Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Male sexual development in the nematode Caenorhabditis elegans requires the genes fem-1, fem-2, and fem-3. The current model of sex determination portrays the FEM proteins as components of a novel signal transduction pathway, but the mechanisms involved in signaling through the pathway are not understood. We report the isolation of fem-2 cDNAs in a yeast two-hybrid screen for clones encoding proteins that interact with FEM-3. Association of FEM-3 and FEM-2 in two independent in vitro binding assays substantiates the interaction detected in the two-hybrid system. FEM-2 is related in sequence to protein serine/threonine phosphatases of Type 2C (PP2C). We demonstrate that FEM-2 exhibits magnesium-dependent casein phosphatase activity, typical of PP2C, in vitro. Point mutations that abolish the casein phosphatase activity of FEM-2 without affecting its FEM-3-binding activity reduce severely its ability to rescue male development in fem-2 mutant nematodes. These results suggest that protein phosphorylation regulates sex determination in C. elegans.
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PMID:Caenorhabditis elegans sex-determining protein FEM-2 is a protein phosphatase that promotes male development and interacts directly with FEM-3. 882 90

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn(2+)-, but not a Ca(2+)- or a Mg(2+)-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.
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PMID:Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa. 1054 61

Myxococcus xanthus is a Gram-negative bacterium with a complex life cycle that includes vegetative swarming on rich medium and, upon starvation, aggregation to form fruiting bodies containing spores. Both of these behaviours require multiple Ser/Thr protein kinases. In this paper, we report the first Ser/Thr protein phosphatase gene, pph1, from M. xanthus. DNA sequence analysis of pph1 indicates that it encodes a protein of 254 residues (Mr = 28 308) with strong homology to eukaryotic PP2C phosphatases and that it belongs to a new group of bacterial protein phosphatases that are distinct from bacterial PP2C phosphatases such as RsbU, RsbX and SpoIIE. Recombinant His-tagged Pph1 was purified from Escherichia coli and shown to have Mn2+ or Mg2+ dependent, okadaic acid-resistant phosphatase activity on a synthetic phosphorylated peptide, RRA(pT)VA, indicating that Pph1 is a PP2C phosphatase. Pph1-expression was observed under both vegetative and developmental conditions, but peaked during early aggregation. A pph1 null mutant showed defects during late vegetative growth, swarming and glycerol spore formation. Under starvation-induced developmental conditions, the mutant showed reduced aggregation and failure to form fruiting bodies with viable spores. Using the yeast two-hybrid system, we have observed a strong interaction between Pph1 and the M. xanthus protein kinase Pkn5, a negative effector of development. These results suggest a functional link between a Pkn2-type protein kinase and a PP2C phosphatase.
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PMID:Pph1 from Myxococcus xanthus is a protein phosphatase involved in vegetative growth and development. 1129 81

Using degenerate oligonucleotide primers, we isolated the Caenorhabditis remanei orthologue of the C. elegans sex-determining phosphatase gene fem-2 as well as two other protein phosphatase homologues. Despite the significant sequence divergence between C. elegans and C. remanei FEM-2, we used RNAi-mediated gene knockdown to demonstrate that at least some aspects of male development require FEM-2 function in C. remanei. Consistent with this functional conservation, the conspecific interaction between the FEM-2 and the FEM-3 proteins observed in C. elegans also occurs in C. remanei. To further explore whether the rapid evolution of FEM-2 and FEM-3 affects their molecular interactions, we tested for cross-species interactions between the proteins from C. elegans, C. briggsae, and C. remanei. Although all FEM-2/FEM-3 pairs from a single species interact, only two out of six interspecific pairs bind each other, showing that FEM-2 and FEM-3 are coevolving. Both interspecific interactions involved C. briggsae FEM-3. We constructed chimeric versions of FEM-2 consisting of various combinations of the C. elegans and C. remanei proteins. C. briggsae FEM-3 interacted with all the chimeras, even those that did not interact with either C. elegans or C. remanei FEM-3. We hypothesize that the promiscuity of C. briggsae FEM-3 reflects an increased reliance on evolutionarily constrained regions of FEM-2 for binding. If so, our data support the notion that the coevolution of two interacting proteins sometimes involves a shift in the domains that contribute to binding.
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PMID:Conspecific and interspecific interactions between the FEM-2 and the FEM-3 sex-determining proteins despite rapid sequence divergence. 1647 23

Type 2C protein phosphatases (PP2C) represent a diversified protein phosphatase family and play various roles in cells. We previously identified and characterized a novel PP2C phosphatase encoded by the CaPTC7 gene in the human fungal pathogen Candida albicans. The CaPtc7p has 365 amino acids with a PP2C core domain at the C terminus and an additional 116-residue N-terminal sequence containing a mitochondrion-targeting sequence. Here, we show that CaPtc7p is indeed localized in the mitochondrion, the only eukaryotic PP2C phosphatase that has been directly shown to reside in the mitochondrion, suggesting its potential role in the regulation of mitochondrial physiology. Furthermore, we show that the expression of CaPTC7 at both transcriptional and protein levels is developmentally regulated during the serum-induced morphogenesis of C. albicans cells. However, disruption of the two alleles of CaPTC7 does not affect cell viability or filamentous development in C. albicans.
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PMID:Expression of CaPTC7 is developmentally regulated during serum-induced morphogenesis in the human fungal pathogen Candida albicans. 1749 72

Protein phosphorylation by protein kinases can be reversed by the action of protein phosphatases. In plants, the Ser/Thr-specific phosphatases dominate among the protein phosphatase families with the type 2C protein phosphatases (PP2Cs) being the most abundant among them. PP2Cs are monomeric enzymes that require metal cations for their activity and are insensitive to known phosphatase inhibitors. PP2Cs were shown to counteract the mitogen-activated protein kinase (MAP kinase/MAPK) activities in plants and to regulate developmental and stress signaling pathways. Studies of PP2C activities can be performed in vitro using recombinant proteins. The potential substrates of PP2Cs can be tested for dephosphorylation by the phosphatase in vitro. We have found that the stress-induced PP2Cs from alfalfa and Arabidopsis interact with stress-activated MAPKs in yeast two-hybrid (Y2H) screens. Consequently, recombinant MAPKs were employed as substrates for dephosphorylation by selected PP2Cs from different family clusters. The members of the PP2C phosphatase family demonstrated specificity toward the substrate already in vitro, supporting the notion that protein phosphatases are specific enzymes. The PP2C from Arabidopsis thaliana cluster B, Arabidopsis PP2C-type phosphatase (AP2C1), and its homolog from Medicago sativa, Medicago PP2C-type phosphatase (MP2C), were able to dephosphorylate and inactivate MAPKs, whereas the ABSCISIC ACID (ABA)-INSENSITIVE 2 (ABI2) and HOMOLOGY TO ABI1 (HAB1) PP2Cs from the distinct Arabidopsis cluster A were not able to do so. The method described here can be used for the determination of PP2C protein activity and for studying the effect of mutations introduced into their catalytic domains.
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PMID:Substrate analysis of Arabidopsis PP2C-type protein phosphatases. 2183 65

Protein phosphorylation, regulated by protein kinases and protein phosphatases, is crucial for protein structure and function in eukaryotic organisms. Type 2C protein phosphatase (PP2C) belongs to the serine/threonine phosphatase family and its activities require the presence of a divalent magnesium or manganese ion. In the present study, a potential PP2C phosphatase (SjPtc1) was identified in Schistosoma japonicum. The SjPTC1 gene was found to be highly expressed in adult worms. A recombinant SjPtc1 protein showed typical PP2C phosphatase activity. Heterologous SjPTC1 expression reversed the sensitivity of yeast ptc1 null mutants toward H₂O₂, ZnCl₂, cisplatin, and rapamycin. Collectively, the results suggest that SjPtc1 may take part in the regulation of cellular responses to oxidative stress, DNA damage stress, and the TOR (target of rapamycin) signaling pathway.
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PMID:Identification and characterization of PP2C phosphatase SjPtc1 in Schistosoma japonicum. 2918 18

Intracellular signal transduction is built on the basis of the subtle balance between phosphorylation and dephosphorylation. Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F/POPX2) and CaMKP-N (PPM1E/POPX1) are Ser/Thr phosphatases that belong to the PPM (protein phosphatase, Mg²⁺/Mn²⁺-dependent) family. The former was discovered in rat brain as a novel protein phosphatase regulating Ca²⁺/calmodulin-dependent protein kinases (CaMKs), whereas the latter was first identified in human cDNA databases using the rat CaMKP sequence. Subsequent studies have revealed that they are involved in various cellular functions through regulation of not only CaMKs but also other protein kinases such as AMP-activated protein kinase. Furthermore, accumulating evidence shows possible involvement of CaMKP and CaMKP-N in the pathogenesis of various diseases including cancer. Therefore, the biochemistry of CaMKP and CaMKP-N largely contributes to molecular medicine targeting these phosphatases. In this review, we summarized recent progress in the enzymology and biology of CaMKP and CaMKP-N. We also focused on etiology studies in which CaMKP and CaMKP-N are involved. Based on the emerging evidence, future perspectives of studies on these phosphatases and related issues to be elucidated are discussed.
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PMID:Functions and dysfunctions of Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and CaMKP-N/PPM1E. 2931 28

Chlamydiae are obligate intracellular Gram-negative bacterial pathogens that undergo an essential, but poorly understood, biphasic developmental cycle transitioning between the infectious elementary body and the replicative reticulate body. Ser/Thr/Tyr phosphorylation has been increasingly recognized for its role in regulating bacterial physiology. Chlamydia spp. encode two Hanks'-type kinases in addition to a type 2C protein phosphatase (PP2C; CppA) and appears capable of global protein phosphorylation. While these findings substantiate the importance of protein phosphorylation in Chlamydia, the physiological impact of protein phosphorylation remains enigmatic. In this study, we investigated the in vivo role of CppA by using recombinant protein point mutants and small-molecule inhibitors. Recombinant CppA (rCppA) amino acid point mutants based upon missense mutations identified in growth-deficient Chlamydia trachomatis strains exhibited reduced, but not a complete loss of, phosphatase activity toward p-nitrophenyl phosphate (pNPP) and phosphopeptides. To more directly explore the importance of CppA in chlamydial development, we implemented a chemical "knockout" approach using derivatives of 5,5'-methylenedisalicylic acid (MDSA). Several MDSA derivatives significantly reduced CppA activity in vitro and the growth of C. trachomatis L2, C. trachomatis D, and Chlamydia muridarum in a cell culture infection model. The inhibition of C. trachomatis L2 growth was more pronounced when treated at earlier infection time points, and the removal of the inhibitors after 12 h postinfection did not rescue progeny production. Our findings revealed that altered CppA activity reduces chlamydial growth and that CppA function is likely crucial for early differentiation events. Collectively, our findings further support the importance of the protein phosphorylation network in chlamydial development.IMPORTANCEChlamydia is a significant cause of disease in humans, including sexually transmitted infections, the ocular infection trachoma, and pneumonia. Despite the critical roles of protein phosphatases in bacterial physiology, their function in pathogenesis is less clear. Our findings demonstrate that CppA, a broad-specificity type 2C protein phosphatase (PP2C), is critical for chlamydial development and further substantiate reversible phosphorylation as a key regulatory mechanism in Chlamydia Additionally, our work highlights the potential of CppA to serve as a novel target for future therapeutic strategies and supports the feasibility of designing more potent PP2C phosphatase inhibitors for Chlamydia and other pathogenic bacteria.
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PMID:Inhibition of the Protein Phosphatase CppA Alters Development of Chlamydia trachomatis. 3003 48

Plants maintain a dynamic balance between plant growth and stress tolerance to optimise their fitness and ensure survival. Here, we investigated the roles of a clade A type 2C protein phosphatase (PP2C)-encoding gene, OsPP2C09, in regulating the trade-off between plant growth and drought tolerance in rice (Oryza sativa L.). The OsPP2C09 protein interacted with the core components of abscisic acid (ABA) signalling and showed PP2C phosphatase activity in vitro. OsPP2C09 positively affected plant growth but acted as a negative regulator of drought tolerance through ABA signalling. Transcript and protein levels of OsPP2C09 were rapidly induced by exogenous ABA treatments, which suppressed excessive ABA signalling and plant growth arrest. OsPP2C09 transcript levels in roots were much higher than those in shoots under normal conditions. After ABA, polyethylene glycol and dehydration treatments, the accumulation rate of OsPP2C09 transcripts in roots was more rapid and greater than that in shoots. This differential expression between the roots and shoots may increase the plant's root-to-shoot ratio under drought-stress conditions. This study sheds new light on the roles of OsPP2C09 in coordinating plant growth and drought tolerance. In particular, we propose that OsPP2C09-mediated ABA desensitisation contributes to root elongation under drought-stress conditions in rice.
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PMID:OsPP2C09, a negative regulatory factor in abscisic acid signalling, plays an essential role in balancing plant growth and drought tolerance in rice. 3243 75


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