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Recent progress in understanding signal transduction owes much to new genetic approaches, first by unraveling the molecular basis of classic mutations, and then by the use of gene targeting. Recent studies have examined mammalian signal transduction from cell surface to nucleus, especially ligand-receptor systems and cytosolic signal transducers.
In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951-962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (Kd approximately 5 nM), only CT elicited a cAMP-dependent Cl- secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.
Each cytokine which utilizes the Jak-Stat signal transduction pathway activates a distinct combination of members of the Jak and Stat families. Thus, either the Jaks, the Stats, or both could contribute to the specificity of ligand action. With the use of chimeric receptors involving the interferon gamma receptor (IFN-gammaR) complex as a model system, we demonstrate that Jak2 activation is not an absolute requirement for IFN-gamma signaling. Other members of the Jak family can functionally substitute for Jak2. IFN-gamma can signal through the activation of Jak family members other than Jak2 as measured by Statlalpha homodimerization and major histocompatibility complex class I antigen expression. This indicates that Jaks are interchangeable and indiscriminative in the Jak-Stat signal transduction pathway. The necessity for the activation of one particular kinase during signaling can be overcome by recruiting another kinase to the receptor complex. The results may suggest that the Jaks do not contribute to the specificity of signal transduction in the Jak-Stat pathway to the same degree as Stats.
The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the completion of the genome sequence of Arabidopsis thaliana just around the corner, we can expect even faster progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis and function, and researchers focused on questions such as, how do proteins enter chloroplasts? or, what is the structure of the macromolecules of the cell wall and how are these molecules secreted? Signal transduction dealt primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction are merging because signals pass between organelles and a single signal transduction pathway usually involves multiple organelles or cellular structures. Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure? This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of all these cellular structures need to be coordinated during ABA-induced stomatal closure. For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic structures, consider how plants mount defense responses against pathogens. Elicitors produced by pathogens bind to receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose locations remain to be determined. The movement of transcription factors into the nucleus to activate the defense genes requires their release from cytosolic anchors and passage through the nuclear pore complexes of the nuclear envelope. This review does not cover all the recent progress in plant signal transduction and cell biology; it is confined to the topics that were discussed at a recent (November 1998) workshop held in Santiago at which lecturers from Chile, the USA and the UK presented recent results from their laboratories.
This study examined adenylyl cyclase (AC) signal transduction in alcohol-sensitive brain regions of rats selectively bred for high (HAD) and low (LAD) alcohol drinking and correlated these findings with differences in sensitivity and tolerance to alcohol-induced sedation found within these lines. LAD rats were more sensitive to the sedative effects of alcohol than were HAD rats as evidenced by a shorter latency to lose the righting response (RR) after a single alcohol challenge. When time to recover the RR was compared after each of two alcohol challenges, HAD rats recovered the RR more rapidly following the second challenge compared to the first, indicating that the HAD rats rapidly developed tolerance to the sedative effects of alcohol. Tolerance did not develop in rats of the LAD line. Two months after completion of behavioral testing, adenylyl cyclase (AC) signal transduction was examined in alcohol-sensitive brain regions of rats from both lines. Immunoblot analyses indicated that LAD rats had greater Gs alpha expression in the frontal cortex (FC) and hippocampus (HIP) compared to HAD rats. Rats with the highest HIP and FC Gs alpha levels were more rapidly affected by the sedative properties of alcohol than were rats with lower Gs alpha levels. G protein expression and AC activity in the FC, HIP, cerebellum (CERE), and nucleus accumbens (ACB) were also correlated with sensitivity to the sedative properties of alcohol and with the rapid development of tolerance to this alcohol effect. The results suggest that sensitivity and tolerance to alcohol-induced sedation may be mediated in part through AC signal transduction.
Historically, two strategies have been used to dissect the insulin signal transduction pathway. One was to start at the insulin receptor and work down the signal transduction pathway from the plasma membrane. The other was to select a physiological action of insulin, namely the mechanism by which insulin stimulates glycogen synthesis, and then work backwards towards the receptor. The hope was that eventually the groups working down from the top of the insulin signalling pathway would meet up with those working upwards from the bottom of the pathway. This has now happened, and in this lecture I will describe the recent advances that have linked the research from both ends of the insulin signal transduction pathway. I will also discuss how these findings have enabled pharmaceutical companies to embark on novel programmes to develop improved therapies for the treatment of diabetes in the future.
Spatial and temporal organization of signal transduction is essential in determining the speed and precision by which signaling events occur. Adaptor proteins are key to organizing signaling enzymes near their select substrates and away from others in order to optimize precision and speed of response. Here, we describe the role of adaptor proteins in determining the specific function of individual protein kinase C (PKC) isozymes. These isozyme-selective proteins were called collectively RACKs (receptors for activated C-kinase). The role of RACKs in PKC-mediated signaling was determined using isozyme-specific inhibitors and activators of the binding of each isozyme to its respective RACK. In addition to anchoring activated PKC isozymes, RACKs anchor other signaling enzymes. RACK1, the anchoring protein for activated betaIIPKC, binds for example, Src tyrosine kinase, integrin, and phosphodiesterase. RACK2, the epsilonPKC-specific RACK, is a coated-vesicle protein and thus is involved in vesicular release and cell-cell communication. Therefore, RACKs are not only adaptors for PKC, but also serve as adaptor proteins for several other signaling enzymes. Because at least some of the proteins that bind to RACKs, including PKC itself, regulate cell growth, modulating their interactions with RACKs may help elucidate signaling pathways leading to carcinogenesis and could result in the identification of novel therapeutic targets.
Throughout this symposium, recurrent themes were highlighted that may provide important clues to the pathogenesis of mucosal inflammation and IBD. First, the mucosal immune system is unique: Studies describing signaling paradigms in peripheral immunocytes should be re-explored in the gut where the rules that govern cell signaling may not be the same. Paradigms are a point of departure to characterize similarities and differences in mucosal immunity. A good example is a differential requirement for costimulation through CD2 in lamina propria T cells compared with peripheral T cells. Furthermore, a new definition of T-cell "costimulation" is beginning to emerge. Costimulatory molecules may function to overcome physical barriers by allowing cognate interactions between other molecules or by targeting signaling complexes to membrane microdomains. This concept also relates to another recurrent theme: Interactions between signaling pathways and the cytoskeleton are functionally important. Finally, we were introduced to the novel concept of metabolic parameters as a readout for signal transduction in the immune system. In the recent past, cell signaling has been viewed as a linear exercise, connecting a cell surface receptor to a series of intermediate molecules to a program of gene expression. However, signal transduction is in fact a three-dimensional exercise in cell biology. The future challenge, as pointed out in the keynote address, is to integrate reductionist models into reality and describe networks of signal transduction pathways in complex biosystems. "Threshold" responses were emphasized, with a small incremental increase or decrease in enzymatic activity leading to an on-off phenomenon referred to as a "molecular switch." In IBD, minute genetically determined differences in enzymatic activity may be critical. This point emphasizes the power of a genetic approach in IBD. Without strong genetic evidence, it is unlikely that fuctional assays will clarify the importance of small differences in enzymatic activity that may have dramatic biologic consequences. This symposium identified recently described signal transduction molecules that may be attractive therapeutic targets in IBD. Characterization of signaling molecules such as SLP-76, SLAM, SAP, and Fyb in the mucosal immune system will be an important area of future research. Ultimately, well-developed scientific hypotheses need to be tested in human beings. This paradigm was perhaps best illustrated by PPARgamma, where reductionist models and mouse experiments have recently lead to small trials suggesting proof of concept in human IBD. This meeting also emphasized a renewed interest in innate immunity in IBD and inflammation research. The role of enteric flora in initiating and perpetuating inflammation in animal models of IBD suggests at some level the importance of the innate immune response. The role of TLRs and bacterial interactions were discussed, as was NF-kappaB as the prominent transcription factor target of innate immune activation. Numerous bridges between innate and adaptive immunity were highlighted, including IL-10, IL-12, IL-18, and IFN-gamma. Their production during an innate immune response can profoundly affect functional T-cell responses in humans. In conclusion, the challenge of understanding signal transduction in IBD is one of integrating well-characterized inflammatory pathways into a complex biologic system that is inhabited by diverse cell types that communicate, and is characterized by interactions with a complex microbial environment. Making sense of this complexity is a daunting task that will require a multifactorial approach utilizing reductionist systems, mouse models, genetic studies, and ultimately human clinical trials.
Bacteria have devised phosphotransfer signaling mechanisms for eliciting a variety of adaptive responses to their environment. These mechanisms are collectively referred to as two-component regulatory systems. Each system generally consists of a sensor protein histidine kinase, which is anchored in the cell membrane, and a cytoplasmic response regulator, whose activity is modulated by the sensor. Most response regulators are transcription factors. In this review, we briefly introduce the established concept on bacterial two-component regulatory systems, using the Agrobacterium VirA-VirG system as an example, and give the evidence for the existence of quite similar systems in higher plants, such as the signal transduction induced by the phytohormone cytokinin. The Arabidopsis CRE1 histidine kinase and its related proteins AHK2 and AHK3 perceive cytokinins in the environment and transduce a signal, presumably through the AHP bridge components that carry the histidine-containing phosphotransfer (HPt) domain, to the ARR1 response regulator that transcriptionally activates genes immediately responsive to cytokinins. In addition, this signal transfer process appears to participate in cross-talk with signaling systems that respond to daylight and another phytohormone, ethylene, through an intracellular pool of several ARR1-like molecular species and the AHP components.
BACKGROUND: Electrical stimulation is used to treat nonunions and to augment spinal fusions. We studied the biochemical pathways that are activated in signal transduction when various types of electrical stimulation are applied to bone cells. METHODS: Cultured MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields at appropriate field strengths for thirty minutes and for two, six, and twenty-four hours. The DNA content of each dish was determined. Other cultures of MC3T3-E1 bone cells were exposed to capacitive coupling, inductive coupling, or combined electromagnetic fields for two hours in the presence of various inhibitors of signal transduction, with or without electrical stimulation, and the DNA content of each dish was determined. RESULTS: All three signals produced a significant increase in DNA content per dish compared with that in the controls at all time-points (p < 0.05), but only exposure to capacitive coupling resulted in a significant, ever-increasing DNA production at each time-period beyond thirty minutes. The use of specific metabolic inhibitors indicated that, with capacitive coupling, signal transduction was by means of influx of Ca(2+) through voltage-gated calcium channels leading to an increase in cytosolic Ca(2+) (blocked by verapamil), cytoskeletal calmodulin (blocked by W-7), and prostaglandin E2 (blocked by indomethacin). With inductive coupling and combined electromagnetic fields, signal transduction was by means of intracellular release of Ca(2+) leading to an increase in cytosolic Ca(2+) (blocked by TMB-8) and an increase in activated cytoskeletal calmodulin (blocked by W-7). CONCLUSIONS: The initial events in signal transduction were found to be different when capacitive coupling was compared with inductive coupling and with combined electromagnetic fields; the initial event with capacitive coupling is Ca(2+) ion translocation through cell-membrane voltage-gated calcium channels, whereas the initial event with inductive coupling and with combined electromagnetic fields is the release of Ca(2+) from intracellular stores. The final pathway, however, is the same for all three signals-that is, there is an increase in cytosolic Ca(2+) and an increase in activated cytoskeletal calmodulin.
SOCS-1 (suppressor of cytokine signaling-1) is a representative of a family of negative regulators of cytokine signaling (SOCS-1 to SOCS-7 and CIS) characterized by a highly conserved C-terminal SOCS box preceded by an SH2 domain. This study comprehensively examined the ability of several SOCS family members to negatively regulate the gp130 signaling pathway. SOCS-1 and SOCS-3 inhibited both interleukin-6 (IL-6)- and leukemia inhibitory factor (LIF)-induced macrophage differentiation of murine monocytic leukemic M1 cells and LIF induction of a Stat3-responsive reporter construct in 293T fibroblasts. Deletion of amino acids 51-78 in the N-terminal region of SOCS-1 prevented inhibition of LIF signaling. The SOCS-1 and SOCS-3 N-terminal regions were functionally interchangeable, but this did not extend to other SOCS family members. Mutation of SH2 domains abrogated the ability of both SOCS-1 and SOCS-3 to inhibit LIF signal transduction. Unlike SOCS-1, SOCS-3 was unable to inhibit JAK kinase activity in vitro, suggesting that SOCS-1 and SOCS-3 act on the JAK-STAT pathway in different ways. Thus, although inhibition of signaling by SOCS-1 and SOCS-3 requires both the SH2 and N-terminal domains, their mechanisms of action appear to be biochemically different.
In general, protein phosphorylation is one of the most widely used mechanisms for regulating biological processes, including intracellular signal transduction. In eukaryotes, the cascades of protein phosphorylation and dephosphorylation events involving a number of protein tyrosine or serine/threonine kinases have been well studied. In contrast, recent intensive studies revealed that bacteria have devised a quite different phosphotransfer signaling mechanism for eliciting a variety of adaptive responses to their environment. Such a bacterial signal transduction mechanism was originally referred to as a "two-component regulatory system." The mode of molecular communication between a "sensor kinase" and its cognate phospho-accepting "response regulator" is principally based on histidine-to-aspartate (His-Asp) phosphotransfer. In Escherichia coli, for example, at least 30 different sensor-regulator pairs operate in a wide variety of adaptive responses. This particular signal transduction mechanism was once thought to be restricted to prokaryotes. However, many instances have recently been uncovered in diverse eukaryotic species. Furthermore, recent studies suggested that the molecular mechanism underlying the bacterial signal transduction is not simple as, and, in fact, is more sophisticated than thought previously. The new concept should be referred to as the "multi-step His-Asp phosphotransfer signaling mechanism."
The alphabeta TCR is a multimeric protein complex comprising ligand-binding and signal-transducing subunits. The signal transduction processes are mediated by the immunoreceptor tyrosine-based activation motifs (ITAMs), and up to 10 ITAMs are present within a single TCR complex. This multiplicity may allow for signal amplification and/or the formation of qualitatively distinct intracellular signals. Notably, the TCR-zeta subunit contains three ITAMs, and exists as a disulfide-linked homodimer in the TCR complex. In normal murine thymocytes and peripheral T cells, a proportion of TCR-zeta molecules is constitutively tyrosine phosphorylated and associated with the ZAP-70 protein tyrosine kinase. We examined the contribution of the different TCR-zeta ITAMs in regulating the constitutive phosphorylation of the TCR-zeta subunit in thymocytes by analyzing TCR-zeta-deficient mice that had been reconstituted with either full-length or single ITAM-containing TCR-zeta subunits. We report in this work that in the absence of a full-length TCR-zeta subunit, there is no apparent constitutive phosphorylation of the remaining TCR/CD3 ITAMs. Following TCR ligation, all of the CD3 ITAMs become inducibly phosphorylated and associate with the ZAP-70 protein tyrosine kinase. Regardless of the number of TCR-zeta ITAMs present in the TCR complex, we report that a number of molecules involved in downstream signaling events, such as ZAP-70, SLP-76, and pp36, are all inducibly tyrosine phosphorylated following TCR ligation. These results support the notion that the different TCR ITAMs function in a quantitative rather than qualitative manner.
BACKGROUND: The phytohormone ethylene is a key regulator of plant growth and development. Components of the pathway for ethylene signal transduction were identified by genetic approaches in Arabidopsis and have now been shown to function in agronomically important plants as well. SCOPE: This review focuses on recent advances in our knowledge on ethylene signal transduction, in particular on recently proposed components of the pathway, on the interaction between the pathway components and on the roles of transcriptional and post-transcriptional regulation in ethylene signalling. CONCLUSIONS: Data indicate that the site of ethylene perception is at the endoplasmic reticulum and point to the importance of protein complexes in mediating the initial steps in ethylene signal transduction. The expression level of pathway components is regulated by both transcriptional and post-transcriptional mechanisms, degradation of the transcription factor EIN3 being a primary means by which the sensitivity of plants to ethylene is regulated. EIN3 also represents a control point for cross-talk with other signalling pathways, as exemplified by the effects of glucose upon its expression level. Amplification of the initial ethylene signal is likely to play a significant role in signal transduction and several mechanisms exist by which this may occur based on properties of known pathway components. Signal output from the pathway is mediated in part by carefully orchestrated changes in gene expression, the breadth of these changes now becoming clear through expression analysis using microarrays.
Signal transduction pathways mediate cell-cell interactions and integrate signals from the extracellular environment through specific receptors at the cell membrane. They play a pivotal role in regulating cellular growth and differentiation and in mediating many physiological and pathological processes, such as apoptosis, inflammation, and tumor development. The mitogen- activated protein kinases (MAPKs) constitute a cascade of phosphorylation events that transmit extracellular growth signals through membrane-bound Ras to the nucleus of the cell. In this chapter, detailed protocols for analyzing the kinase activities of the key components of the MAPKs pathway MEK1, ERK1, JNK, and p38 MAPK are described. A brief introduction to the chemical inhibitors to the MAPKs pathway is provided in the method section of each kinase assay. Inhibitors of other signaling pathways are summarized in Table 1. The reporter assay of cyclin D1, a key downstream target gene of MAPKs pathway, is also described in detail.
Two peptide segments designated LLP1 (residues 828-855) and LLP2 (residues 768-788) of the HIV-1 transmembrane (TM) envelope protein display structural and functional properties of calmodulin (CaM) binding. These LLP segments may contribute to cytopathogenesis by binding cellular CaM and inhibiting normal CaM-regulated signal transduction pathways. To determine whether these peptides could interrupt signal transduction in vivo, a cellular assay which uses a reporter gene linked to the nuclear factor of activated T cells (NF-AT) was used. Signal transduction perturbation was tested by exogenous addition of LLPs, W-7 or ionomycin; the LLPs inhibited NF-AT-mediated signal transduction as measured by reduced reporter activity. The LLP inhibition profile of NF-AT-driven luciferase activity was similar to the CaM inhibitor W-7. This was in direct contrast to ionomycin, a mobile calcium ion carrier which caused a significant increase in luciferase activity. These findings are consistent with the hypothesis that the CaM-binding properties of TM may contribute to defects in signal transduction leading to the T-cell anergy observed in patients infected with HIV-1.
We report here experiments on the analysis of cellular signal transduction in a series of patients with chronic B cell disorders (B cell chronic lymphocytic leukemia [B-CLL] and prolymphocytic leukemia). We compared the response of the leukemic cells with primary external signals (interleukin 2 [IL-2] or B cell differentiation factors [BCDF or IL-6]) with their response to secondary inducers (the phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA] or the calcium ionophore A23187) that circumvent the first part of the signal transduction pathway by directly activating the key enzyme protein kinase C. One BCDF was synthesized by mitogen-activated peripheral blood B lymphocytes; a second BCDF was constitutively produced by the human bladder carcinoma cell line T24. Changes in morphology, Tac (IL-2 receptor) expression, RNA synthesis measured by 3H-uridine uptake, and immunoglobulin production tested by enzyme-linked immunosorbent assay were used as parameters of successful signal transduction. TPA alone and TPA plus A23187 (synergistically) effectively initiated differentiation in all the leukemia cases. Neither IL-2 nor BCDF (singly or in combinations) caused equivalent responses. On the other hand, IL-2 and BCDF produced a substantial differentiation effect on normal B lymphocytes. Our data suggest that (a) B-CLL cells are able to respond to direct stimulation of the second messenger pathway (through protein kinase C) but not to the physiological stimuli IL-2 or BCDF; (b) the defect in signal transduction appears to be located upstream of protein kinase C (a possible candidate is a G protein); (c) malignant B cells may spontaneously or after treatment with inducers express the IL-2 receptor (Tac antigen) in the absence of a functional differentiating response to IL-2; and (d) signs of proliferation/differentiation in B-CLL samples after incubation with IL-2 or BCDF might be due to contamination of the cell populations with residual normal B cells.
The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region and elicits cellular signal transduction through this motif. To investigate the role of K1 signal transduction in KSHV replication, we expressed full-length K1 and CD8-K1 chimeras in BCBL1 cells. Unlike its strong signaling activity in uninfected B lymphocytes, K1 did not induce intracellular calcium mobilization or NF-AT activation at detectable levels in KSHV-infected BCBL1 cells. Instead, K1 signaling dramatically suppressed KSHV lytic reactivation induced by tetradecanoyl phorbol acetate (TPA) stimulation, but not by ORF50 ectopic expression. Mutational analysis showed that the cytoplasmic ITAM sequence of K1 was required for this suppression. Viral microarray and immunoblot analyses demonstrated that K1 signaling suppressed the TPA-mediated increase in the expression of a large subset of viral lytic genes in KSHV-infected BCBL1 cells. Furthermore, electrophoretic mobility shift assays demonstrated that TPA-induced activation of AP-1, NF-kappaB, and Oct-1 activities was severely diminished in BCBL1 cells expressing the K1 cytoplasmic domain. The reduced activities of these transcription factors may confer the observed reduction in viral lytic gene expression. These results demonstrate that K1-mediated signal transduction in KSHV-infected cells is profoundly different from that in KSHV-negative cells. Furthermore, K1 signal transduction efficiently suppresses TPA-mediated viral reactivation in an ITAM-dependent manner, and this suppression may contribute to the establishment and/or maintenance of KSHV latency in vivo.