Regulation of the osmoregulatory HOG MAPK cascade in yeast

The budding yeast Saccharomyces cerevisiae has at least five signal pathways containing a MAP kinase (MAPK) cascade. The high osmolarity glycerol (HOG) MAPK pathway is essential for yeast survival in high osmolarity environment. This mini-review surveys recent developments in regulation of the HOG pathway with specific emphasis on the roles of protein phosphatases and protein subcellular localization. The Hog1 MAPK in the HOG pathway is negatively regulated jointly by the protein tyrosine phosphatases Ptp2/Ptp3 and the type 2 protein phosphatases Ptc1/Ptc2/Ptc3. Specificities of these phosphatases are determined by docking interactions as well as their cellular localizations. The subcellular localizations of the osmosensors (Sln1 and Sho1), kinases (Pbs2, Hog1), and phosphatases in the HOG pathway are intricately regulated to achieve their specific functions.     

Histoenzymological studies on the distribution of certain phosphatases in the retinae of owlet (Athene brama) and house sparrow (Passer domesticus)

A comparative study on the distribution of alkaline phosphatase (AlP), acid phosphatase (AcP) and 5'-nucleotidase (5-N) amongst the different constituents of retinae of owlet and house sparrow revealed some interesting aspects of the localization of such phosphatases in both the cases. The outer segment of photoreceptors, where light strikes first, are positive for all the phosphatases. Further, areas composed of synapses, reveal activity of the three enzymes. Another interesting aspect is related to the total absence of the activity of AlP and 5-N in the ganglion cells of both the animals. Other sites of phosphatases in various layers have been also identified. The possible metabolic roles of various phosphatases at different sites have been discussed.     

Phosphatase of Chlamydomonas reinhardi: biochemical and cytochemical approach with specific mutants.

The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate.     

SHIP family inositol phosphatases interact with and negatively regulate the Tec tyrosine kinase

The Tec family of protein-tyrosine kinases (PTKs), that includes Tec, Itk, Btk, Bmx, and Txk, plays an essential role in phospholipase Cgamma (PLCgamma) activation following antigen receptor stimulation. This function requires activation of phosphatidylinositol 3-kinase (PI 3-kinase), which promotes Tec membrane localization through phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P(3)) generation. The mechanism of negative regulation of Tec family PTKs is poorly understood. In this study, we show that the inositol 5' phosphatases SHIP1 and SHIP2 interact preferentially with Tec, compared with other Tec family members. Four lines of evidence suggest that SHIP phosphatases are negative regulators of Tec. First, SHIP1 and SHIP2 are potent inhibitors of Tec activity. Second, inactivation of the Tec SH3 domain, which is necessary and sufficient for SHIP binding, generates a hyperactive form of Tec. Third, SHIP1 inhibits Tec membrane localization. Finally, constitutively targeting Tec to the membrane relieves SHIP1-mediated inhibition. These data suggest that SHIP phosphatases can interact with and functionally inactivate Tec by de-phosphorylation of local PtdIns 3,4,5-P(3) and inhibition of Tec membrane localization.     

Acid Phosphatase Complex from the Freshwater Snail Viviparus viviparus L. under Standard Conditions and Intoxication by Cadmium Ions

Acid phosphatases differing in both subcellular localization and substrate specificity were isolated for the first time from the liver of the freshwater snail Viviparus viviparus L. by preparative isoelectrofocusing. One of five characterized phosphatases is highly specific to ADP and the others can hydrolyze (at variable rate) a series of natural substrates. A scheme is proposed for the involvement of the studied phosphatases in carbohydrate metabolism. We have also studied some peculiarities of the effect of Cd2+ in vitro and in vivo on the activities of individual components of the acid phosphatase complex and corresponding changes in metabolism of the freshwater snail as a new test-object allowing the estimation of toxicity in water.     

Histochemical Localization of Alkaline Phosphatases and {beta}-Glucosidases in Cucurbitaceae

The distribution of alkaline phosphatases and ß-glucosidasesin the tissues of the stems of Colocynthls citrullus, Cucumissativus and Cucurbita pepo was studied. There was similarityin the pattern of distribution for these two groups of enzymesand that of acid phosphatases. The activities of these enzymesvaried not only from plant to plant but also from tissue totissue. However both enzymes showed increasing activity withplant age and localization was correspondingly more restricted.The significance of these localizations is discussed.     

A new procedure for the ultrastructural localization of acid phosphatases

Summary The synthesis and properties of two new lead-containing diazonium chlorides are described. The use of these reagents for the electron microscopical localization of acid phosphatases in animal cells has been assessed.     

Subcellular localization and possible functions of acid β-glycerophosphatases and naphthol esterases in plant cells

Summary The subcellular localization of esterases and acid phosphatases in described for cells from roots of Vicia faba. The possible mode of transport of the esterases from a cytoplasmic site of synthesis to the cell wall is discussed.     

Protein tyrosine phosphatases: regulatory mechanisms

Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and dimerization. Here, we review the regulatory mechanisms found to control the classical protein-tyrosine phosphatases.     

Mass spectrometry approaches to study mammalian kinase and phosphatase associated proteins

Reversible phosphorylation events regulate critical aspects of cellular biology by affecting protein conformation, cellular localization, enzymatic activity and associations with interaction partners. Kinases and phosphatases interact not only with their substrates but also with regulatory subunits and other proteins, including scaffolds. In recent years, affinity purification coupled to mass spectrometry (AP-MS) has proven to be a powerful tool to identify protein-protein interactions (PPIs) involving kinases and phosphatases. In this review we outline general considerations for successful AP-MS, and describe strategies that we have used to characterize the interactions of kinases and phosphatases in human cells.     

Localization of alkaline phosphatase isozymes in mouse duodenum by immunofluorescence microscopy

Summary Recent studies on the alkaline phosphatases of the mouse duodenum have revealed the presence of two classes of isozymes, differing in immunochemical, electrophoretic, chromatographic, and kinetic properties. We have now examined the localization of these two types of phosphatases by the immunofluorescence technique. The use of antisera prepared against both types reveals that the isozymes of both classes are localized in the microvilli of the epithelial cells, and both are found from the villi tips to their bases. No significant intracellular localization of either class of phosphatase was observed.Supported by research grant HD03490 from the National Institute of Child Health and Human Development, U.S. Public Health Service.     

The activity of some phosphatases in tissues of adult Hymenolepis nana Siebold (Csetoda)

Histochemical methods were used to study the localization and activity of acid and alkaline phosphatases, ATP-ase, 5-nucleotidase, and glucose-6-phosphatase in tissues of the mature form of Hymenolepis nana. Considerable differences in activity and localization of particular enzymes were observed in the organs of the parasite. The results obtained permit the statement that the integument is the most active enzymatically; in connection with the literature data, this gives grounds for the thesis that the integument of the cestodes functions as an absorbent-digestive organ.     

Sperm development and motility are regulated by PP1 phosphatases in Caenorhabditis elegans

Sperm from different species have evolved distinctive motility structures, including tubulin-based flagella in mammals and major sperm protein (MSP)-based pseudopods in nematodes. Despite such divergence, we show that sperm-specific PP1 phosphatases, which are required for male fertility in mouse, function in multiple processes in the development and motility of Caenorhabditis elegans amoeboid sperm. We used live-imaging analysis to show the PP1 phosphatases GSP-3 and GSP-4 (GSP-3/4) are required to partition chromosomes during sperm meiosis. Postmeiosis, tracking fluorescently labeled sperm revealed that both male and hermaphrodite sperm lacking GSP-3/4 are immotile. Genetic and in vitro activation assays show lack of GSP-3/4 causes defects in pseudopod development and the rate of pseudopodial treadmilling. Further, GSP-3/4 are required for the localization dynamics of MSP. GSP-3/4 shift localization in concert with MSP from fibrous bodies that sequester MSP at the base of the pseudopod, where directed MSP disassembly facilitates pseudopod contraction. Consistent with a role for GSP-3/4 as a spatial regulator of MSP disassembly, MSP is mislocalized in sperm lacking GSP-3/4. Although a requirement for PP1 phosphatases in nematode and mammalian sperm suggests evolutionary conservation, we show PP1s have independently evolved sperm-specific paralogs in separate lineages. Thus PP1 phosphatases are highly adaptable and employed across a broad range of sexually reproducing species to regulate male fertility.     

Localization of acid and alkaline phosphatases in Myxococcus coralloides D

Myxococcus coralloides produces two different phosphatases, one acid and the other alkaline. Both enzymes were localized by physical and biochemical techniques. Spheroplasts from M. coralloides released 20–30% of the phosphatase activities. Osmotic shock or treatment with high MgCl₂ or LiCl concentrations did not produce a greater release. Cytochemical localization situated the phosphatases in the outer membrane and the periplasmic space. Separation of the cytoplasmic membrane and outer membrane of the cells by sucrose gradient centrifugation showed that phosphatases are located primarily in the outer membrane. membrane.     

Helicobacter pylori CheZHP and ChePep form a novel chemotaxis‐regulatory complex distinct from the core chemotaxis signaling proteins and the flagellar motor

Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non‐phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZ_HP, localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZ_HP localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZ_HP for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZ_HP determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZ_HP and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities.     

A lead phthalocyanin method for the demonstration of acid hydrolases in plant and animal tissues

Summary A new method of synthesis of the lead phthalocyanin and the chemical properties of its diazotate are described. The use of this reagent for high resolution studies of the localization of intra- and extralysosomal acid phosphatases and esterases in plant and animal cells has been assessed.     

Protein phosphatases in MAPK signalling: we keep learning from yeast

Because of their key role in cell signalling, a rigorous regulation of mitogen-activated protein kinases (MAPKs) is essential in eukaryotic physiology. Whereas the use of binding motifs and scaffold proteins guarantees the selective activation of a specific MAPK pathway, activating kinases and downregulating phosphatases control the appropriate intensity and timing of MAPK activation. Tyrosine, serine/threonine and dual-specificity phosphatases co-ordinately dephosphorylate and thereby inactivate MAPKs. In budding yeast, enzymes that belong to these three types of phosphatases have been shown to counteract the MAPKs that govern the cellular response to varied extracellular stimuli. Studies carried out with these yeast phosphatases have expanded our knowledge of essential key aspects of the biology of these negative regulators, such as their function, the mechanisms that operate in their modulation by MAPK pathways and their binding to MAPK substrates. Furthermore, yeast MAPK phosphatases have been shown to play additional and essential roles in MAPK-mediated signalling, controlling MAPK localization or cross-talk among pathways. This review stresses the importance of these negative regulators in eukaryotic signalling by discussing the recent developments and perspectives in the study of yeast MAPK phosphatases.     

The tumor suppressor CDKN3 controls mitosis

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2^pThr-161 at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKβ phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.     

The Mechanisms and Time Factor of the Enzyme Structure of a Paleosoil

The data on the enzyme activity of paleosoils of archaeological sites are given. It is shown that the activity of phosphatases and urease in soils of ancient settlements is significantly higher than in modern soils: 1.5–2.0 times for urease and 7–15 times for phosphatase in some cultural layers of the Bronze Age. This is related to a large amount of organic material (garbage, rubbish, excrement, and urea), which entered the soil in ancient times and stimulated soil microorganisms to produce a greater amount of enzymes, whose high activity has been preserved for 4000 years. The location of the enzymes was determined by soil fumigation using chloroform and activation of extracellular enzymes by glycine. The release of intracellular enzymes as a result of fumigation caused a significant increase in phosphatase activity in modern soils and soils of ancient settlements in contrast to the virgin paleosoil of the Bronze Age. The treatment by glycine exerted a smaller effect on the activity of phosphatases, but caused a significant increase in urease activity. This may indicate the predominating extracellular localization of urease in paleosoils of ancient settlements, while phosphatase is characterized by both extra- and intracellular localization.