A number of thiol-dependent oxidoreductases are released from cells and act on the cell surface. Correspondingly, several cell-surface processes appear to depend on catalyzed thiol-disulfide exchange, including integrin activation and the fusion of viral particles with the host membrane. Tumor cells frequently increase the abundance of secreted and cell-surface forms of particular oxidoreductases, and evidence suggests that oxidoreductases released from tumor cells promote growth and contribute to the remodeling of the cellular microenvironment. Few cell-surface or membrane proteins that are targeted by extracellular redox enzymes have been identified. One major reason for this slow progress is the highly transient nature of thiol-disulfide exchange, making its detection by conventional techniques difficult or impossible. Here we describe the application of an activity-based proteomics approach, also known as "mechanism-based kinetic trapping," to identify individual cell-surface target proteins that engage in disulfide exchange with thiol-dependent oxidoreductases. Although we have applied this approach to thioredoxin-1, it should also be applicable to other members of the thioredoxin superfamily whose activity is based on the CXXC active-site motif.
Cell behaviors are regulated by signaling pathways triggered by the activation of cell surface receptors. A key aspect of receptor signaling is the location of these receptors relative to their ligands and to other receptors, particularly in epithelia, whose cells are polarized by tight junction barriers into apical and basolateral membrane regions. In polarized epithelia, the co-receptor ErbB2 is often localized to the apical surface by its intramembrane ligand Muc4, thus segregating it from its partner ErbB3, which is sequestered at the lateral surface, co-localized with cadherin junctions. The ErbB2-ErbB3 receptor heterodimer, when activated, is a potent stimulator of cell proliferation; thus, the segregation mechanism helps maintain these cells in a differentiated state. Similarly, epidermal growth factor, the ligand for ErbB1, which is present in the apical fluid of some epithelia, is segregated from its receptor by the tight junction barrier. Loss of cell polarity and the tight junction barrier facilitates the interaction of ErbB2 with the hemidesmosome integrin α 6 β 4 . This integrin acts as a docking site for signaling pathways to promote cell proliferation and further disruption of cell junctions. The ultimate dissolution of tight junctions may result from activation of transforming growth factor–β receptors, one subunit of which is directly associated with the junction. This activation triggers degradation of critical tight junction components. These sequestration and segregation phenomena provide a model by which overexpression of the ErbB2 receptor kinase may trigger oncogenesis by initiating junction breakdown. Equally important, these mechanisms may act as a sensor for epithelial damage that can activate repair mechanisms.
The cytoplasmic CARD-containing DExD/H box RNA helicases RIG-I and MDA5 act as sensors of viral infections through recognition of viral double-stranded (ds) RNAs. They both associate with the mitochondrial adaptor IPS-1 (also referred to as MAVS, VISA, and CARDIF) through homotypic CARD-CARD interactions. IPS-1, in turn, triggers signaling pathways, including activation of the protein kinases TBK1 and IKKε, responsible for the phosphorylation of IRF3, a key transcription factor involved in interferon (IFN) synthesis, one essential element of the innate immune response. RIG-I remains in an autoinhibited state in the absence of dsRNA, through an internal repressor domain (RD) that binds within both its CARD and its RNA helicase domains and therefore acts in cis to control its multimerization and interaction with IPS-1. Ectopic expression of the RD prevents signaling and increases cell permissiveness to viruses, including hepatitis C virus. LGP2, which is another DExD/H RNA helicase of the RIG-I and MDA5 family and which is devoid of CARD domain, negatively controls IFN induction at different levels: by sequestering dsRNA, by blocking RIG-I’s multimerization in trans through a domain analogous to the RIG-I RD, and by competing with the protein kinase IKKε for a common interaction site on IPS-1. The ability of RIG-I and LGP2 to exert such a feedback control at the earliest steps of IFN synthesis allows the cells to exert a tight regulation of the induction of the innate immune response.
During chemotaxis, phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) accumulates at the leading edge of a eukaryotic cell, where it induces the formation of pseudopodia. PIP 3 has been suggested to be the compass of cells navigating in gradients of signaling molecules. Recent observations suggest that chemotaxis is more complex than previously anticipated. Complete inhibition of all PIP 3 signaling has little effect, and alternative pathways have been identified. In addition, selective pseudopod growth and retraction are more important in directing cell movement than is the place where new pseudopodia are formed.
In the process of pollination, haploid pollen germinates on the stigma surface and a pollen tube grows through the diploid tissues of the pistil toward the ovary. The pistil has two basic functions: to prevent unwanted pollen from gaining access to the ovary and to support the growth of desirable pollen. Pollen-pistil signaling allows these different types of pollen to be distinguished. Self-incompatibility (SI) systems, controlled by the S locus, are the best-understood pollen-pistil signaling systems. Other SI systems have been investigated at the molecular level, but the physiology of pollen tube rejection is best understood in the field poppy, Papaver rhoeas . This species has a gametophytic SI system: Pollen is rejected when its S haplotype is the same as either of the two S haplotypes expressed in the diploid pistil. Recent advances reveal new ways that SI controls pollen tube metabolism. A soluble pyrophosphatase is down-regulated as part of the rapid SI response, and, over the long term, perturbations of the actin cytoskeleton lead to programmed cell death in incompatible pollen tubes. Manipulating incompatible pollen tube metabolism in this way may leave more resources available for supporting the growth of compatible pollen tubes, the complementary function of the pistil.
Cytokines are soluble factors that regulate intercellular communication by binding to specific cell-surface receptors and activating cellular responses. A small subset of cytokines, however, has been recognized to act in an intracrine manner without being secreted. These molecules enter the nucleus and regulate gene transcription by binding nuclear coactivators or repressors. Interleukin-33 (IL-33), a cytokine with high sequence and structural similarity to IL-1 and IL-18, has now been identified as another member of this group of "double agents." The activity of IL-33, however, appears to be the opposite of other dual-activity molecules such as the proinflammatory molecules, IL-1α and HMBG1 (high-mobility group box 1). Soluble IL-33 binds the Toll-interleukin 1 (IL-1) receptor (TIR) domain-containing receptor ST2 and has T helper 2 (Th2) immunoregulatory activity. ST2 also inhibits the activity of Toll-like receptors (TLRs) by sequestering the TLR adaptor molecules MyD88 and Mal. The HMBG1 receptor pairs with TLRs and helps drive responses to infections, raising the possibility that ST2, acting as a coreceptor for TLRs, could modulate and perhaps limit immune responses to pathogens. The nuclear targets of IL-33 are still unknown, but the expression of IL-33 in inflamed tissues, its nuclear repressor activity, and the antagonistic properties of ST2 suggest that it could decrease inflammation, opposing the activity of factors like IL-1. If this holds true, IL-33 has potential as a novel therapeutic in autoimmune and inflammatory diseases.
Photoperiodic influences on the relations between levels of a steroid hormone and aggressive behavior add a layer of complexity to our ideas about how genes influence behavior. The effects of estrogens on aggression are modulated by day length and involve both genomic and nongenomic routes of hormone action.
Signaling downstream of classical seven-transmembrane domain receptors (7TMRs) had generally been thought to recruit factors that are in large part separate from those recruited by atypical 7TMRs, such as Frizzleds (Fzs), receptors for the Wnt family of glycoproteins. Classical 7TMRs are also known as G protein–coupled receptors (GPCRs) and are mediated by signaling factors such as heterotrimeric guanine nucleotide–binding proteins (G proteins), GPCR kinases (GRKs), and β-arrestins. Over the past few years, it has become increasingly apparent that classical and atypical 7TMRs share these factors, which are often associated with mediating classical 7TMR signaling, as well as the scaffolding proteins that were initially thought to be involved in transmitting atypical 7TMR signals. This sharing of signaling components by agonists that bind classical 7TMRs and those binding to atypical 7TMRs establishes the possibility of extensive crosstalk between these receptor classes. We discuss the evidence for, and against, crosstalk, and examine mechanisms by which this can occur.
The mechanisms by which morphogens, such as Sonic hedgehog (Shh), specify distinct cell fates in a concentration-dependent manner are not fully understood. Shh signaling is regulated by a feedback network that comprises Shh-binding factors, the expression of which is controlled by the Hedgehog pathway itself. Recent studies have identified the hedgehog-binding protein growth arrest–specific gene 1 (Gas1) as a component of this network. Gas1 binds Shh to promote signaling, but its expression is subsequently inhibited by pathway activity. Gas1−/− mice display Shh dosage–dependent phenotypes in the neural tube, midface, and digits. Ectopic expression and in vitro assays indicate that Gas1 binds Shh synergistically with the Hedgehog receptor Patched1 and promotes signaling in a cell-autonomous fashion. Furthermore, Gas1 cooperates with another component of the feedback network, Cdo, in patterning the neural tube and midface. The coordinate regulation of the activity and expression of several different positively and negatively acting Shh binding proteins should result in fine-tuned modulation of graded Shh signaling.
Neurons are highly polarized cells, typically with a long axon and relatively short dendrites. A wealth of recent data has identified a number of signaling molecules that are involved in neuronal polarization. Kinesin superfamily proteins (KIFs) contribute to the establishment and maintenance of neuronal polarity by selectively transporting various proteins and vesicles to either the axon or dendrites. Now evidence is emerging that KIFs also play an important role in axonal formation, the initial event of neuronal polarization. In particular, KIF13B transports phosphatidylinositol (3,4,5)-trisphosphate, which, based on current hypotheses, is one of the most upstream molecules in the intracellular signaling cascades involved in axonal formation.
A wide range of extracellular signals are transduced by G protein–coupled receptors (GPCRs). When activated by ligands, GPCRs can activate associated heterotrimeric guanine nucleotide–binding proteins (G proteins), which in turn act on various effectors. Increasing evidence indicates that GPCRs also signal independently of heterotrimeric G proteins. Several GPCRs directly interact with Src-family kinases. Here, we discuss the evidence for direct interaction and activation of Src-family kinases by GPCRs and data that suggest that agonist dosage provides a mechanism by which GPCRs can switch between G protein–dependent and G protein–independent signaling.
Cytokinins are essential plant hormones that control cell division, shoot meristem initiation, leaf and root differentiation, vasculature patterning, chloroplast biogenesis, photomorphogenesis, fertility, seed development, senescence, and stress tolerance. The Arabidopsis cytokinin signal transduction pathway involves hybrid histidine protein kinases [AHK2, AHK3, and AHK4 (also known as CRE1or WOL)] as cytokinin receptors, histidine phosphotransfer proteins (AHPs), and nuclear response regulators (ARRs) that serve as transcriptional regulators. There are four major steps in the cytokinin phosphorelay: (i) AHK sensing and signaling, (ii) AHP nuclear translocation, (iii) ARR-dependent transcriptional activation, and (iv) a negative-feedback loop through cytokinin-inducible ARR gene products. Each step is executed by components encoded by multigene families. The effects of cytokinin depend on cell type, environment, and developmental stage. The response is frequently the outcome of interactions with other plant signaling pathways.
The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor that mediates most of the toxic and carcinogenic effects of drugs and environmental toxins collectively known as xenobiotics. Ligand activation of the AhR stimulates the transcription of genes that encode several xenobiotic-metabolizing enzymes. The molecular mechanisms and signaling pathways evoked by the activation of the AhR are becoming increasingly understood and underscore the participation of the AhR in crucial processes, including cellular stress response, proliferation, differentiation, inflammation, and carcinogenesis. Studies now implicate the AhR as an integral part of the multifaceted signal transduction pathway initiated by the exposure of keratinocytes to ultraviolet B radiation (UVB), which is the most ubiquitous hazard to human skin and the principal risk factor for skin cancer. Ligand-dependent activation of the AhR in the cytosol provides a molecular bridge that links cytoplasmic events to nuclear signals, thus unmasking a previously unknown role for this transcription factor in the complex cellular response to UVB.
The nuclear pore complex functions both to separate and to connect the nucleus and the cytoplasm. Minute-to-minute changes in gene expression depend on rapid translocation of transcription factors and other regulatory proteins from the cytosol into the nucleus. However, a controversy exists as to whether cell signaling allows large molecules to enter the nucleus through tightly regulated facilitated transport or by the opening of a floodgate. A recent report suggesting that some hormones increase nuclear permeability through changes in intracellular Ca(2+) concentration has reignited this debate. Here, I consider both the basic permeability of the nuclear membrane under resting conditions and the effects of Ca(2+) on the permeability of the nuclear pore. I discuss facilitated transport through the nuclear pore complex, with particular attention to the nuclear transport of Ca(2+)-CaM signaling complexes. Finally, I weigh the arguments in favor of a generic increase in permeability versus stimulation of facilitated transport as possible mechanisms for mediating cell signaling to the nucleus.
Toll-like receptor (TLR) activation is primarily thought to affect antigen-presenting cells (APCs) by inducing an innate immune response that can subsequently activate the adaptive immune system. However, there are increasing data that demonstrate expression and activation of TLRs on T cells, thus providing evidence for a direct role for TLRs in the activation of an adaptive immune response. A study recently demonstrated that Pam3CSK { N -palmitoyl- S -[2,3-bis(palmitoloxy)-(2 RS )-propyl]-Cys-Ser-Lys 4 }, a TLR2 agonist lipopeptide, activates T helper 1 (T H 1) cells and induces interferon-γ (IFN-γ) production, even in the absence of TLR1, which differs from its mechanism of activation of APCs. Moreover, whereas Pam3CSK-stimulated IFN-γ production by T H 1 cells is ablated in the absence of both myeloid differentiation marker 88 (MyD88), an adaptor protein in the TLR pathway, and interleukin-1 receptor (IL-1R)–associated kinase–4 (IRAK4), the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase (JNK) are still phosphorylated. These data suggest that TLR2 activation of T H 1 cells occurs through a mechanism different from that described for APCs and provides further evidence of direct TLR activation of the adaptive immune system.
The Tec-family protein tyrosine kinase IL-2–inducible T cell kinase (ITK) mediates T cell activation, as does the adaptor protein SLP-76 (SH2-domain–containing leukocyte protein of 76 kD), which forms a complex with ITK and other intracellular signaling enzymes. One of these enzymes is phospholipase C–γ1 (PLC-γ1), which mediates T cell receptor (TCR)–stimulated intracellular calcium mobilization leading to the activation of transcription factors such as nuclear factor of activated T cells. The Src-family tyrosine kinase Lck and the Syk-family tyrosine kinase ζ chain–associated protein kinase of 70 kD (ZAP-70), together with ITK, are necessary for the phosphorylation of PLC-γ1 in response to TCR stimulation. ITK is thought to phosphorylate a specific tyrosine residue of PLC-γ1 that is required for its activation. The mechanism of activation of ITK appears to involve the interaction between SLP-76 and ITK, which not only initiates ITK activity but is also important to maintain the kinase activity of ITK. This suggests that SLP-76 acts as more than a neutral adaptor in mediating T cell activation; SLP-76 also directly influences the kinase activity of ITK, allowing ITK to phosphorylate PLC-γ1.
Focal adhesions provide physical linkages between the interior of an adhesive cell and the extracellular matrix (ECM). They may be involved in mediating such functions as cell migration, anchorage, mechanical interactions with the ECM, and the detection of physical cues in the environment. Cell biologists have long struggled to piece together the complex array of components found at focal adhesions, and the equally complex network of interactions among these components, into a "machine" that performs these putative functions. Two recent studies, however, indicate that focal adhesions may be more amorphous and dynamic than previously envisioned. These studies found different degrees of correlated retrograde movement of various focal adhesion proteins with actin filaments, while integrins remained largely stationary. Such differential movements appear inconsistent with a precisely engineered machine and may reflect a slippage clutch for transmitting a variable amount of contractile force to the substrate for migration, a gauge of physical interactions at adhesive sites, or a mechanism for releasing signals from the adhesive sites to the interior of the cell.
To truly understand signal transduction, we will ultimately need to understand the dynamics and kinetics of individual proteins as they perform their functions in a single cell. Groundbreaking advances in single-molecule biophysics now allow us to follow the motion of many individual proteins on the cell surface with the use of fluorescent probes, such as quantum dots. However, discriminating the directed movement of single molecules from their natural Brownian motion remains a challenge. A recent paper provides a powerful statistical approach for distinguishing periods of directed motion of individual γ-aminobutyric acid (GABA) receptors from periods during which they undergo Brownian motion. This new methodology should help single-molecule researchers determine the dynamics of individual proteins participating in signaling cascades.
Microfluidic devices are revolutionizing bioanalysis, and designs capable of detecting single protein molecules are now available. Two recently described microfluidic devices provide information on the number of β 2 -adrenergic receptors in individual cultured insect cells and measure the degradation of phycobilisomes in individual cyanobacteria, respectively. This latter experiment, which included the analysis of three single cells in parallel, heralds a bright future for high-throughput single-cell analyzers. These devices could greatly advance research in signal transduction and studies of the effects of environmental stimuli or xenobiotics on cellular responses.
Hexokinase1 (HXK1) is an evolutionarily conserved glucose sensor in plants. However, the molecular mechanism through which HXK1 controls the expression of genes encoding proteins involved in photosynthesis is a mystery. Recent research demonstrates that a previously unknown HXK1 nuclear complex controls the expression of specific photosynthetic genes, a process that is independent of glucose metabolism but requires two unexpected partners, VHA-B1 and RPT5B. Both VHA-B1 and RPT5B have well-established and conserved functions in processes that are seemingly unrelated to glucose-dependent regulation of gene expression, and neither of them is a predominantly nuclear protein. Biochemical, genetic, and molecular evidence demonstrates that VHA-B1 and RPT5B directly interact with HXK1 in the nucleus and that the HXK1 complex binds to the cis-acting elements of chlorophyll a/b binding protein 2, a photosynthetic gene that is transcriptionally suppressed by glucose. The identification of the HXK1 nuclear complex reveals an unexpected glucose-signaling mechanism and reinforces the notion that metabolic enzymes can play unique roles in signal transduction by directly controlling gene expression in the nucleus.
