Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.

Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.

Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis.

DNA-damaging agents induce accumulation of the tumor suppressor and G1 checkpoint protein p53, leading cells to either growth arrest in G1 or apoptosis (programmed cell death). The p53-dependent G1 arrest involves induction of p21 (also called WAF1/CIP1/SDI1), which prevents cyclin kinase-mediated phosphorylation of retinoblastoma protein (RB). Recent studies suggest a p53-independent G1 checkpoint as well; however, little is known about its molecular mechanisms. We report that induction of a protein-serine/threonine phosphatase activity by DNA damage signals is at least one of the mechanisms responsible for p53-independent, RB-mediated G1 arrest and consequent apoptosis. When two p53-null human leukemic cell lines (HL-60 and U-937) were treated with a variety of anticancer agents, RB became hypophosphorylated, accompanied with G1 arrest. This was followed immediately (in less than 30 min) by apoptosis, as determined by the accumulation of pre-G1 apoptotic cells and the internucleosomal fragmentation of DNA. Addition of calyculin A or okadaic acid (specific serine/threonine phosphatase inhibitors) or zinc chloride (apoptosis inhibitor) prevented the G1 arrest- and apoptosis-specific RB dephosphorylation. The levels of cyclin E- and cyclin A-associated kinase activities remained high during RB dephosphorylation, supporting the involvement of a chemotherapy-induced serine/threonine phosphatase(s) rather than p21. Furthermore, the induced phosphatase activity coimmunoprecipitated with the hyperphosphorylated RB and was active in a cell-free system that reproduced the growth arrest- and apoptosis-specific RB dephosphorylation, which was inhibitable by calyculin A but not zinc. We propose that the RB phosphatase(s) might be one of the p53-independent G1 checkpoint regulators.

Effect of salinity on growth, pigmentation, N2 fixation and alkaline phosphatase activity of cultured Trichodesmium sp

Trichodesmium sp. isolated from the Great Barrier Reef lagoon was cultured in artificial seawater media containing a range of salinities. Trichodesmium sp. actively grew over a wide range of salinities (22 to 43 psu) and hence can be classed as euryhaline. Maximum growth occurred with salinities in the range 33 to 37 psu. Chl a content and alkaline phosphatase activity were found to increase with salinity over the range 22 to 43 psu, but the N-2 fixation rate was reduced at salinities below and above the range for maximum growth. Growth in media exhibiting maximum growth was characterised by well-dispersed cultures of filaments, while significant aggregations of filaments formed in other media. It is proposed that the tendency for Trichodesmium filaments to aggregate in media with salinities outside the range for maximum growth is an opportunistic response to a deficiency of cellular nitrogen, which results from the reduced N-2 fixation rates, and the aggregation occurs in order to enhance the uptake of combined N released within the aggregates and/or the N-2 fixation within the aggregates.

High alkaline phosphatase activity in phosphate replete waters: The case of two macrotidal estuaries

The occurrence of alkaline phosphatase activity (APA) that hydrolyses organic phosphorus into phosphate (PO4) is commonly related to PO4 deficiency of oceanic, coastal and fresh waters. APA is almost never investigated in PO4-rich estuaries, since very low activities are expected to occur. As a consequence, microbial mineralization of organic phosphorus into PO4 has often been ignored in estuaries. In this study, we examined the importance of potential APA and the associated microbial dynamics in two estuaries, the Aulne and the Elorn (Northwestern France), presenting two different levels of PO4 concentrations. Unexpected high potential APA was observed in both estuaries. Values ranged from 50 to 506 nmol L−1 h−1, which range is usually found in very phosphorus-limited environments. High potential APA values were observed in the oligohaline zone (salinity 5–15) in spring and summer, corresponding to a PO4 peak and a maximum bacterial production of particle-attached bacteria. In all cases, high potential APA was associated with high suspended particulate matter and total particulate phosphorus. The low contribution of the 0.2–1 μm fraction to total APA, the strong correlation between particulate APA and bacterial biomass, and the close relationship between the production of particle-attached bacteria and APA, suggested that high potential APA is mainly due to particle-attached bacteria. These results suggest that the microbial mineralization of organic phosphorus may contribute to an estuarine PO4 production in spring and summer besides physicochemical processes.

Metals and linear alkylbenzene sulphonate as inhibitors of the algae Pseudokirchneriella subcapitata acid phosphatase activity.

Sewage sludge applied to soils as a fertilizer often contains metals and linear alkylbenzene sulphonate (LAS) as contaminants. These pollutants can be transported to the aquatic environment where they can alter the phosphatase activity in living organisms. The acid phosphatase of algae plays important roles in metabolism such as decomposing organic phosphate into free phosphate and autophagic digestive processes. The order of in vitro inhi- bition of Pseudokirchneriella subcapitata acid phosphatase at the highest concentration tested was LAS[Hg2? = Al 3?[Se4? = Pb2?[Cd2?. A non-competitive inhibi- tion mechanism was obtained for Hg2? (Ki = 0.040 mM) and a competitive inhibition for LAS (Ki = 0.007 mM). In vivo studies with treated algae cultures showed that the inhibition of specific activity was observed in algae exposed during 7 days, in contrast to short term (24 h) treatments with both these chemicals. Our results suggest that the inhibition parameters in vitro did not markedly differ between the two chemicals. On the other hand, in vivo evaluations showed strong differences between both pollu- tants regarding the concentration values and the degree of response.

Inter-domain interactions influence the stability and catalytic activity of the bi-domain protein tyrosine phosphatase PTP99A

The two protein tyrosine phosphatase (PTP) domains in bi-domain PTPs share high sequence and structural similarity. However, only one of the two PIP domains is catalytically active. Here we describe biochemical studies on the two tandem PTP domains of the bi-domain PTP, PTP99A. Phosphatase activity, monitored using small molecule as well as peptide substrates, revealed that the inactive (D2) domain activates the catalytic (D1) domain. Thermodynamic measurements suggest that the inactive D2 domain stabilizes the bi-domain (D1-D2) protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by few interactions at the inter-domain interface. In particular, mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent location to the so-called allosteric site of the canonical single domain PIP, PTP1B. These observations suggest functional optimization in bi-domain PTPs whereby the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme. (C) 2012 Elsevier B.V. All rights reserved.

Contributions of phosphatase and microbial activity to internal phosphorus loading and their relation to lake eutrophication

Phosphatase may accelerate the process of lake eutrophication through improving phosphorus bioavailability. This mechanism was studied in three Chinese eutrophic shallow lakes (Lake Taihu, Lake Longyang,and Lake Lianhua). Phosphatase activity was related to the concentration of soluble reactive phosphorus (SRP) and chlorophyll a. Stability of dissolved phosphatase in reverse micelles may be attributed to molecular size, conformation and active residues of the enzyme. At the site with Microcystis bloomed in Lake Taihu, dissolved phosphatase activity was higher and more stable in micelles, SRP concentrations were lower in interstitial water, the contents of different forms of phosphorus and the amounts of aerobic bacteria were lower while respiration efficiency was higher in sediments. Phosphobacteria, both inorganic and organic and other microorganisms were abundant in surface water but rare in sediments. Therefore, internal phosphorus may substantially flux into water column by enzymatic hydrolysis and anaerobic release, together with mobility of bacteria, thereby initiating the bloom. In short, biological mechanism may act in concert with physical and chemical factors to drive the internal phosphorus release and accelerate lake eutrophication.

Cell nucleus activity during post-embryonic development of Apis melliferaL. (Hymenoptera: Apidae). Intranuclear acid phosphatase

We report nuclear acid phosphatase activity in the somatic (intra-ovariolar and stromatic) and germ cells of differentiating honey bee worker ovaries, as well as in the midgut cells of metamorphosing bees. There was heterogeneity in the intensity and distribution of electron dense deposits of lead phosphate, indicative of acid phosphatase activity in the nuclei of these tissues, during different phases of post-embryonic bee development. This heterogeneity was interpreted as a variation of the nuclear functional state, related to the cell functions in these tissues.

Phosphatase and tensin homolog regulates stability and activity of EphB1 receptor

Deregulation of receptor tyrosine kinases (RTKs) is linked to a broad range of cancers, stressing the necessity of studying their regulatory pathways. We and others demonstrated previously that c-Cbl is necessary for the lysosomal degradation of erythropoietin-producing hepatocellular B1 (EphB1) carcinoma and epidermal growth factor receptor (EGFR) RTKs. Moreover, the tumor suppressor phosphatase and tensin homolog (PTEN) was shown to modulate c-Cbl-dependent EGFR degradation. We therefore investigated the involvement of PTEN in EphB1 signaling and degradation. We used PTEN mutants, PTEN, and NHERF1 small interfering RNA in CHO-EphB1 and SW480 cells endogenously expressing EphB1 to delineate EphB1-PTEN interactions. PTEN was constitutively associated with c-Cbl, protecting it from degradation. EphB1 stimulation triggered ∼50% serine-threonine PTEN dephosphorylation and PTEN-Cbl complex disruption, a process requiring PTEN protein phosphatase activity. Both proteins independently translocated to EphB1, with PTEN in association with the scaffold protein NHERF1. Biologically, PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis. This study demonstrates for the first time in mammalian cells that the Eph receptor and PTEN associate and influence their signaling. Moreover, it contributes to the emerging concept that PTEN regulates expression of RTKs through modulation of their degradation. Finally, it reveals a new role for PTEN protein phosphatase activity involved in this process.

(Table 1) Activity of alkali phosphatase in suspended matter at 20°C and environmental temperatures, and recycling time of organic phosphorus in the Barents and Norwegian seas

Alkali phosphatase activity and hydrochemical structure of waters in the Barents and Norwegian seas were investigated. In a sea with the seasonal bioproduction cycle alkali phosphatase activity is also seasonal, rising with trophic level of waters. At the end of hydrological and biological winter activity is practically zero. Alkali phosphatase activity is especially important in summer, when plankton has consumed winter supply of phosphate in the euphotic layer and nutrient limitation of primary production begins. In summer production and destruction cycle, apparent time for recycling of phosphorus by phosphatase in suspended matter in the euphotic layer of the Barents Sea and Norwegian Sea averages from 7 to 30 hours.

Activity of ETS enzymes and phosphatase in the Sea of Okhotsk and Kuril region in summer 1992 and 1993

An estimate of rate of transformation of organic matter and regeneration of nutrients (in particular phosphorus) was calculated for different regions of the Sea of Okhotsk. The rate was estimated by means of rate of complete oxidation of organic matter to CO2 and H2O catalyzed by enzymes of the electron transport system (ETS) and rate of hydrolytic splitting of phosphate from organic phosphorus compounds catalyzed by alkaline phosphatase. Organic matter destruction rate was at its maximum on the shelf of Kamchatka and Sakhalin, as well as in the layer of maximum oxygen gradients in deep waters. It was found that zones of intensive primary production were characterized by high rates of phosphorus regeneration, which provided for 80% of primary production when concentration of mineral phosphorus was low.

Residue-specific investigation of the relationship between oxidation and activity of a dual specificity phosphatase by mass spectrometry

Redox regulation of signalling pathways is critical in proliferation and apoptosis; redox imbalance can lead to pathologies such as inflammation and cancer. Vaccinia H1-related protein (VHR; DUSP3) is a dual-specificity phosphatase important in controlling MAP kinase activity during cell cycle. the active-site motif contains a cysteine that acts as a nucleophile during catalysis. We used VHR to investigate the effect of oxidation in vitro on phosphatase activity, with the aim of determining how the profile of site-specific modification related to catalytic activity. Recombinant human VHR was expressed in E. coli and purified using a GST-tag. Protein was subjected to oxidation with various concentrations of SIN-1 or tetranitromethane (TNM) as nitrating agents, or HOCl. the activity was assayed using either 3-O-methylfluorescein phosphate with fluorescence detection or PIP3 by phosphate release with malachite green. the sites of oxidation were mapped using HPLC coupled to tandem mass spectrometry on an ABSciex 5600TripleTOF following in-gel digestion. More than 25 different concentration-dependent oxidative modifications to the protein were detected, including oxidations of methionine, cysteine, histidine, lysine, proline and tyrosine, and the % oxidized peptide (versus unmodified peptide) was determined from the extracted ion chromatograms. Unsurprisingly, methionine residues were very susceptible to oxidation, but there was a significant different in the extent of their oxidation. Similarly, tyrosine residues varied greatly in their modifications: Y85 and Y138 were readily nitrated, whereas Y38, Y78 and Y101 showed little modification. Y138 must be phosphorylated for MAPK phosphatase activity, so this susceptibility impacts on signalling pathways. Di- and tri- oxidations of cysteine residues were observed, but did not correlate directly with loss of activity. Overall, the catalytic activity did not correlate with redox state of any individual residue, but the total oxidative load correlated with treatment concentration and activity. This study provides the first comprehensive analysis of oxidation modifications of VHR, and demonstrates both heterogenous oxidant effects and differential residue susceptibility in a signalling phosphatase.

Effect of a novel compound from Lycopodium obscurum L. on osteogenic activity of osteoblasts in vitro

We investigated the potential of an extract of Lycopodium obscurum L.; stigmastane-3-oxo-21-oic acid (SA), to enhance osteogensis of mouse osteoblastic MC3T3-E1 cells. SA at a concentration of 16 µM was found to have no significant effect upon the viability of the cells, thus concentrations of 8 µM and 16 µM of SA were used in all further experiments. Both concentrations of SA had an inhibitory affect upon alkaline phosphatase activity (ALP) after 8 days incubation, however, after 16 days activity was restored to control levels. However Alizarin red S staining showed increased levels of mineralization for both concentrations after 16 days culture. Real time PCR showed inhibition of genes Runx2 and Osterix genes responsible for the up-regulation of ALP. However early time point (8 days) up-regulation of bone matrix mineralization genes OPN and OCN, and late time point (16 days) up-regulation of both Jun-D and Fra-2 mRNA expression was significantly enhanced. These results suggest a potential me-chanism of SA in enhancing bone fracture healing is through the up-regulating bone matrix minera-lization.

Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana

Lysophosphatidic acid (LPA) acts as a signaling molecule that regulates diverse cellular processes and it can rapidly be metabolized by phosphatase and acyltransferase LPA phosphatase gene has not been identified and characterized in plants so far The BLAST search revealed that the At3g03520 is similar to phospholipase family. and distantly related to bacterial phosphatases The conserved motif. (J)4XXXNXSFD, was identified in both At3g03520 like phospholipases and acid phosphatases In silico expression analysis of At3g03520 revealed a high expression during phosphate starvation and abiotic stresses. This gene was overexpressed in Escherichia coli and shown to posses LPA specific phosphatase activity These results Suggest that this gene possibly plays a role in signal transduction and storage lipid synthesis.