been known to regulate appressorium formation and infection hyphal growth of P. oryzae Interestingly, phosphorylation was detected at the site Ser240 of Pmp1, which is a putative protein phosphatase highly conserved in filamentous fungi but not characterized. We thus generated Δpmp1 deletion mutants and dominant allele PMP1S240D mutants. Phenotyping analyses indicated that Pmp1 is required for virulence, conidiation and mycelial growth. Further, we observed that phosphorylation level of Pmk1 in mycelia was significantly increased in the Δpmp1 mutant, but decreased in the PMP1S240D mutant in comparison with the wild type, demonstrating that Pmp1 phosphorylated at Ser240 is important for regulating phosphorylation of Pmk1. To our surprise, phosphorylation of Mps1, another MAP kinase required for cell wall integrity and appressorium formation of P. oryzae, was also significantly enhanced in the Δpmp1 mutant, but decreased in the PMP1S240D mutant. In addition, we found that Pmp1 directly interacts with Mps1 and the region AA180-230 of Pmp1 is required for the interaction. In summary, this study sheds new lights on the protein phosphorylation mediated regulatory networks in P. oryzae.

The Putative Protein Phosphatase MoYvh1 Functions Upstream of MoPdeH to Regulate the Development and Pathogenicity in Magnaporthe oryzae.

Protein phosphatases are critical regulators in eukaryotic cells. For example, the budding yeast Saccharomyces cerevisiae dual specificity protein phosphatase (DSP) ScYvh1 regulates growth, sporulation, and glycogen accumulation. Despite such importance, functions of Yvh1 proteins in filamentous fungi are not well understood. In this study, we characterized putative protein phosphatase MoYvh1, an Yvh1 homolog in the rice blast fungus Magnaporthe oryzae. Deletion of the MoYVH1 gene resulted in significant reductions in vegetative growth, conidial production, and virulence. The ΔMoyvh1 mutant also displayed defects in cell-wall integrity and was hyposensitive to the exogenous osmotic stress. Further examination revealed that the ΔMoyvh1 mutant had defects in appressorium function and invasive hyphae growth, resulting attenuated pathogenicity. Interestingly, we found that MoYvh1 affects the scavenging of host-derived reactive oxygen species that promotes M. oryzae infection. Finally, overexpression of the phosphodiesterase MoPDEH suppressed the defects in conidia formation and pathogenicity of the ΔMoyvh1 mutant, suggesting MoYvh1 could regulate MoPDEH for its function. Our study reveals not only the importance of MoYvh1 proteins in growth, differentiation, and virulence of the rice blast fungus but, also, a genetic link between MoYvh1 and MoPDEH-cAMP signaling in this fungus.

Towards understanding peroxisomal phosphoregulation in Arabidopsis thaliana.

This work identifies new protein phosphatases and phosphatase-related proteins targeting peroxisomes, and raises the question of a novel protein import pathway from ER to peroxisomes involving peroxisomal targeting signal type 1 (PTS1) Plant peroxisomes are essential for several processes, for example lipid metabolism, free radical detoxification, development, and stress-related functions. Although research on peroxisomes has been intensified, reversible phosphorylation as a control mechanism in peroxisomes is barely studied. Therefore, it is crucial to identify all peroxisomal proteins involved in phosphoregulation. We here started with protein phosphatases, and searched the Arabidopsis thaliana genome for phosphatase-related proteins with putative peroxisomal targeting signals (PTS). Five potential peroxisomal candidates were detected, from which four were confirmed to target peroxisomes or have a functional PTS. The highly conserved Ser-Ser-Met> was validated for two protein phosphatase 2C (PP2C) family members (POL like phosphatases, PLL2 and PLL3) as a functional peroxisomal targeting signal type 1 (PTS1). Full-length PLL2 and PLL3 fused with a reporter protein targeted peroxisomes in two plant expression systems. A putative protein phosphatase, purple acid phosphatase 7 (PAP7), was found to be dually targeted to ER and peroxisomes and experiments indicated a possible trafficking to peroxisomes via the ER depending on peroxisomal PTS1. In addition, a protein phosphatase 2A regulator (TIP41) was validated to harbor a functional PTS1 (Ser-Lys-Val>), but the full-length protein targeted cytosol and nucleus. Reverse genetics indicated a role for TIP41 in senescence signaling. Mass spectrometry of whole seedlings and isolated peroxisomes was employed, and identified new putative phosphorylated peroxisomal proteins. Previously, only one protein phosphatase, belonging to the phospho-protein phosphatase (PPP) family, was identified as a peroxisomal protein. The present work implies that members of two other main protein phosphatase families, i.e. PP2C and PAP, are also targeting peroxisomes.

BRI2 and BRI3 are functionally distinct phosphoproteins

Three BRI protein family members have been identified. Among these are BRI3 and BRI2, the latter is associated with Familial Danish and Familial British dementias. ‘In silico’ sequence analysis identified putative PP1 binding sites in BRI2 and BRI3. This is singularly important, given that protein phosphorylation is a major mechanism regulating intracellular processes. Protein phosphatase 1 (PP1) interacting proteins (PIPs) are fundamental in determining substrate specificity and subcellular localization of this phosphatase. More than 200 PIPs have thus far been reported.Both BRI2 and BRI3 are type II transmembrane glycoproteins relevant in neuronal systems. Using Myc-BRI2 and Myc-BRI3, wild type and PP1 binding mutant constructs, it was possible to show, for the first time, that in fact BRI2 and BRI3 bind PP1. The complexes BRI2:PP1 and BRI3:PP1 were validated in vitro and in vivo. The subcellular distribution of BRI2 and BRI3 is similar; both localize to the perinuclear area and Golgi apparatus in non-neuronal cells. However, in SH-SY5Y cells, BRI2 and BRI3 could also be detected in elongated cellular projections (‘processes’) and in rat cortical neurons both are broadly distributed throughout the cell body, neuritis and the nucleus. Consistently, co-localization of BRI2 and BRI3 with PP1 was evident. The functional significance of these complexes is apparent given that both BRI proteins are substrates of PP1, thus simultaneously this is the first report of BRI2 and BRI3 as phosphoproteins. Moreover, we show that when BRI2 is phosphorylated a significant increase in neuronal outgrowth and differentiation is evident.Interestingly, the Alzheimer's amyloid precursor protein (APP), forms a trimeric complex composed of PP1 and Fe65, with PP1 having the capacity to dephosphorylate APP at Thr668 residue. The emerging consensus appears to be that PP1 containing complexes are crucial in regulating signaling events underlying neuropathological conditions.

IBR5 Modulates Temperature-Dependent, R Protein CHS3-Mediated Defense Responses in Arabidopsis.

Plant responses to low temperature are tightly associated with defense responses. We previously characterized the chilling-sensitive mutant chs3-1 resulting from the activation of the Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR)-type resistance (R) protein harboring a C-terminal LIM (Lin-11, Isl-1 and Mec-3 domains) domain. Here we report the identification of a suppressor of chs3, ibr5-7 (indole-3-butyric acid response 5), which largely suppresses chilling-activated defense responses. IBR5 encodes a putative dual-specificity protein phosphatase. The accumulation of CHS3 protein at chilling temperatures is inhibited by the IBR5 mutation. Moreover, chs3-conferred defense phenotypes were synergistically suppressed by mutations in HSP90 and IBR5. Further analysis showed that IBR5, with holdase activity, physically associates with CHS3, HSP90 and SGT1b (Suppressor of the G2 allele of skp1) to form a complex that protects CHS3. In addition to the positive role of IBR5 in regulating CHS3, IBR5 is also involved in defense responses mediated by R genes, including SNC1 (Suppressor of npr1-1, Constitutive 1), RPS4 (Resistance to P. syringae 4) and RPM1 (Resistance to Pseudomonas syringae pv. maculicola 1). Thus, the results of the present study reveal a role for IBR5 in the regulation of multiple R protein-mediated defense responses.

PP1 Anchoring onto NCX1 Facilitates Dephosphorylation of P-SER68-PLM

Introduction: The cardiac Na+/Ca2+ exchanger 1 (NCX1) modulates excitation-contraction coupling and contributes to Ca2+ removal in cardiomyocytes. Altered expression and activity of NCX1 is linked to dysfunctional Ca2+ handling in chronic heart disease. Consequently, modulation of NCX1 activity is proposed as a therapeutic target. The cytosolic loop of NCX1, ∼550 amino acids, comprises several important docking and regulation sites. In particular, phospholemman (PLM) has been shown to interact and inhibit NCX1 activity when phosphorylated at serine 68 (pSer68-PLM). Importantly, pSer68-PLM has been shown as a substrate for protein phosphatase 1 (PP1) in the context of PLM regulation of the sodium-potassium pump. PP1 regulation of NCX1 is unknown.Hypothesis: PP1 binds to NCX1 and regulates its activity by dephosphorylating pSer68-PLM.Methods and Results: Using co-immunoprecipitation in rat heart lysates we have shown that NCX1 exists in a macromolecular complex with PP1 and PLM. This facilitates specific control of NCX function. Bioinformatic analysis revealed three putative PP1 binding sites on NCX1. Co-localization studies, co-immunoprecipitations, pull down, mutation- and peptide overlay assays indicated that PP1 bound directly to the consensus sequence R/KVxF in calcium binding domain 1 (CBD1) of NCX1. The reciprocal NCX1 binding site in PP1 was identified within residues 235-260, a region which harbours important anchoring sites. A peptide docking model was generated showing how the R/KVxF peptide may bind in the hydrophobic pocket of PP1. Surface plasmon resonance analysis indicated that the NCX1-PP1 binding is strong and stable. This binding does not inhibit PP1 activity. Co-expression of NCX1 with PLM and PP1 in HEK293 down regulates pSer68-PLM, indicating that PP1-R/KVxF- binding is a prerequisite for dephosphorylation of pSer68-PLM.Conclusion: R/KVxF motif in NCX1-CBD1 anchors PP1, does not change the activity of the enzyme and facilitates dephosphorylation of pSer68-PLM.

The unique serine/threonine phosphatase from the minimal bacterium Mycoplasma synoviae: biochemical characterization and metal dependence.

Serine/threonine protein phosphatases have been described in many pathogenic bacteria as essential enzymes involved in phosphorylation-dependent signal transduction pathways and frequently associated with the virulence of these organisms. An inspection of Mycoplasma synoviae genome revealed the presence of a gene (prpC) encoding a putative protein phosphatase of the protein phosphatase 2C (PP2C) subfamily. Here, we report a complete biochemical characterization of M. synoviae phosphatase (PrpC) and the particular role of metal ions in the structure-function relationship of this enzyme. PrpC amino acid sequence analysis revealed that all the residues involved in the dinuclear metal center and the putative third metal ion-coordinating residues, conserved in PP2C phosphatases, are present in PrpC. PrpC is a monomeric protein able to dephosphorylate phospho-substrates with Mn(2+) ions' dependence. Thermal stability analysis demonstrated the enzyme stability at mild temperatures and the influence of Mn(2+) ions in this property. Mass spectrometry analysis suggested that three metal ions bind to PrpC, two of which with an apparent high-affinity constant. Mutational analysis of the putative third metal-coordinating residues, Asp122 and Arg164, revealed that these variants exhibited a weaker binding of manganese ions, and that both mutations affected PrpC phosphatase activity. According to these results, PrpC is a metal-dependent protein phosphatase member with an improved stability in the holo form and with Asp122, possibly implicated in the third metal-binding site, essential to catalytic activity.

Ppg1, a PP2A-type protein phosphatase, controls filament extension and virulence in Candida albicans.

Candida albicans, a major human fungal pathogen, is the primary cause of invasive candidiasis in a wide array of immunocompromised patients. C. albicans virulence requires the ability to undergo a reversible morphological transition from yeast to filaments in response to a variety of host environmental cues. These cues are sensed by the pathogen and activate multiple signal transduction pathways to induce filamentation. Reversible phosphorylation events are critical for regulation of many of these pathways. While a variety of protein kinases are known to function as components of C. albicans filamentous growth signal transduction pathways, considerably little is known about the role of phosphatases. Here we demonstrate that PPG1, encoding a putative type 2A-related protein phosphatase, is important for C. albicans filament extension, invasion, and virulence in a mouse model of systemic candidiasis. PPG1 is also important for downregulation of NRG1, a key transcriptional repressor of C. albicans filamentous growth, and is shown to affect the expression of several filament-specific target genes. An epistasis analysis suggests that PPG1 controls C. albicans filamentation via the cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway. We demonstrate that Ppg1 possesses phosphatase activity and that a ppg1 catalytic mutant shows nearly equivalent filamentation, invasion, and virulence defects compared to those of a ppg1Δ/Δ strain. Overall, our results suggest that phosphatases, such as Ppg1, play critical roles in controlling and fine-tuning C. albicans filament extension and virulence as well as signal transduction pathways, transcriptional regulators, and target genes associated with these processes.

Function of arsATorf7orf8 of Bacillus sp. CDB3 in arsenic resistance

Bacillus sp. CDB3 isolated from an arsenic contaminated cattle dip site possesses an uncommon arsenic resistance (ars) operon bearing eight genes in the order of arsRYCDATorf7orf8. We investigated the functions of arsA, arsT, orf7 and orf8 in arsenic resistance using a plasmid-based gene knockout approach in the ars gene deficient Escherichia coli strain AW3110. The CDB3 arsA gene was shown to play a significant role in resistance, suggesting that the encoded ArsA may couple with the arsenite transporter, forming an ArsAY complex that can enhance arsenite extrusion efficiency. The disruption of either arsT or orf7 was not observed to affect arsenic resistance in the heterologous E. coli host, but their involvement in arsenic resistance can not be excluded. The orf8 gene is predicted to encode a putative dual-specificity protein phosphatase which also shares certain homology to arsenate reductases. The function loss of orf8 resulted in a remarkable decrease in resistance to arsenate, though not arsenite. To examine if this effect was due to the reduction of arsenate by orf8, the arsC gene within the 8-gene operon was disrupted. The resulting abolishment of arsenate resistance suggests that the involvement of orf8 in arsenic resistance is not via reductase activity.

Atypical Mitogen-Activated Protein Kinase Phosphatase Implicated in Regulating Transition from Pre-S-Phase Asexual Intraerythrocytic Development of Plasmodium falciparum

Intraerythrocytic development of the human malaria parasite Plasmodium falciparum appears as a continuous flow through growth and proliferation. To develop a greater understanding of the critical regulatory events, we utilized piggyBac insertional mutagenesis to randomly disrupt genes. Screening a collection of piggyBac mutants for slow growth, we isolated the attenuated parasite C9, which carried a single insertion disrupting the open reading frame (ORF) of PF3D7_1305500. This gene encodes a protein structurally similar to a mitogen-activated protein kinase (MAPK) phosphatase, except for two notable characteristics that alter the signature motif of the dual-specificity phosphatase domain, suggesting that it may be a low-activity phosphatase or pseudophosphatase. C9 parasites demonstrated a significantly lower growth rate with delayed entry into the S/M phase of the cell cycle, which follows the stage of maximum PF3D7_1305500 expression in intact parasites. Genetic complementation with the full-length PF3D7_1305500 rescued the wild-type phenotype of C9, validating the importance of the putative protein phosphatase PF3D7_1305500 as a regulator of pre-S-phase cell cycle progression in P. falciparum.