Effects of tyrosine kinase and phosphatase inhibitors on microtubules in Arabidopsis root cells

To investigate the role of tyrosine phosphorylation/dephosphorylation processes in plant cells the morphology of Arabidopsis thaliana primary roots and the organization of cortical microtubules (MTs) were studied after inhibition of protein tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs). It was found that all tested types of PTKs inhibitors (herbimycin A, genistein and tyrphostin AG 18) altered root hair growth and development, probably as a result of their significant influences on MTs organization in root hairs. The treatment also led to MTs reorientation and disruption in epidermis and cortex cells of both elongation and differentiation zones of primary roots. Enhanced tyrosine phosphorylation after treatment with a PTPs inhibitor (sodium orthovanadate) resulted in intense induction of root hair development and growth and caused a significant shortening of the elongation zone. It also led to changes of MTs orientation from transverse to longitudinal in epidermis and cortex cells of the elongation and differentiation zones of the root. From the data obtained we can suppose that tyrosine phosphorylation can be involved in the dynamics and organization of MTs in different types of plant cells.     

Rapid modulation of nitrate reductase in pea roots

The regulatory properties of nitrate reductase (NR; EC 1.6.6.1) in root extracts from hydroponically grown pea (Pisum sativum L. cv. Kleine Rheinländerin) plants were examined and compared with known properties of NR from spinach and pea leaves. Nitrate-reductase activity (NRA) extracted from pea roots decreased slowly when plants were kept in the dark, or when illuminated plants were detopped, with a half-time of about 4 h (= slow modulation in vivo). In contrast, the half-time for the dark-inactivation of NR from pea leaves was only 10 min. However, when root tip segments were transferred from aerobic to anaerobic conditions or vice versa, changes in NRA were as rapid as in leaves (= rapid modulation in vivo). Nitrate-reductase activity was low when extracted from roots kept in solutions flushed with air or pure oxygen, and high in nitrogen. Okadaic acid, a specific inhibitor of type-1 and type-2A protein phosphatases, totally prevented the in vivo activation by anaerobiosis of NR, indicating that rapid activation of root NR involved protein dephosphorylation. Under aerobic conditions, the low NRA in roots was also rapidly increased by incubating the roots with either uncouplers or mannose. Under these conditions, and also under anaerobiosis, ATP levels in roots were much lower than in aerated control roots. Thus, whenever ATP levels in roots were artificially decreased, NRA increased rapidly. The highly active NR extracted from anaerobic roots could be partially inactivated in vitro by preincubation of desalted root extracts with MgATP (2 mM), with a half-time of about 20 min. It was reactivated by subsequently incubating the extracts with excess AMP (2 mM). Thus, pea root NR shares many of the previously described properties of NR from spinach leaves, suggesting that the root enzyme, like the leaf enzyme, can be rapidly modulated, probably by reversible protein phosphorylation/ dephosphorylation.     

Growth arrest induced by transforming growth factor beta 1 is accompanied by protein phosphatase activation in human keratinocytes.

Protein phosphorylation and dephosphorylation are involved in regulation of cell growth. We tested the hypothesis that the growth inhibitory effect of transforming growth factor beta 1 (TGF-beta 1) involves activation of protein phosphatases. Exposure of human keratinocytes in culture to 400 pM TGF-beta 1 for 48 h led to 80% inhibition of DNA synthesis as measured by nuclear labeling. Incubation of cultured keratinocytes with 400 pM TGF-beta 1 rapidly activated (within 30 min) protein serine/threonine phosphatase, measured using phosphorylase as a substrate. Based on several criteria, including neutralization of activity with specific antibodies and inhibitor-2, TGF-beta 1-activated phosphorylase phosphatase was identified as protein phosphatase 1. TGF-beta 1 did not have rapid effects on protein serine/threonine phosphatase activity (type 2A) measured with histone phosphorylated by protein kinase C or on protein tyrosine phosphatase activity. However, protein tyrosine phosphatase was activated at 48 h, coincident with growth arrest. Differentiation, induced by the combination of TGF-beta 1 plus calcium or by serum, was not accompanied by further serine/threonine or tyrosine phosphatase activation. We conclude that induction of growth arrest in keratinocytes by TGF-beta 1 involves acute activation of protein phosphatase 1, while activation of protein tyrosine phosphatase may represent an additional mechanism for maintaining cells in a growth-arrested state.     

Effects of redox agents on protein tyrosine phosphorylation in pea roots

Posttranslational protein modifications and their interaction are an important way for the regulation of activities of proteins and their supramolecular complexes. More than 50 soluble proteins phosphorylated on tyrosine were detected in pea (Pisum sativum L., cv. Truzhenik) roots by one- and two-dimensional electrophoresis and immunoblotting with a highly specific antibody (PY20). The level of tyrosine phosphorylation of these proteins was changed under in situ action of redox and alkylating agents.     

Tyrosine kinase inhibitors reverse butyrate stimulation of human Caco-2 intestinal epithelial cell alkaline phosphatase but not butyrate promotion of dipeptidyl dipeptidase

Short chain fatty acids such as sodium butyrate are concentrated in the colonic lumen and may protect against colon carcinogenesis by maintaining colonocytic differentiation, but the mechanisms by which they act are not fully understood. It has recently been suggested that short chain fatty acids modulate cellular tyrosine kinase activity in addition to altering chromatin structure via regulation of histone acetylation and DNA methylation. Therefore, the authors evaluated the influence of tyrosine kinase inhibition on the effects of 10 mM butyrate on human Caco-2 intestinal epithelial differentiation, using alkaline phosphatase and dipeptidyl dipeptidase specific activity as markers of differentiation, and two tyrosine kinase inhibitors, of different mechanisms of action and different effects on Caco-2 brush border enzyme specific activity, to block tyrosine kinase activity. As expected, butyrate stimulated both alkaline phosphatase and dipeptidyl dipeptidase specific activity. The tyrosine kinase inhibitors prevented, and indeed one inhibitor reversed the effects of butyrate on alkaline phosphatase specific activity. However, tyrosine kinase inhibition did not prevent butyrate stimulation of dipeptidyl dipeptidase specific activity. Different pathways are likely to regulate the effects of butyrate on expression of these two brush border enzymes. Butyrate stimulation of alkaline phosphatase, but not dipeptidyl dipeptidase, may involve tyrosine phosphorylation signaling.     

The nitrate reductase inhibitor,tungsten, disrupts actin microfilaments in Zea mays L.

Tungsten is a widely used inhibitor of nitrate reductase, applied to diminish the nitric oxide levels in plants. It was recently shown that tungsten also has heavy metal attributes. Since information about the toxic effects of tungsten on actin is limited, and considering that actin microfilaments are involved in the entry of tungsten inside plant cells, the effects of tungsten on them were studied in Zea mays seedlings. Treatments with sodium tungstate for 3, 6, 12 or 24 h were performed on intact seedlings and seedlings with truncated roots. Afterwards, actin microfilaments in meristematic root and leaf tissues were stained with fluorescent phalloidin, and the specimens were examined by confocal laser scanning microscopy. While the actin microfilament network was well organized in untreated seedlings, in tungstate-treated ones it was disrupted in a time-dependent manner. In protodermal root cells, the effects of tungsten were stronger as cortical microfilaments were almost completely depolymerized and the intracellular ones appeared highly bundled. Fluorescence intensity measurements confirmed the above results. In the meristematic leaf tissue of intact seedlings, no depolymerization of actin microfilaments was noticed. However, when root tips were severed prior to tungstate application, both cortical and endoplasmic actin networks of leaf cells were disrupted and bundled after 24 h of treatment. The differential response of root and leaf tissues to tungsten toxicity may be due to differential penetration and absorption, while the effects on actin microfilaments could not be attributed to the nitric oxide depletion by tungsten.     

Effects of sodium tungstate on the ultrastructure and growth of pea (Pisum sativum) and cotton (Gossypium hirsutum) seedlings

Pea (Pisum sativum L. cv. Onmard) and cotton (Gossypium hirsutum L. cv. Campo) seedlings were treated with two concentrations (200 and 500 mg/l) of sodium tungstate (Na₂WO₄) and the developmental effects were investigated. Tungstate retarded seedling growth rate and stopped root elongation in both species. Seedling growth recovered when tungstate was removed, but primary roots continued to be stunted, while lateral root initiation and growth were stimulated. Tungstate induced premature vacuolation in cells of the root apical meristem, with vacuoles having an unusual semi-circular or cap-like shape around the nucleus. In control roots, the nuclei were spherical with prominent nucleoli bearing several randomly distributed fibrillar centres. In the tungstate-treated cells nuclei contained spherical nucleoli with a big nucleolar vacuole. Occasionally, cytoplasmic components, such as mitochondria, were entrapped in the nucleoplasm of interphasic cells of the treated roots. In these roots, most cell plates were fused to only one lateral parental wall suggesting a non-uniform centrifugal extension. The vesicles in these cell plates were dark and fused to each other at a much lower rate than in the dividing cells of the untreated seedlings. Phragmoplast and cortical microtubules were abundant in the untreated cells, but scarcely detected in the treated ones. All these observations are consistent with the view that tungstate causes considerable toxic effects to pea and cotton seedlings.     

PTP inhibitor IV protects JNK kinase activity by inhibiting dual-specificity phosphatase 14 (DUSP14)

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.     

L1 and N-CAM Antibodies Trigger Protein Phosphatase Activity in Growth Cone-Enriched Membranes

Abstract: Triggering of the cell adhesion molecules L1 or N-CAM in a nerve growth cone membrane fraction from fetal rat brain with purified L1 or N-CAM or specific antibodies decreases the steady-state levels of protein tyrosine phosphorylation in the membranes. Here we report that triggering of L1 and N-CAM in the growth cone-enriched membrane fraction with a subset of antibodies directed against the extracellular region of L1 and N-CAM elicited dephosphorylation of endogenous protein substrates, indicating the presence of a cell adhesion molecule-activated phosphatase. The most prominent substrates were a membrane-associated 200-kDa protein and tubulin, both of which were dephosphorylated on tyrosine and serine/threonine residues in response to L1 or N-CAM triggering. The antibody-induced phosphatase was inhibited by agents that blocked tyrosine and serine/threonine phosphatases, including sodium orthovanadate, vanadyl sulfate, zinc cations, heparin, and sodium pyrophosphate. Purified L1 and N-CAM fragments and other antibodies reacting with the extracellular region of these adhesion molecules did not activate the phosphatase but did inhibit tyrosine phosphorylation. These properties suggested that triggering of L1 and N-CAM can lead to either phosphatase activation or tyrosine kinase inhibition in growth cone membranes. These findings implicate protein phosphatases in addition to tyrosine kinases as components of L1 and N-CAM intracellular signaling pathways in growth cones.     

Early signalling pathways in rice roots under vanadate stress

Vanadate is beneficial to plant growth at low concentration. However, plant exposure to high concentrations of vanadate has been shown to arrest cell growth and lead to cell death. We are interested in understanding the signalling pathways of rice roots in response to vanadate stress. In this study, we demonstrated that vanadate induced rice root cell death and suppressed root growth. In addition, we found that vanadate induced ROS accumulation, increased lipid peroxidation and elicited a remarkable increase of MAPKs and CDPKs activities in rice roots. In contrast, pre-treatment of rice roots with ROS scavenger (sodium benzoate), serine/threonine protein phosphatase inhibitor (endothall), and CDPK antagonist (W7), reduced the vanadate-induced MAPKs activation. Furthermore, the expression of a MAPK gene (OsMPK3) and four tyrosine phosphatase genes (OsDSP3, OsDSP5, OsDSP6, and OsDSP10) were regulated by vanadate in rice roots. Collectively, these results strongly suggest that ROS, protein phosphatase, and CDPK may function in the vanadate-triggered MAPK signalling pathway cause cell death and retarded growth in rice roots.     

Protein tyrosine phosphatase activity modulation by endothelin-1 in rabbit platelets

Protein tyrosine phosphorylation, modulated by the rate of both protein tyrosine kinase and protein tyrosine phosphatase activities, is critical for cellular signal transduction cascades. We report that endothelin-1 stimulation of rabbit platelets resulted in a dose- and time-dependent tyrosine phosphorylation of four groups of proteins in the molecular mass ranges of 50, 60, 70–100 and 100–200 kDa and that one of these corresponds to focal adhesion kinase. This effect is also related to the approximately 60% decrease in protein tyrosine phosphatase activity. Moreover, this inhibited activity was less sensitive to orthovanadate. In the presence of forskolin that increases the cAMP level a dose-dependent inhibition of the endothelin-stimulated tyrosine phosphorylation of different protein substrates and a correlation with an increase in the protein tyrosine phosphatase activity (11.6-fold compared to control) have been found. Further studies by immunoblotting of immunoprecipitated soluble fraction with anti-protein tyrosine phosphatase-1C from endothelin-stimulated platelets have demonstrated that the tyrosine phosphorylation of platelet protein tyrosine phosphatase-1C is correlated with the decrease in its phosphatase activity. As a consequence, modulation and regulation by endothelin-1 in rabbit platelets can be proposed through a cAMP-dependent pathway and a tyrosine phosphorylation process that may affect some relevant proteins such as focal adhesion kinase.     

Growth inhibition and protein tyrosine phosphorylation in MCF 7 breast cancer cells by a novel K vitamin

We have now found that the most potent, Cpd 5 [2-(2-mercaptoethanol)-3-methyl-1, 4-napthoquinone], inhibits growth of doxorubicin-resistant and doxorubicin-sensitive breast cancer cells (MCF 7r and MCF 7w) in culture. Growth inhibition by Cpd 5 was antagonized by the thiol antioxidants glutathione and cysteine, but not by catalase or superoxide dismutase, suggesting that growth inhibition is probably via conjugation of cellular thiols. In support of this, we found that Cpd 5 inhibited the activity of thiol containing cellular protein tyrosine phosphatase (PTP) enzyme, with consequent induction of various tyrosine phosphoproteins, but not serine or tyrosine phosphoproteins. The tyrosine phosphorylation was also inhibited by exogenous glutathione or cysteine and could be enhanced by depletion of cellular glutathione by BSO. This effect of Cpd 5 on protein tyrosine phosphorylation was highly selective, however. Tyrosine phosphorylation of EGF-R, Erb-B2, and ERK1/2 was increased, but not that of Insulin-R or JNK. ERK1/2 tyrosine phosphorylation and growth inhibition increased with increasing concentrations of Cpd 5. Furthermore, suppression of Cpd 5-mediated ERK1/2 phosphorylation by an ERK-kinase inhibitor antagonized growth inhibition. These results suggest a strong correlation between ERK1/2 phosphorylation by Cpd 5 and growth inhibition. This novel K-vitamin analog thus inhibits MCF 7 cell growth and induces selective protein tyrosine phosphorylation.     

Receptor type protein tyrosine phosphatase zeta-pleiotrophin signaling controls endocytic trafficking of DNER that regulates neuritogenesis

Protein tyrosine phosphatase zeta (PTPzeta) is a receptor type protein tyrosine phosphatase that uses pleiotrophin as a ligand. Pleiotrophin inactivates the phosphatase activity of PTPzeta, resulting in the increase of tyrosine phosphorylation levels of its substrates. We studied the functional interaction between PTPzeta and DNER, a Notch-related transmembrane protein highly expressed in cerebellar Purkinje cells. PTPzeta and DNER displayed patchy colocalization in the dendrites of Purkinje cells, and immunoprecipitation experiments indicated that these proteins formed complexes. Several tyrosine residues in and adjacent to the tyrosine-based and the second C-terminal sorting motifs of DNER were phosphorylated and were dephosphorylated by PTPzeta, and phosphorylation of these tyrosine residues resulted in the accumulation of DNER on the plasma membrane. DNER mutants lacking sorting motifs accumulated on the plasma membrane of Purkinje cells and Neuro-2A cells and induced their process extension. While normal DNER was actively endocytosed and inhibited the retinoic-acid-induced neurite outgrowth of Neuro-2A cells, pleiotrophin stimulation increased the tyrosine phosphorylation level of DNER and suppressed the endocytosis of this protein, which led to the reversal of this inhibition, thus allowing neurite extension. These observations suggest that pleiotrophin-PTPzeta signaling controls subcellular localization of DNER and thereby regulates neuritogenesis.     

A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo

Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.     

Effect of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on tyrosine phosphorylation of pea proteins

Changes in tyrosine phosphorylation of soluble polypeptides of pea (Pisum sativum L.) roots were revealed under the action of exogenous hydrogen peroxide in situ and in vitro. The polypeptides whose tyrosine phosphorylation in situ was vanadate-sensitive were identified. A thiol agent dithiothreitol and the antioxidant ascorbic acid reversed the effect of hydrogen peroxide in vitro. The results indicate that tyrosine phosphorylation of pea proteins is a subject to redox regulation.     

Root hydrotropism of an agravitropic pea mutant, ageotropum

We have partially characterized root hydrotropism of an agravitropic pea mutant, ageotropum (from Pisum sativum L. cv. Weibull's Weitor), without interference of gravitropism. Lowering the atmospheric air humidity inhibited root elongation and caused root curvature toward the moisture-saturated substrate in ageotropum pea. Removal of root tips approximately 1.5 mm in length blocked the hydrotropic response. A computer-assisted image analysis showed that the hydrotropic curvature in the roots of ageotropum pea was chiefly due to a greater inhibition of elongation on the humid side than the dry side of the roots. Similarly, gravitropic curvature of Alaska pea roots resulted from inhibition of elongation on the lower side of the horizontally placed roots, while the upper side of the roots maintained a normal growth rate. Gravitropic bending of Alaska pea roots was apparent 30 min after stimulation, whereas differential growth as well as curvature in positive root hydrotropism of ageotropum pea became visible 4–5 h after the continuous hydrostimulation. Application of 2,3,5-triiodobenzoic acid or ethyleneglycol-bis-( β -aminoethylether)-N,N,N',N'-tetraacetic acid was inhibitory to both root hydrotropism of ageotropum pea and root gravitropism of Alaska pea. Some mutual response mechanism for both hydrotropism and gravitropism may exist in roots, although the stimulusperception mechanisms differ from one another.     

Preface

Biological properties of soil are not only essential for the maintenance of soil fertility and the sustainability of the plant-soil ecosystems, but also indicators of land reclamation of contaminated or disturbed soils. This experiment involves two plants (barley and field pea) growing in four soils with different hydrocarbon contents. The objective was to study the effect of hydrocarbons on plant growth and microbial activity, and to evaluate the acid phosphatase activity as an indicator of reclamation of hydrocarbon-contaminated soils. Barley root mass decreased with the increase of the hydrocarbon content but field pea roots were not sensitive to the hydrocarbon content in this experiment. The hydrocarbon contamination reduced the plant growth but increased the microbial activity. The acid phosphatase activity was controlled by both plant root production and microbial activity, therefore it was not a good indicator of the reclamation of oil-contaminated soils.     

The impact of heavy metals on the activity of some enzymes along the barley root

Barley seedlings 48 h after the onset of germination on filter paper treated for 24 h by 1 mM cadmium (Cd), 3 mM nickel (Ni) or 0.5 mM mercury (Hg) showed similar approximately 45% root growth inhibition. Although root growth inhibition was similar, loss of cell viability evaluated, as Evans blue uptake was distinct among Cd, Ni and Hg treated roots. While Cd and Hg caused cell death along the whole barley root (0–8 mm), Ni induced significant loss of cell viability only in root cells 6–8 mm distance from the root tip. Our results suggest that different metabolic processes are activated in different parts of barley root in relation to distance from the root tip during heavy metal (HM) treatment. Some of them are characteristic for several HMs such as inhibition of ascorbic acid oxidase or glutathione-S-transferase stimulation, while others are specific for individual HMs, e.g. activation of acid phosphatase and lipoxygenase by Cd and Hg, or inhibition of ascorbate peroxidase by Ni and Hg treatment.     

Calpain controls the balance between protein tyrosine kinase and tyrosine phosphatase activities during platelet activation.

Protein phosphorylation was studied during platelet stimulation in two ranges of ionized [Ca2+]. At ionized [Ca2+]i< or = 1 microM, proteins were phosphorylated. At ionized [Ca2+]i > or = 4 microM, phosphoproteins disappeared. Protein dephosphorylation was prevented by the combined action of calpeptin and phosphatase inhibitors. Protein tyrosine phosphatase activity was stimulated regardless of the ionized [Ca2+] level. Protein tyrosine kinase activity was stimulated at ionized [Ca2+]i < or =1 microM, whereas at ionized [Ca2+]i > or =4 microM, no protein tyrosine kinase activity was observed except in the presence of calpeptin. Thus, the massive tyrosine phosphoprotein disappearance observed at a high ionized [Ca2+]i resulted not only in protein tyrosine phosphatase activation, but also in calpain-induced protein tyrosine kinase inactivation.     

N-cadherin is an in vivo substrate for protein tyrosine phosphatase sigma (PTPsigma) and participates in PTPsigma-mediated inhibition of axon growth

Protein tyrosine phosphatase sigma (PTPsigma) belongs to the LAR family of receptor tyrosine phosphatases and was previously shown to negatively regulate axon growth. The substrate for PTPsigma and the effector(s) mediating this inhibitory effect were unknown. Here we report the identification of N-cadherin as an in vivo substrate for PTPsigma. Using brain lysates from PTPsigma knockout mice, in combination with substrate trapping, we identified a hyper-tyrosine-phosphorylated protein of approximately 120 kDa in the knockout animals (relative to sibling controls), which was identified by mass spectrometry and immunoblotting as N-cadherin. beta-Catenin also precipitated in the complex and was also a substrate for PTPsigma. Dorsal root ganglion (DRG) neurons, which highly express endogenous N-cadherin and PTPsigma, exhibited a faster growth rate in the knockout mice than in the sibling controls when grown on laminin or N-cadherin substrata. However, when N-cadherin function was disrupted by an inhibitory peptide or lowering calcium concentrations, the differential growth rate between the knockout and sibling control mice was greatly diminished. These results suggest that the elevated tyrosine phosphorylation of N-cadherin in the PTPsigma(-/-) mice likely disrupted N-cadherin function, resulting in accelerated DRG nerve growth. We conclude that N-cadherin is a physiological substrate for PTPsigma and that N-cadherin (and likely beta-catenin) participates in PTPsigma-mediated inhibition of axon growth.