Effects of staurosporine, okadaic acid and sodium fluoride on protein phosphorylation in graviresponding oat shoot pulvini.

This study focuses on the characterization of protein phosphorylation in the gravitropic response in oat shoot pulvini through the use of inhibitors of this process, namely staurosporine, okadaic acid and sodium fluoride. These three inhibitors reduce gravitropic curvature and cause changes in the phosphorylation of 38- and 50-kDa soluble proteins which show different levels of phosphorylation between lower and upper halves of gravistimulated pulvini. A kinetic analysis of phosphorylation shows that the 38- and 50-kDa soluble proteins exhibit different levels of phosphorylation between lower and upper halves of graviresponsive pulvini at 5 min after initiation of gravistimulation of stems. In addition, the phosphorylation of 63- and 70-kDa proteins from a total membrane preparation increases in lower halves of the pulvini following gravistimulation. These phosphoproteins are not found in the plasma membrane fraction. Taken together, at least four kinds of phosphoproteins are gravi-related. Of these, the 38- and 50-kDa soluble phosphoproteins may be involved in the regulation of early stages of the gravitropic response.     

Changes in starch content in oat (Avena sativa) shoot pulvini during the gravitropic response.

In order to determine if components of the signal transduction pathway are involved in starch metabolism during the gravitropic response, the effects of inhibitors of phosphoprotein phosphatases and protein kinases (OA), and calcium channel blockers (LaCl3), on gravitropic bending and starch levels in gravisensitive node/pulvini of oat shoots were examined. Among the compounds tested, okadaic acid (OA) and lanthanum chloride (LaCl3) showed the strongest inhibitory effects on the negative gravitropic curvature response in oat shoot node/pulvini. At the same time, they caused a rapid loss of starch in graviresponding pulvini based on a quantitative analysis of starch levels in the bending tissues over 48 h periods. These two compounds act initially to block the net increase in starch content that occurs during the early stages (0-9 h) in graviresponding oat shoot pulvini. As a result, starch levels drop precipitously in shoots treated with OA and LaCl3, starting at time zero of gravistimulation by reorientation. These findings suggest that protein dephosphorylation and calcium play a role in starch metabolism in oat shoot pulvini in response to a gravistimulation signal. They also indicate that the amount of starch present in the chloroplast gravisensors in oat shoot pulvini may determine the rate of upward bending in graviresponding pulvini.     

A role for inositol 1,4,5-trisphosphate in gravitropic signaling and the retention of cold-perceived gravistimulation of oat shoot pulvini

Plants sense positional changes relative to the gravity vector. To date, the signaling processes by which the perception of a gravistimulus is linked to the initiation of differential growth are poorly defined. We have investigated the role of inositol 1,4,5-trisphosphate (InsP(3)) in the gravitropic response of oat (Avena sativa) shoot pulvini. Within 15 s of gravistimulation, InsP(3) levels increased 3-fold over vertical controls in upper and lower pulvinus halves and fluctuated in both pulvinus halves over the first minutes. Between 10 and 30 min of gravistimulation, InsP(3) levels in the lower pulvinus half increased 3-fold over the upper. Changes in InsP(3) were confined to the pulvinus and were not detected in internodal tissue, highlighting the importance of the pulvinus for both graviperception and response. Inhibition of phospholipase C blocked the long-term increase in InsP(3), and reduced gravitropic bending by 65%. Short-term changes in InsP(3) were unimpaired by the inhibitor. Gravitropic bending of oat plants is inhibited at 4 degrees C; however, the plants retain the information of a positional change and respond at room temperature. Both short- and long-term changes in InsP(3) were present at 4 degrees C. We propose a role for InsP(3) in the establishment of tissue polarity during the gravitropic response of oat pulvini. InsP(3) may be involved in the retention of cold-perceived gravistimulation by providing positional information in the pulvini prior to the redistribution of auxin.     

Changes in starch and inositol 1,4,5-trisphosphate levels and auxin transport are interrelated in graviresponding oat (Avena sativa) shoots

This study was conducted to unravel a mechanism for the gravitropic curvature response in oat (Avena sativa) shoot pulvini. For this purpose, we examined the downward movement of starch-filled chloroplast gravisensors, differential changes in inositol 1,4,5-trisphosphate (IP(3)) levels, transport of indole-3-acetic acid (IAA) and gravitropic curvature. Upon gravistimulation, the ratio for IAA levels in lower halves versus those in upper halves (L/U) increased from 1.0 at 0 h and reached a maximum value of 1.45 at 8 h. When shoots were grown in the dark for 10 d, to deplete starch in the chloroplast, the gravity-induced L/U of IAA was reduced to 1.0. N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both auxin transport inhibitors, significantly reduced the amount of gravitropic curvature and gravity-induced lateral IAA transport, but did not reduce the gravity-induced late change in the L/U ratio of IP(3) levels. U73122, a specific phospholipase C (PLC) inhibitor, decreased gravity-induced curvature. Because U73122 reduced the ratio of L/U of IAA imposed by gravistimulation, it is clear that IAA transport is correlated with changes in IP(3) levels upon gravistimulation. These results indicate that gravistimulation-induced differential lateral IAA transport may result from the onset of graviperception in the chloroplast gravisensors coupled with gravity-induced asymmetric changes in IP(3) levels in oat shoot pulvini.     

Changes in the ultrastructure of cell walls, cellulose synthesis, and glucan synthase activity from gravistimulated pulvini of oat (Avena sativa)

Ultrastructural analyses of the cell walls from top and bottom halves of gravistimulated pulvini from oat leaves show a decrease in the density of material within the cell walls from the lower halves of pulvini after 24 h of gravistimulation. Assays of cellulose synthesis with a 14C-sucrose pulse-chase experiment indicate no difference in the amount of new cellulose synthesized in top compared with bottom halves of gravistimulated pulvini. The highest rate of cellulose synthesis occurs with 12-24 h of gravistimulation. Treatment of graviresponding pulvini with 2,6-dichlorobenzonitrile (DCBN) had only a minor effect on segment gravitropic curvature. We also found that there is no difference in the activities of either glucan synthase I or glucan synthase II in top halves as compared with bottom halves of gravistimulated pulvini. We conclude that the graviresponse in oat stems is not driven by new cell wall synthesis but, rather, by changes in cell wall plasticity and osmotic potential.     

Auxin-induced delay of abscission: The involvement of calcium ions and protein phosphorylation in bean explants

Treatment with Ca²⁺ channel blockers such as lanthanum chloride and verapamil promoted abscission in pulvinar explants of bean ( Phaseolus vulgaris L. ev. Pirate). In addition, auxin-induced delay of abscission was markedly reduced in the presence of Ca²⁺ channel blockers. In vitro phosphorylation studies were performed using membrane preparations (130000 g pellet) from freshly excised as well as auxintreated and control (minus auxin) pulvinar sections. Auxin-treated sections showed a 66 kDa phosphoprotein as well as Ca²⁺ -dependent phosphorylation that were not observed in control explants.
Coomassie blue stammg of soluble proteins (130000 g supernatent) separated on SDS-PAGE revealed the presence of 62. 55 and 47 kDa polypeptides only in the freshly excised pulvini. However. no distinct changes were observed in soluble protein profile between auxin-treated and control explants. When soluble proteins were phosphorylated in vitro, Ca²⁺ promoted the phosphorylation of 92, 55. 40 and I7 kDa polypeptides only in freshly excised pulvmi. Ca²⁺-dependent phosphorylation of soluble proteins was not observed in either the control or auxin-treated explants. In addition. in vivo phosphorylation studies were performed using freshly excised. auxrn-treated and control explants. Freshly excused segments and auxin-treated ex-plants showed similar phosphoproteins, which were different from those observed mcontrol.     

Increased levels of reactive oxygen species and expression of a cytoplasmic aconitase/iron regulatory protein 1 homolog during the early response of maize pulvini to gravistimulation

The maize (Zea mays L.) stem pulvinus is a disc of tissue located apical to each node that functions to return a tipped stem to a more upright position via increased cell elongation on its lower side. We investigated the possibility that reactive oxygen species (ROS) and hydrogen peroxide (H2O2), in particular, are involved in the gravitropic response of the pulvinus prior to initiation of the growth response by employing the cytochemical stain 3,3'-diaminobenzidine (DAB). DAB polymers were found in the bundle sheath cells of gravistimulated pulvini in association with amyloplasts after 1 min of gravistimulation, and the signal spread throughout the cytosol of these cells by 30 min. Furthermore, treatment of maize stem explants containing pulvini with 1 mm H2O2 on their upper sides caused reversal of bending polarity. Similar, though less dramatic, results were obtained via application of 1 mm ascorbic acid to the lower side of the explants. In addition, we determined that a maize cytoplasmic aconitase/iron regulatory protein 1 (IRP1) homolog is up-regulated in the pulvinus bundle sheath cells after gravistimulation using suppressive subtractive hybridization PCR (SSH PCR), real-time RT-PCR and in situ hybridization. Although we do not yet know the role of the IRP1 homolog in the pulvinus, the protein is known to be a redox sensor in other systems. Collectively, our results point to an increase in ROS quite early in the gravitropic signalling pathway and its possible role in determining the direction of bending of the pulvini. We speculate that an ROS burst may serve to link the physical phenomenon of amyloplast sedimentation to the changes in cellular biochemistry and gene expression that facilitate directional growth.     

Characterization of a CK II protein kinase from etiolated oat seedlings

Protein kinases play a central role in controlling the cellular metabolism of living organisms. A protein kinase was purified from etiolated oat seedlings by several steps of ion-exchange and affinity chromatographies. The kinase was a 150-kDa tetrameric protein and composed of three subunits of 34, 37, and 40 kDa proteins. The 34 and 40 kDa proteins had ATP binding sites, suggesting that they are catalytic subunits and that the 37-kDa protein is a regulatory subunit. In the in vitro phosphorylation of a crude oat cell extract, it intensively phosphorylated a serine residue of a 110-kDa protein. The 110-kDa protein was tentatively identified as a DNA topoisomerase I, based on an amino acid sequence homology. Phosphorylation of the 110-kDa protein by the kinase required ATP or GTP as a phosphoryl group donor. The kinase activity was inhibited by 50% at a concentration of 0.05 microg/ml heparin. These results, therefore, indicate that the purified kinase is a CK II protein kinase and may be involved in the regulation of DNA topoisomerase I activity.     

Induction of tyrosine phosphorylated proteins in THP-1 cells by Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae porins

The effect of porins purified from Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae on induction of tyrosine phosphorylation in THP-1 cells and C3H/HeJ macrophage was investigated. Incubation of porins at concentration of 1.0-5.0 microg ml(-1) with either THP-1 or macrophage from C3H/HeJ mice resulted in tyrosine phosphorylation of specific host cell proteins. After porin stimulation a pattern of tyrosine phosphorylated proteins appeared in the soluble cytoplasmic fraction, in the membrane fraction and in the insoluble protein fraction. The observed effects were dependent on the porin concentrations; they reached a maximal expression at 3 min and declined at 60 min. Porin and lipopolysaccharide treatments induce a similar phosphorylation pattern in all of the three cellular fractions studied. A difference can be observed in the cytoplasmic fraction bands of 50-60 kDa, which are more evident after treatment with lipopolysaccharide, and in the insoluble fraction band of 80 kDa and the cytoplasmic fraction band of 250 kDa, which are more evident after treatment with porins. The events of tyrosine protein phosphorylation were present in macrophage from lipopolysaccaride-hyporesponsive C3H/HeJ mice stimulated with porins, while they were markedly reduced when the cells were stimulated with lipopolysaccharide. Staurosporine, genistein and cytochalasin D induced in the three cellular fractions a different inhibition pattern of tyrosine protein phosphorylation in porin stimulated cells. Porins extracted from the three bacterial species investigated behave in a similar way as stimuli more or less potent; Hib porin seems to be the most powerful stimulator and, moreover, it specifically induces phosphorylation of a 55 kDa band.     

Low water potentials affect expression of genes encoding vegetative storage proteins and plasma membrane proton ATPase in soybean

We have examined growth, water status and gene expression in dark-grown soybean (Glycine max L. Merr.) seedlings in response to water deficit (low water potentials) during the first days following germination. The genes encoded the plasma membrane proton ATPase and two proteins of 28 kDa and 31 kDa putatively involved in vegetative storage. Water potentials of stems and roots decreased when 2-day-old seedlings were transferred to water-saturated air. Stem growth was inhibited immediately. Root growth continued at control rates for one day and then was totally inhibited when the normal root-stem water potential gradient was reversed.Expression of mRNA for the 28 kDa and 31 kDa proteins, measured independently using specific 3-end probes, occurred about equally in stems. However, only the mRNA for the 31 kDa protein was detected in roots and at a lower abundance than in stems. Low water potentials increased the mRNA only for the 28 kDa protein in stems and the 31 kDa protein in roots. This differential expression followed the inhibition of stem growth but preceded the inhibition of root growth.The expression of the message for the ATPase, measured using a probe synthesized from a partial oat ATPase clone, was low in stems and roots but there was a 6-fold increase at low water potentials in roots. The increase followed the inhibition of root growth. This appears to be the first instance of regulation of ATPase gene expression in plants and the first demonstration of differential expression of the 28 kDa, 31 kDa, and ATPase messages. The correlation with the differential growth responses of the stems and roots raises the possibility that the differential gene expression could be involved in the growth response to low water potentials.     

Calcium Requirement for α-MSH Action on Melanophores: Studies with Forskolin

Abstract

α-MSH-induced pigment dispersion in melanophores shows an absolute requirement for extracellular Ca²⁺. To localize Ca²⁺ sites involved in the mechanism of action of α-MSH we studied the effects of Ca²⁺ deprivation on α-MSH and forskolin-induced melanophore responses. In an in vitro melanophore system employing ventral tailfins of Xenopus tadpoles, melanophore responses were assayed in terms of pigment dispersion and the phosphorylation state of a 53 kDa melanophore-specific protein. In the same melanophore system α-MSH has been shown to specifically increase the phosphorylation of this 53 kDa protein.

Forskolin induces a dose-dependent pigment dispersion (EC₅₀ 7 × 10^?7 M). In contrast to the dispersion induced by α-MSH forskolin-induced dispersion does not require extracellular Ca²⁺. Moreover, in a Ca²⁺-free medium melanophores with permanently activated MSH-receptors aggregate, but can be redispersed by the addition of forskolin. Forskolin increases 53 kDa phosphorylation in a dosedependent manner. Maximal stimulation with forskolin (10^?5 M) is four-fold and equals maximal 53 kDa phosphorylation obtainable with α-MSH. The MSH-induced increase in 53 kDa phosphorylation is inhibited by Ca²⁺ deprivation, whereas the forskolin-induced increase is unaffected. Our results suggest that α-MSH and forskolin stimulate melanophores through a common pathway and confirm that cAMP is a second messenger in α-MSH action in this system. We conclude that the Ca²⁺ sites in the mechanism of α-MSH action on melanophores precede adenylate cyclase activation.     

Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle cells

Aberrant vascular smooth muscle cell (VSMC) hyperplasia is the hallmark of atherosclerosis and restenosis seen after vascular surgery. Heparin inhibits VSMC proliferation in animal models and in cell culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering phosphorylation of key mitogenic signaling proteins in VSMC, we examined tyrosine phosphorylation of cellular proteins in quiescent VSMC stimulated with serum in the presence or absence of heparin. Western blot analysis with anti-phosphotyrosine antibodies shows that heparin specifically alters the tyrosine phosphorylation of only two proteins (42 kDa and 200 kDa). The 200 kDa protein (p200) is dephosphorylated within 2.5 min after heparin treatment with an IC50 that closely parallels the IC50 for growth inhibition. Studies using the tyrosine phosphatase inhibitor, sodium orthovanadate, indicate that heparin blocks p200 phosphorylation by inhibiting a kinase. Phosphorylation of p200 is not altered in heparin-resistant cells, supporting a role for p200 in mediating the antiproliferative effect of heparin. Purification and sequence analysis indicate that p200 exhibits very high homology to the heavy chain of nonmuscle myosin IIA. The 42 kDa protein, identified as mitogen activated protein kinase (MAPK), undergoes dephosphorylation within 15 min after heparin treatment, and this effect is also not seen in heparin-resistant cells. The identification of only two heparin-regulated tyrosine phosphoproteins suggests that they may be key mediators of the antiproliferative effect of heparin.     

Do Starch Statoliths Act as the Gravisensors in Cereal Grass Pulvini?

To determine if starch statoliths do, in fact, act as gravisensors in cereal grass shoots, starch was removed from the starch statoliths by placing 45-day-old intact barley plants (Hordeum vulgare cv `Larker') in the dark at 25°C for 5 days. Evidence from staining with I₂-KI, scanning electron microscopy, and transmission electron microscopy indicated that starch grains were no longer present in plastids in the pulvini of plants placed in the dark for 5 days. Furthermore, gravitropic curvature response in these pulvini was reduced to zero, even though pulvini from vertically oriented plants were still capable of elongating in response to applied auxin plus gibberellic acid. However, when 0.1 molar sucrose was fed to the dark pretreated, starch statolith-free pulvini during gravistimulation in the dark, they not only reformed starch grains in the starch-depleted plastids in the pulvini, but they also showed an upward bending response. Starch grain reformation appeared to precede reappearance of the graviresponse in these sucrose-fed pulvini. These results strongly support the view that starch statoliths do indeed serve as the gravisensors in cereal grass shoots.     

Evidence for autophosphorylation of hyaluronate binding protein and its enhanced phosphorylation in rat histiocytoma.

This report documents for the first time the in vitro autophosphorylation of purified 68 kDa hyaluronate binding protein in presence of [32P] ATP. The rate of phosphorylation is proportional to the concentration of protein and to the time of incubation up to 5 min. By both phosphoamino acid and western blot analysis with antiphosphotyrosine antibodies, we have confirmed that the phosphorylation occurs at tyrosine residues. Immunoprecipitation with anti HA binding protein antibody shows a 5 fold increase in the phosphorylation in macrophage histiocytoma compared to normal macrophage. Supplementing hyaluronate with hyaluronate binding protein in the medium is further shown to enhance total protein phosphorylation in rat histiocytoma.     

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.     

Effects of neurotensin on caudate nucleus protein phosphorylation.

The tridecapeptide, neurotensin (NT), is heterogenously distributed in the mammalian central nervous system and exhibits many neurotransmitter-like characteristics. However, the molecular mechanisms of NT signal transduction remain obscure. In this report, we demonstrate NT-induced stimulation of specific protein substrate phosphorylation in the rat caudate nucleus. Rat caudate nucleus was dissected, a P2 fraction prepared and proteins phosphorylated in vitro with [32P]ATP for 1 min. Phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiograms prepared. NT preincubation in the absence of calcium resulted in markedly increased phosphorylation in vitro of proteins with apparent molecular weights of 80,000 and 50,000. These effects were not observed if calcium was present during the NT preincubation period. Both calcium and cAMP enhanced phosphorylation of the 80 kDa protein, but phosphorylation of the 50 kDa protein was responsive only to calcium.     

Protein kinase(s) in bovine brain coated vesicles

Purified bovine brain coated vesicles contain protein kinase activity which phosphorylates 165, 54 and 50 kDa protein substrates. These phosphorylations do not seem to be induced by a unique protein kinase: indeed, the three substrates present different localizations in coated vesicles, the phosphorylation sites are either serine or threonine residues and vanadate and ATP[gamma S] have different effects on 32P incorporation in the substrates. Comparison of the coated vesicle protein and phosphorylation patterns from different tissues and animal origins shows that only the 50 kDa protein phosphorylation is always observed, compared to the great diversity in other minor phosphorylations which are observed or not in the various coated vesicles. The possible presence of a 50 kDa phosphoprotein phosphatase is also discussed. It is suggested that the 50 kDa protein with its connected specific kinase and phosphatase seems to constitute a regulatory system present in coated vesicles.