Protein phosphatase 2C (PP2C) function in higher plants

In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases. Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals. This review will focus on the plant PP2C family of protein phosphatases. Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes. For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction. Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA. Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways. Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway. Thus, the plant PP2Cs function as regulators of various signal transduction pathways.     

An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene expression in barley aleurone layers

The phytohormone abscisic acid (ABA) induces genes-encoding proteins involved in desiccation tolerance and dormancy in seeds, but ABA also suppresses gibberellin (GA)-responsive genes encoding hydrolytic enzymes essential for postgermination growth. A unique serine/threonine protein kinase, PKABA1 mRNA, up-regulated by ABA in seeds, has been identified. In this report, the effect of PKABA1 on the signal transduction pathway mediating ABA induction and suppression of genes has been determined in aleurone layers of barley seeds. Two groups of gene constructs were introduced to barley aleurone layers by using particle bombardment: the reporter constructs containing the coding sequence of beta-glucuronidase gene linked to hormone-responsive promoters and the effector constructs containing the coding region of protein kinases linked to a constitutive promoter. Constitutive expression of PKABA1 drastically suppressed expression of low- and high-pI alpha-amylase and protease genes induced by GA. However, the presence of PKABA1 had only a small effect on the ABA induction of a gene encoding a late embryogenesis abundant protein, HVA1. Our results indicate that PKABA1 acts as a key intermediate in the signal transduction pathway leading to the suppression of GA-inducible gene expression in cereal aleurone layers.     

A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana

The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, the maintenance of seed dormancy, and the inhibition of plant growth. All three responses are affected in the ABA-insensitive mutant abi1 of Arabidopsis thaliana, suggesting that an early step in the signaling of ABA is controlled by the ABI1 locus. The ABI1 gene was cloned by chromosome walking, and a missense mutation was identified in the structural gene of the abi1 mutant. The ABI1 gene encodes a protein with high similarity to protein serine or threonine phosphatases of type 2C with the novel feature of a putative Ca2+ binding site. Thus, the control of the phosphorylation state of cell signaling components by the ABI1 product could mediate pleiotropic hormone responses.     

Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose

Abscisic acid (ABA) is a plant hormone involved in the response of plants to reduced water availability. Reduction of guard cell turgor by ABA diminishes the aperture of the stomatal pore and thereby contributes to the ability of the plant to conserve water during periods of drought. Previous work has demonstrated that cytosolic Ca2+ is involved in the signal transduction pathway that mediates the reduction in guard cell turgor elicited by ABA. Here we report that ABA uses a Ca2+-mobilization pathway that involves cyclic adenosine 5'-diphosphoribose (cADPR). Microinjection of cADPR into guard cells caused reductions in turgor that were preceded by increases in the concentration of free Ca2+ in the cytosol. Patch clamp measurements of isolated guard cell vacuoles revealed the presence of a cADPR-elicited Ca2+-selective current that was inhibited at cytosolic Ca2+ >/= 600 nM. Furthermore, microinjection of the cADPR antagonist 8-NH2-cADPR caused a reduction in the rate of turgor loss in response to ABA in 54% of cells tested, and nicotinamide, an antagonist of cADPR production, elicited a dose-dependent block of ABA-induced stomatal closure. Our data provide definitive evidence for a physiological role for cADPR and illustrate one mechanism of stimulus-specific Ca2+ mobilization in higher plants. Taken together with other recent data [Wu, Y., Kuzma, J., Marechal, E., Graeff, R., Lee, H. C., Foster, R. & Chua, N.-H. (1997) Science 278, 2126-2130], these results establish cADPR as a key player in ABA signal transduction pathways in plants.     

Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as alpha-amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.     

Pharmacokinetics of fusidic acid after a single dose of a new paediatric suspension

Cell proliferation in response to growth factors is mediated by specific high affinity receptors. Ligand-binding by receptors of the protein tyrosine kinase family results in the stimulation of several intracellular signal transduction pathways. Key signalling enzymes are recruited to the plasma membrane through the formation of stable complexes with activated receptors. These interactions are mediated by the conserved, non-catalytic SH2 domains present in the signalling molecules, which bind with high affinity and specificity to tyrosine-phosphorylated sequences on the receptors. The assembly of enzyme complexes is emerging as a major mechanism of signal transduction and may regulate the pleiotropic effects of growth factors.     

Gene structure and expression analysis of the drought- and abscisic acid-responsive CDeT11-24 gene family from the resurrection plant Craterostigma plantagineum Hochst

In order to understand the molecular mechanisms which are responsible for desiccation tolerance in the resurrection plant Craterostigma plantagineum Hochst. a thorough analysis of the CDeT11-24 gene family was performed. CDeT11-24 comprises a small gene family whose genes are expressed in response to dehydration, salt stress and abscisic acid (ABA) treatment in leaves. The gene products are constitutively expressed in roots and disappear only when the plants are transferred to water. It is therefore suggested that the proteins are involved in sensing water status. The predicted proteins are very hydrophilic; they share some features with late-embryogenesis-abundant proteins, and sequence similarities were found with two ABA- and drought-regulated Arabidopsis genes. The analysis of beta-glucuronidase reporter genes driven by the CDeT11-24 promoter showed high activity in mature seeds in both transgenic Arabidopsis and tobacco. In vegetative tissues the promoter activity in response to ABA was restricted to young Arabidosis seedlings. The responsiveness to ABA during later developmental stages was regained in the presence of the Arabidopsis gene product ABI3. Dehydration-induced promoter activity was only observed in Arabidopsis leaves at a particular developmental stage. This analysis indicates that some components in the signal transduction pathway of the resurrection plant are not active in tobacco or Arabidopsis.     

C-ABI3, the carrot homologue of the Arabidopsis ABI3, is expressed during both zygotic and somatic embryogenesis and functions in the regulation of embryo-specific ABA-inducible genes

A carrot gene homologous to the ABI3 gene of Arabidopsis was isolated from a carrot somatic embryo cDNA library and designated C-ABI3. The sequence of C-ABI3 was very similar to those of ABI3 of Arabidopsis and VP1 of maize in certain conserved regions. The expression of C-ABI3 was detected specifically in embryogenic cells, somatic embryos and developing seeds. Thus, expression of C-ABI3 was limited to tissues that acquired desiccation tolerance in response to endogenous or exogenous abscisic acid (ABA). Endogenous levels of ABA in seeds increased transiently and then desiccation of seeds started. The expression of C-ABI3 in developing seeds was observed prior to the increase in levels of endogenous ABA that was followed by desiccation of seeds. In transgenic mature leaves in which C-ABI3 was ectopically expressed, expression of ECP31, ECP63 and ECP40 was induced by treatment with ABA, which indicates that the expression of ECP genes was controlled by the pathway(s) that involved C-ABI3 and ABA. This suggests that C-ABI3 has the same function as VP1/ABI3 factor in carrot somatic embryos.     

Hedgehog and its patched-smoothened receptor complex: a novel signalling mechanism at the cell surface

Pattern formation and morphogenesis depend on the careful execution of complex genetic programs, which are conserved in multicellular organisms. An important signal in some of these programs in Drosophila and vertebrates is the secreted Hedgehog (Hh) protein, which primarily functions as an inducer of morphogenetic signals. The Hh signal plays a decisive role in such critical developmental processes as neurulation and somite and limb formation. The Hh signalling pathway exhibits a novel mechanism of signal reception and transduction. In the absence of the Hh signal, the membrane protein Patched (Ptc) represses the constitutive signalling activity of a second membrane protein, Smoothened (Smo), by virtue of its ability to form a Ptc-Smo complex. Hence, mutations within the ptc gene that result in the failure of Ptc to inhibit Smo lead to constitutive activity of the Hh signalling pathway and to cancer, such as basal cell carcinoma. For activation of Hh-target genes, the N-terminal signalling domain of Hh binds to the Ptc-Smo receptor complex to activate two parallel signalling pathways. Furthermore, Hh limits its own range of action by impeding its diffusion through (i) covalent linkage of its N-terminal signalling moiety to cholesterol, mediated by the cholesterol transferase activity of its C-terminal moiety, and (ii) induction of, and sequestration by, its antagonist, Ptc.     

A higher plant seven-transmembrane receptor that influences sensitivity to cytokinins

BACKGROUND: All organisms perceive and respond to a profusion of environmental and endogenous signals that influence growth, development and behavior. The G-protein signalling pathway is a highly conserved mechanism for transducing extracellular signals, and the superfamily of receptors that have seven transmembrane (7TM) domains is a primary element of this pathway. Evidence that heterotrimeric G proteins are involved in signal transduction in plants is accumulating, prompting speculation that plant 7TM receptors might exist. RESULTS: Using information in the dbEST database of expressed sequence tags, we isolated an Arabidopsis thaliana gene (GCR1) that encodes a protein with seven predicted membrane-spanning domains and other features characteristic of 7TM receptors. The protein shows 18-23% amino-acid identity (46-53% similarity) to, and good colinear alignment with, 7TM receptors from three different families. Its highest sequence identity is with the Dictyostelium cAMP receptors. GCR1 is expressed at very low levels in the roots, stems and leaves of Arabidopsis; it is a single-copy gene which maps close to the restriction fragment length polymorphism marker m291 on chromosome 5. Transgenic Arabidopsis expressing antisense GCR1 under the control of the constitutive cauliflower mosaic virus 35S promoter have reduced sensitivity to cytokinins in roots and shoots, yet respond normally to all other plant hormones. This suggests a functional role for GCR1 in cytokinin signal transduction. CONCLUSIONS: GCR1 encodes the first 7TM receptor homologue identified in higher plants and is involved in cytokinin signal transduction. This discovery suggests that 7TM receptors are ancient and predate the divergence of plants and animals.     

Synthesis and radiopharmacology of O-(2-[18F]fluoroethyl)-L-tyrosine for tumor imaging

The impact of simultaneous environmental stresses on plants and how they respond to combined stresses compared with single stresses is largely unclear. By using a transgene (RD29A-LUC) consisting of the firefly luciferase coding sequence (LUC) driven by the stress-responsive RD29A promoter, we investigated the interactive effects of temperature, osmotic stress, and the phytohormone abscisic acid (ABA) in the regulation of gene expression in Arabidopsis seedlings. Results indicated that both positive and negative interactions exist among the studied stress factors in regulating gene expression. At a normal growth temperature (22 degrees C), osmotic stress and ABA act synergistically to induce the transgene expression. Low temperature inhibits the response to osmotic stress or to combined treatment of osmotic stress and ABA, whereas low temperature and ABA treatments are additive in inducing transgene expression. Although high temperature alone does not activate the transgene, it significantly amplifies the effects of ABA and osmotic stress. The effect of multiple stresses in the regulation of RD29A-LUC expression in signal transduction mutants was also studied. The results are discussed in the context of cold and osmotic stress signal transduction pathways.     

Participation of phosphoinositide-specific phospholipase Cgamma1 and STAT1 protein in the formation of latent complexes of signal proteins

It is known that the growth factor activates appropriate membrane receptors which become starting points of cascades of protein-protein interactions leading to cellular response. Recent data suggest that different signalling pathways may cross-talk during the cellular response. Here we show that phosphoinositide-specific phospholipase C gamma 1, one of the key elements in phosphoinositide pathway of signal transduction, is physically associated with members of the STAT pathway. The precipitation of phospholipase C gamma 1, using polyclonal antibody in A-431 cells, leads to co-immunoprecipitation of STAT1 alpha and STAT1 beta, as well as STAT3. The formation of such complexes was observed in both unstimulated and EGF stimulated cells. The participation of SH3-domains in the formation of such complexes is discussed.     

A subset of notch functions during Drosophila eye development require Su(H) and the E(spl) gene complex

The Notch signalling pathway is involved in many processes where cell fate is decided. Previous work showed that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signalling represses atonal expression during lateral specification. In this paper we investigate which known components of the Notch pathway are involved in each signalling process. Using clonal analysis we show that a ligand of Notch, Delta, is required along with Notch for both proneural enhancement and lateral specification, while the downstream components Suppressor-of-Hairless and Enhancer-of-Split are involved only in lateral specification. Our data point to a distinct signal transduction pathway during proneural enhancement by Notch. Using misexpression experiments we also show that particular Enhancer-of-split bHLH genes can differ greatly in their contribution to lateral specification.     

The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease

The Saccharomyces cerevisiae targets of rapamycin, TOR1 and TOR2, signal activation of cell growth in response to nutrient availability. Loss of TOR or rapamycin treatment causes yeast cells to arrest growth in early G1 and to express several other physiological properties of starved (G0) cells. As part of this starvation response, high affinity amino acid permeases such as the tryptophan permease TAT2 are targeted to the vacuole and degraded. Here we show that the TOR signalling pathway phosphorylates the Ser/Thr kinase NPR1 and thereby inhibits the starvation-induced turnover of TAT2. Overexpression of NPR1 inhibits growth and induces the degradation of TAT2, whereas loss of NPR1 confers resistance to rapamycin and to FK506, an inhibitor of amino acid import. NPR1 is controlled by TOR and the type 2A phosphatase-associated protein TAP42. First, overexpression of NPR1 is toxic only when TOR function is reduced. Secondly, NPR1 is rapidly dephosphorylated in the absence of TOR. Thirdly, NPR1 dephosphorylation does not occur in a rapamycin-resistant tap42 mutant. Thus, the TOR nutrient signalling pathway also controls growth by inhibiting a stationary phase (G0) programme. The control of NPR1 by TOR is analogous to the control of p70 s6 kinase and 4E-BP1 by mTOR in mammalian cells.     

Assessment of the toxicity of TAH on the cell lines of SO-Rb50 and SO-Rb70]

Appropriate cell-to-substrate adhesion together with SGF stimulation is necessary to initiate and continue cell cycle progression of growth arrested cells. Adhesion-dependent signaling events, which likely occur through integrin receptors specifically organized with cytoskeletal components within focal contacts, can induce expression of specific genes and stimulate quiescent cells into the growth cycle. The mechanisms as to how: (1) cell-to-substrate adhesion complexes are formed and maintained, (2) adhesion-dependent signal transduction events interface with SGF initiated signalling events, (3) adhesion influences expression of growth-state regulated genes, and (4) an appropriate cytoarchitectural environment may coordinate these events to regulate cellular growth are unclear. While it is apparent that defining these mechanisms would be critical to understanding the basic events which control cell growth, many of the mechanisms are just beginning to be addressed and understood.     

Ethanol inhibits hepatocyte proliferation in insulin receptor substrate 1 transgenic mice

BACKGROUND & AIMS: Long-term ethanol consumption is known to impair the ability of the liver to regenerate, but the molecular mechanisms are poorly understood. Multiple growth factors promote hepatocyte proliferation, some of which involve the insulin receptor substrate 1 (IRS-1)-mediated signal transduction pathway. To explore effects of ethanol on the IRS-1 signal liver growth in vivo, studies in transgenic mice overexpressing IRS-1 in the liver were performed because these mice show constitutive activation of the downstream signal transduction pathways leading to enhanced hepatocyte proliferation. METHODS: Tyrosyl phosphorylation of IRS-1 and subsequent protein-protein interactions were examined in liver lysates from animals fed ethanol or control diet. Activity of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) was assessed by specific enzymatic assays. Hepatocyte proliferation was measured by incorporation of [3H]thymidine into liver DNA. RESULTS: Tyrosyl phosphorylation of IRS-1, association of IRS-1 with PI3K, and activation of downstream PI3K and MAPK pathways were greatly reduced as a result of long-term ethanol consumption. Ethanol virtually abolished the enhanced hepatocyte DNA synthesis induced by expression of the IRS-1 transgene. CONCLUSIONS: Altered transmission of growth signals through the IRS-1-mediated signal transduction cascade may represent a molecular mechanism of how ethanol inhibits hepatocyte proliferation.