Signalling of abscisic acid to regulate plant growth

Abscisic acid (ABA) mediated growth control is a fundamental response of plants to adverse environmental cues. The linkage between ABA perception and growth control is currently being unravelled by using different experimental approaches such as mutant analysis and microinjection experiments. So far, two protein phosphatases, ABI1 and ABI2, cADPR, pH, and Ca2+ have been identified as main components of the ABA signalling pathway. Here, the ABA signal transduction pathway is compared to signalling cascades from yeast and mammalian cells. A model for a bifurcated ABA signal transduction pathway exerting a positive and negative control mechanism is proposed.     

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.     

A molecular switch changes the signalling pathway used by the Fc gamma RI antibody receptor to mobilise calcium

BACKGROUND: Leukocytes express Fc gamma receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody-directed cellular cytotoxicity. The nature of the myeloid response to Fc gamma receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability. RESULTS: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fc gamma receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor Fc gamma RI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the gamma chain, an Fc gamma RI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of Fc gamma RI resulted in the Fc gamma RIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated. CONCLUSIONS: The switch in Fc gamma RI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by Fc gamma RI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.     

TNF-related ligands and their receptors

Multicellular organisms have the challenging task of coordinating the activities of many distinct cell types. This coordination is accomplished largely by cell-associated and soluble signalling molecules that act locally or distantly to alter target-cell physiology. The tumour necrosis factor family of cytokines are type II transmembrane proteins that are important regulators of homeostasis and have been implicated as mediators of disease. These molecules serve as ligands for a family of cell-surface receptors termed the tumour necrosis factor/nerve growth factor (TNF/NGF) receptor family. The receptors are type I transmembrane proteins capable of mediating a wide range of responses in vitro and in vivo. Signal transduction is mediated by several newly discovered cytoplasmic proteins that couple these receptors to downstream signalling events. The elucidation and use of spontaneously occurring mutants in TNF-related ligands and receptors in addition to gene-targeting experiments have begun to clarify the diverse biological effects mediated by this superfamily of cytokines.     

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.     

Chemical stability and human plasma pharmacokinetics of reduced folates

The vast majority of signalling pathways in mammalian cells are mediated by heterotrimeric (alpha betagamma) G proteins. Reviewed here is regulation of signal transduction by the betagamma complex at different protein interfaces: subunit-subunit, receptor-G protein and G protein-effector. The role of diverse beta and gamma subunit types in achieving specificity in signalling and potentially unidentified functions for these subunits also are discussed.     

The evolution of haematopoietic cytokine/receptor complexes

The evolutionary expansion of the haematopoietic cytokines and their receptors is characterized by the duplication of both cytokines and receptors. A systematic analysis of primary sequence homology indicates that receptors for gp130-associated cytokines group into signal transducing and non-signal transducing receptors. This observation is consistent with the evolution of the interleukins 6, 11 and 12, granulocyte colony stimulating factor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M, and the ciliary neurotrophic factor complexes from a common ancestral complex which included a homodimer of gp130-like signalling receptors and an interleukin 6 receptor-like non-signalling receptor. Alterations in the components of the complex are proposed to have arisen by receptor duplication and divergence to allow signal transduction via a LIF receptor/gp130 heterodimer, and loss of the non-signalling receptor component in the G-CSF and the LIF lineage. The short-chain haematopoietins and their receptors do not group clearly, although interleukins 4 and 13 grouped together, as did 2 and 10. Internal duplication of the ligand-binding domain appears to have occurred independently in three separate lineages. These observations have implications for the classification of cytokines and receptors, and for the modelling by homology of their structures and interactions.     

Activation of the signal transducer glycoprotein 130 by both IL-6 and IL-11 requires two distinct binding epitopes

The coordination and regulation of immune responses are primarily mediated by cytokines that bind to specific cell surface receptors. Glycoprotein 130 (gp130) belongs to the family of class I cytokine receptors and is the common signal-transducing receptor subunit shared by the so-called IL-6 type cytokines (IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and cardiotrophin-1). The inflammatory cytokines IL-6 and IL-11 induce gp130 homodimerization after binding to their specific alpha receptors, which leads to the activation of the Janus kinase/STAT signal transduction pathway. A molecular model of IL-6/IL-6R/gp130, which is based on the structure of the growth hormone/growth hormone receptor complex, allowed the selection of several amino acids located in the cytokine-binding module of gp130 for mutagenesis. The mutants were analyzed with regard to IL-6- or IL-11-induced STAT activation and ligand binding. It was found that Y190 and F191 are essential for the interaction of gp130 with IL-6 as well as IL-11, suggesting a common mode of recognition of helical cytokines by class I cytokine receptors. Furthermore, the requirement of the gp130 N-terminal Ig-like domain for ligand binding and signal transduction was demonstrated by the use of deletion mutants. Thus, besides the observed analogy to the growth hormone/growth hormone receptor complex, there is a substantial difference in the mechanism of receptor engagement by cytokines that signal via gp130.     

Assembly of the Drosophila phototransduction cascade into a signalling complex shapes elementary responses

The subcellular compartmentalization of signalling molecules helps to ensure the selective activation of different signal-transduction cascades within a single cell. Although there are many examples of compartmentalized signalling molecules, there are few examples of entire signalling cascades being organized as distinct signalling complexes. In Drosophila photoreceptors, the InaD protein, which consists of five PDZ domains, functions as a multivalent adaptor that brings together several components of the phototransduction cascade into a macromolecular complex. Here we study single-photon responses in several photoreceptor mutant backgrounds, and show that the InaD macromolecular complex is the unit of signalling that underlies elementary responses. We show that the localized activity of this signalling unit promotes reliable single-photon responses as well as rapid activation and feedback regulation. Finally, we use genetic and electrophysiological tools to illustrate how the assembly of signalling molecules into a transduction complex limits signal amplification in vivo.     

Signalling properties of glycosylphosphatidylinositols and their regulated release from membranes in the turnover of glycosylphosphatidylinositol-anchored proteins

Glycosylphosphatidylinositols are involved in transmembrane signalling, and their signal mediated release from the cell membrane has been demonstrated for several hormones and growth factors. Presently, however, only indirect evidence exists for the enzyme responsible for the signal mediated release of glycosylphosphatidylinositols. Indirect evidence from product identification led to the conclusion that in mammals the hormone sensitive activity is that of a glycosylphosphatidylinositol anchor hydrolyzing phospholipase C. On the other hand a mammalian glycosylphosphatidylinositol anchor hydrolyzing phospholipase D is well-established. This enzyme most likely functions in the intracellular turnover of glycosylphosphatidylinositols, however, its possible relation to the signalling properties of glycosylphosphatidylinositols remains unclear.     

Cellular uptake properties of a 2''-amino/2''-O-methyl-modified chimeric hammerhead ribozyme targeted to the epidermal growth factor receptor mRNA

PURPOSE: Rhabdomyosarcomas (RMS) are heterogeneous in their clinical presentation, histology, and cytogenetics. The growth of some RMS cells has been found to be regulated by the tyrosine kinase insulin-like growth factor (IGF) type I receptor. However, RMS cells exhibit variable sensitivity to inhibitors of tyrosine kinases and IGF receptors. Collectively, these heterogeneous features suggest that differences exist in the growth regulatory pathways of RMS. The objective of this study is to identify active tyrosine kinase signal transduction pathways in embryonal and alveolar RMS cells. METHODS: RMS tumor samples and cell lines representing both embryonal and alveolar histologic subtypes have been analyzed by immunoprecipitation and immunoblotting techniques to characterize phosphotyrosyl protein patterns and to identify tyrosine phosphorylated proteins. RESULTS: RMS cells can be characterized based on the patterns of phosphotyrosyl proteins, including the phosphorylation status of the catenin-like protein Cas1 and the signal adapter protein SHC, and the activation of IGF type I receptor signaling cascades including the formation of SHC-GRB2 signal protein complexes and MAP kinase activation. CONCLUSIONS: Rhabdomyosarcomas, especially the embryonal histologic subtype, are heterogeneous at the level of tyrosine kinase signal transduction. It will be important to characterize the growth regulatory pathways active in individual RMS tumors before targeting molecular therapies to this malignancy.     

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.     

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.     

Tyrosine phosphorylation of the MUC1 breast cancer membrane proteins. Cytokine receptor-like molecules

Phosphorylation on tyrosine residues is a key step in signal transduction pathways mediated by membrane proteins. Although it is known that human breast cancer tissue expresses at least 2 MUC1 type 1 membrane proteins (a polymorphic high molecular weight MUC1 glycoprotein that contains a variable number of tandem 20 amino acid repeat units, and the MUC1/Y protein that is not polymorphic and is lacking this repeat array) their function in the development of human breast cancer has remained elusive. Here it is shown that these MUC1 proteins are extensively phosphorylated, that phosphorylation occurs primarily on tyrosine residues and that following phosphorylation the MUC1 proteins may potentially interact with SH2 domain-containing proteins and thereby initiate a signal transduction cascade. As with cytokine receptors, the MUC1 proteins do not harbor intrinsic tyrosine kinase activity yet are tyrosine phosphorylated and the MUC1/Y protein participates in a cell surface heteromeric complex whose formation is mediated by two cytoplasmically located MUC1 cysteine residues. Furthermore, the MUC1/Y protein demonstrates sequence similarity with sequences present in cytokine receptors that are known to be involved in ligand binding. Our results demonstrate that the two MUC1 isoforms are both likely to function in signal transduction pathways and to be intimately linked to the oncogenetic process and suggest that the MUC1/Y protein may act in a similar fashion to cytokine receptors.     

POP66, a paraneoplastic encephalomyelitis-related antigen, is a marker of adult oligodendrocytes

L-selectin on lymphocytes reacts with glycosylated ligands on high endothelial venule walls in lymphoid organs. Through this carbohydrate-dependent interaction, rolling and initial attachment of lymphocytes to endothelium is mediated. Here we have studied an earlier described L-selectin-induced homotypic aggregation, to further elucidate the events that occur after engagement of L-selectin. It was found that the interaction of L-selectin with fucoidan, but not with other carbohydrates, or with monoclonal antibodies directed against the carbohydrate recognition domain of L-selectin, resulted in homotypic aggregation among both B- or T lymphocytes. Importantly, this aggregation was shown to be both lymphocyte function-associated antigen-1 (LFA-1) and calcium-independent. Furthermore, for aggregation metabolic energy was required, and signalling via protein tyrosine kinase appeared to be involved. Neither de novo protein synthesis, protein kinase C mediated signalling, Gi-protein mediated signal transduction, nor calcium mobilization were required for aggregation. During aggregation, L-selectin was not shed from the lymphocyte's cell surface. Finally, it was found that the lymphocyte binding capacity to high endothelial venules on cryostat sections was not altered upon triggering these lymphocytes via L-selectin. Interestingly, L-selectin-triggered cells showed increased binding to paracortical areas in peripheral lymph nodes. Our data suggest that signals via L-selectin, might lead to altered expression of cell surface molecules, important in interactions other than the first stage of lymphocyte rolling.     

The brain specific Ras exchange factor CDC25 Mm: modulation of its activity through Gi-protein-mediated signals

CDC25Mm is a mouse guanine nucleotide exchange factor specific for Ras, exclusively expressed in the brain. We used a reporter gene containing a Ras-responsive fos-promoter in order to gain information on the role played by this exchange factor in signal transduction. Transient expression of CDC25Mm in CHO cells activates Ras. Moreover serum, but not insulin, can upregulate the response mediated by CDC25Mm and this modulation requires that the CDC25Mm maintains its N-terminal region. NIH3T3 fibroblasts, stably overexpressing this exchange factor, show a partially transformed phenotype, suggesting that the Ras-dependent pathway is constitutively active. In these cells serum and lysophosphatidic acid (LPA) stimulate Ras activity above the basal level while PDGF does not. Both serum and LPA-induced Ras activations in CDC25Mm overexpressing cells can be completely inhibited by pertussis toxin. Moreover, these responses are strongly reduced by coexpression of a truncated version of CDC25Mm lacking the C-terminal catalytic portion. This construct behaves in a dominant negative manner suggesting that it may compete with CDC25Mm by sequestering in an unproductive way signalling components activated by these factors. The data presented indicate that CDC25Mm does not participate in connecting tyrosine kinase receptors with Ras, while it could mediate Ras activation induced by pertussis toxin sensitive Gi-coupled receptors.