Protein phosphatase 1 and 2A inhibitors prolong the switch in the control of glutamate release by group I metabotropic glutamate receptors: characterization of the inhibitory pathway

We have addressed the role of protein phosphatases (PPs) in the modulation of the switch in glutamate release observed after repetitive stimulation of group I metabotropic glutamate receptors (mGluRs). In cerebrocortical nerve terminals the agonist (S:)-3, 5-dihydroxyphenylglycine facilitated evoked glutamate release. However, a second stimulation, 5 min later, reduced rather than facilitated this release. This switch in the control of glutamate release was reversed when a 30-min interval was left between stimulations. Inhibition of the endogenous PPs, PP1 and PP2A, with calyculin A and okadaic acid prevented the recovery of the facilitatory response and maintained the receptor permanently coupled to the inhibitory pathway. The inhibitors of PP2B, cyclosporin A and cypermethrine, had no effect. The inhibition of glutamate release was insensitive to pertussis toxin and was the result of the loss of the release component coupled to N-type Ca(2+) channels. This inhibitory action was suppressed by addition of the protein kinase C activator 4beta-phorbol 12,13-dibutyrate. We conclude that the balance between protein kinase and phosphatase activity at the nerve terminal plays a key role in accommodating the modulation of glutamate release by group I mGluRs.     

Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa

Reversible phosphorylation of serine/threonine residues of cell cycle-regulatory proteins is one of the key molecular mechanisms controlling eukaryotic cell division. In plants, the protein kinase partners (i.e. p34cdc2/CDC28-related kinases) have been extensively studied, while the role of counter-acting protein phosphatases is less well understood. We used endothall (ET) as a cell-permeable inhibitor of serine/threonine-specific protein phosphatases to alter cytological and biochemical characteristics of cell division in cultured alfalfa cells. A high concentration of ET (10 and 50 microM) inhibited both protein phosphatases 1 and 2 (PP1 and PP2A), while a low concentration (1 microM) of ET-treatment primarily reduced the PP2A activity. High concentrations of the inhibitor increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. In contrast, a low concentration of ET did not interfere with chromosomal events but caused significant alterations in the organisation of microtubules. Exposure of cells to 1 microM ET resulted in disturbance of preprophase band formation, increase in the number of nuclei with prophase microtubule assembly, premature polarisation of the spindle, and abnormal phragmoplast maturation. Under the same conditions, the ET-treated cells exhibited an early increase in cdc2MsF kinase activity. These results suggest that PP2A contributes to the control of mitotic kinase activities and microtubule organisation. Normal chromosome condensation and mitotic progression are dependent on both PP1 and PP2A activities. The presented data support the functional role of protein phosphatases in the co-ordination of chromosomal and microtubule events in dividing plant cells.     

Effects of 6-Dimethylaminopurine, 2-Aminopurine, Olomoucine and Sodium Vanadate on DNA Endoreduplication in Primary Roots of Pisum Sativum

The effects of 2-aminopurine, olomoucine, 6-dimethylaminopurine (inhibitors of cyclin-dependent kinases; CDK), and sodium vanadate (a potent inhibitor of protein phosphatases) on DNA endoreduplication were investigated during elongation and differentiation of the primary roots in Pisum sativum. When compared with the untreated control plants, at least one additional round of DNA replication was evidenced to occur within most cells, the majority of which have attained 4C DNA level, and a considerably greater portion of cells represented the endopolyploid state with nuclear DNA content approximating the 8C value. It is concluded that cellular commitment to DNA endoreduplication may appear not only as a consequence of suppression imposed directly upon CDK activity, but also as an indirect output connected with the decreased activity of cdc25 protein phosphatase, an enzyme necessary to turn the switch on for appropriate conformation of the CDK/cyclin B complex. By calculating the absorption profiles of Feulgen-stained nuclei, specific phosphorylation-dependent changes in chromatin condensation of endopolyploid cells have been revealed. It is proposed that acquisition of a certain critical level of chromatin condensation constitutes a prerequisite for additional rounds of DNA synthesis in plants.     

Signal transduction through the T cell receptor is dynamically regulated by balancing kinase and phosphatase activities

Within seconds of T cell receptor engagement, a well-characterized series of tyrosine phosphorylation events mediate cellular activation. It is widely accepted that these initial phosphorylations remain stable until protein tyrosine phosphatases return the cell to its basal level. Based on a model of peripheral blood T cell activation, in which the kinetics of phosphorylation can be modulated, we propose an alternate hypothesis that T cell signaling is highly dynamic. Our results demonstrate that tyrosine phosphorylation and dephosphorylation are occurring co-temporally after T cell receptor cross-linking, regulated by a delicate balance of kinases and phosphatases.     

Vesicular Transport of Extracellular Acid Phosphatases in Yeast Saccharomyces cerevisiae

A method for isolation of secretory vesicles from the yeast Saccharomyces cerevisiae based on the disintegration of protoplasts by osmotic shock followed by separation of the vesicles by centrifugation in a density gradient of Urografin was developed in this study. Two populations of the secretory vesicles that differ in density and shape were separated. Acid phosphatases (EC 3.1.3.2) were used as markers of the secretory vesicles. It was shown that the constitutive acid phosphatase (PHO3 gene product) is mainly transported to the cell surface by a lower density population of vesicles, while the repressible acid phosphatase (a heteromer encoded by PHO5, PHO10, and PHO11 genes) by a vesicle population of higher density. These data provide evidence that at least two pathways of transport of yeast secretory proteins from the place of their synthesis and maturation to the cell surface may exist. To reveal the probable reasons for transport of Pho3p and Pho5p/Pho10p/Pho11p enzymes by two different kinds of vesicles, we isolated vesicles from strains that synthesize the homomeric forms of the repressible acid phosphatase. It was demonstrated that glycoproteins encoded by the PHO10 and/or PHO11 genes could be responsible for the choice of one of the alternative transport pathways of the repressible acid phosphatase. A high correlation coefficient between bud formation and secretion of Pho5p phosphatase and the absence of correlation between bud formation and secretion of minor phosphatases Pho10p and Pho11p suggests different functional roles of the polypeptides that constitute the native repressible acid phosphatase.     

Inhibitory effect of menaquinone-7 (vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro

Heme oxygenase1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular stress. This has led to the realization that HO1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogenactivated protein kinases and other kinases in the mechanism of induction of HO1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO1 gene. Here, we show that a remarkably brief (1–15 min) exposure of normal hepatocytes to low concentrations (0.5–3 M) of PAO produces a marked increase in mRNA and protein of HO1. This increase is comparable to the level obtained by addition of heme (20 M), and occurs without producing changes in cellular glutathione levels or stabilization of HO1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAOmediated induction of HO1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO1 by PAO and by heme may share some common pathways.     

Traits associated with improved P-uptake efficiency in CIMMYT's semidwarf spring bread wheat grown on an acid Andisol in Mexico

Phosphorus deficiency is a major yield limiting constraint in wheat cultivation on acid soils. The plant factors that influence P uptake efficiency (PUPE) are mainly associated with root characteristics. This study was conducted to analyze the genotypic differences and relationships between PUPE, root length density (RLD), colonization by vesicular arbuscular and arbuscular mycorrhizal (V)AM fungi and root excretion of phosphatases in a P-deficient Andisol in the Central Mexican Highlands. Forty-two semidwarf spring-bread-wheat (Triticum aestivumL.) genotypes from CIMMYT were grown without (−P) and with P fertilization (+P), and subsequently in subsets of 30 and 22 genotypes in replicated field trials over 2 and 3 years, respectively. Acid phosphatase activity at the root surface (APASE) was analyzed in accompanying greenhouse experiments in nutrient solution. In this environment, PUPE contributed more than P utilization efficiency, in one experiment almost completely, to the variation of grain yield among genotypes. Late-flowering genotypes were higher yielding, because the postanthesis period of wheat was extended due to the cold weather at the end of the crop cycles, and postanthesis P uptake accounted for 40–45% of total P uptake. PUPE was positively correlated with the numbers of days to anthesis (at −P r=0.57 and at +P r=0.73). The RLD in the upper soil layer (0–20 cm) of the wheat germplasm tested ranged from 0.5 to 2.4 cm cm^-3 at –P and 0.7 to 7.7 at +P. RLD was the most important root trait for improved P absorption, and it was positively genetically correlated with PUPE (at –P r=0.42 and at +P r=0.63) and the number of spikes m^-2 (at –P r=0.58 and at +P r=0.36). RLD in the upper soil layer was more important with P fertilizer application. Without P fertilization, root proliferation in the deeper soil profile secured access to residual, native P in the deeper soil layer. (V)AM-colonisation and APASE were to a lesser degree correlated with PUPE. Among genoptypes, the level of (V)AM-colonisation ranged from 14 to 32% of the RLD in the upper soil layer, and APASE from 0.5 to 1.1 nmol s^-1 plant^-1 10^-2. This revised version was published online in June 2006 with corrections to the Cover Date.     

Disulfide stress: a novel type of oxidative stress in acute pancreatitis

Glutathione oxidation and protein glutathionylation are considered hallmarks of oxidative stress in cells because they reflect thiol redox status in proteins. Our aims were to analyze the redox status of thiols and to identify mixed disulfides and targets of redox signaling in pancreas in experimental acute pancreatitis as a model of acute inflammation associated with glutathione depletion. Glutathione depletion in pancreas in acute pancreatitis is not associated with any increase in oxidized glutathione levels or protein glutathionylation. Cystine and homocystine levels as well as protein cysteinylation and γ-glutamyl cysteinylation markedly rose in pancreas after induction of pancreatitis. Protein cysteinylation was undetectable in pancreas under basal conditions. Targets of disulfide stress were identified by Western blotting, diagonal electrophoresis, and proteomic methods. Cysteinylated albumin was detected. Redox-sensitive PP2A and tyrosine protein phosphatase activities diminished in pancreatitis and this loss was abrogated by N-acetylcysteine. According to our findings, disulfide stress may be considered a specific type of oxidative stress in acute inflammation associated with protein cysteinylation and γ-glutamylcysteinylation and oxidation of the pair cysteine/cystine, but without glutathione oxidation or changes in protein glutathionylation. Two types of targets of disulfide stress were identified: redox buffers, such as ribonuclease inhibitor or albumin, and redox-signaling thiols, which include thioredoxin 1, APE1/Ref1, Keap1, tyrosine and serine/threonine phosphatases, and protein disulfide isomerase. These targets exhibit great relevance in DNA repair, cell proliferation, apoptosis, endoplasmic reticulum stress, and inflammatory response. Disulfide stress would be a specific mechanism of redox signaling independent of glutathione redox status involved in inflammation.