bluuebunny/crossref_metadata_2025 huggingface dataset:
Measuring Peptide Secretion Using the FMRFamide Tagging Technique
Authors: Whim Matthew D.
Published: May 2003

Peptides are transmitters produced by a wide variety of neurons and neuroendocrine cells. They mediate a remarkable range of physiological processes. To better understand the roles played by peptides, a number of methods have been developed that can monitor their secretion. Although each has particular strengths, they cannot rapidly detect the secretion of chemically defined peptides. However, a recently developed approach termed "FMRFamide-tagging" may be useful in this regard. A genetically encoded electrophysiological tag is attached to the peptide prohormone of interest. The "tagged" prohormone together with an ionotropic receptor that binds the tag are expressed in the cell type under investigation. Secretion of the tag (and the co-secreted peptide of interest) are revealed by rapid inward membrane currents that are due to the activation of the tag receptor. In this manner, peptide secretion can be followed on a millisecond time scale. This protocol gives the details of the approach and its potential application to a range of cell types.
The Two Hats of SOS
Authors: Nimnual Anjaruwee , Bar-Sagi Dafna
Published: May 2003

Son of sevenless (SOS) is a guanine nucleotide exchange factor that activates Ras in response to growth factor stimulation. SOS also appears to serve as a guanine nucleotide exchanger for Rac and, thus, may be involved in cytoskeleton reorganization. Nimnual and Bar-Sagi discuss how these two activities of SOS can be regulated and how SOS may be recruited to different cellular locations through interactions with the adaptor proteins Grb2 and E3b1.
Responding to Hypoxia: Lessons From a Model Cell Line
Authors: Seta K. A. , Spicer Z. , Yuan Y. , Lu G. , Millhorn D. E.
Published: May 2003

Mammalian cells require a constant supply of oxygen to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. It is, therefore, not surprising that sophisticated mechanisms have evolved that allow cells to adapt to hypoxia. "Oxygen-sensing" is a special phenotype that functions to detect changes in oxygen tension and to transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in various organisms. Oxygen-sensing cells can be segregated into two distinct cell types: those that functionally depolarize (excitable) and those that do not functionally depolarize (nonexcitable) in response to reduced oxygen. Theoretically, excitable cells have all the same signaling capabilities as the nonexcitable cells, but the nonexcitable cells cannot have all the signaling capabilities as excitable cells. A number of signaling pathways have been identified that regulate gene expression during hypoxia. These include the Ca
2+
-calmodulin pathway, the 3′-5′ adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, the p42 and p44 mitogen-activated protein kinase [(MAPK); also known as the extracellular signal-related kinase (ERK) for ERK1 and ERK2] pathway, the stress-activated protein kinase (SAPK; also known as p38 kinase) pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. In this review, we describe hypoxia-induced signaling in the model O
2
-sensing rat pheochromocytoma (PC12) cell line, the current level of understanding of the major signaling events that are activated by reduced O
2
, and how these signaling events lead to altered gene expression in both excitable and nonexcitable oxygen-sensing cells.
Meeting Report: The Next Interleukin?
Authors: Weber Georg F.
Published: May 2003

A report of the meeting "Third International Conference on Osteopontin and Related Proteins", San Antonio, TX, USA, 10 to 12 May 2002.

At a recent meeting, the structure and biology of the cytokine osteopontin were discussed. The molecule is essential for cellular immune responses and for the remodeling of cardiovasculature and bone. Osteopontin is extensively modified by posttranslational modifications, the consequences of which are only beginning to be understood. In disease, the expression of osteopontin by cancers may enhance their malignant phenotype, possibly reflecting the ability of the cytokine to induce cell migration after ligation of integrins and CD44 variants. A gene family with osteopontin as a member has been characterized.
Confirming Specificity of RNAi in Mammalian Cells
Authors: Lassus Patrice , Rodriguez Joe , Lazebnik Yuri
Published: May 2003

RNA interference (RNAi) is a process of sequence-specific gene silencing. Recent advances in the understanding of RNAi have provided practical tools to silence gene expression in mammalian cells, opening new possibilities for studying the functions of genes and proteins. It is important to ensure that an observed effect of RNAi is due to silencing of the intended target. Indeed, it is possible that an siRNA may silence more than one messenger RNA that is homologous in the region complementary to the siRNA. Considering that we know little about how RNAi works in mammalian cells, other artifacts may be yet to be recognized. Thus, we suggest approaches to rescue the effect of RNAi by ectopically expressing the protein of interest. These approaches involve introducing silent mutations into the complementary DNA of the protein and targeting RNAi to the untranslated regions of the gene.
Bayesian Network Approach to Cell Signaling Pathway Modeling
Authors: Sachs Karen , Gifford David , Jaakkola Tommi , Sorger Peter , Lauffenburger Douglas A.
Published: May 2003

The modeling of cellular signaling pathways is an emerging field. Sachs
et al.
illustrate the application of Bayesian networks to an example cellular pathway involving the activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) in response to fibronectin binding to an integrin. They describe how to use the analysis to select from among proposed models, formulate hypotheses regarding component interactions, and uncover potential dynamic changes in the interactions between these components. Although the data sets currently available for this example problem are too small to definitively point to a particular model, the approach and results provide a glimpse into the power that these methods will achieve once the technology for obtaining the necessary data becomes readily available.
Modeling the Cell's Guidance System
Authors: Iglesias Pablo A. , Levchenko Andre
Published: May 2003

Cell locomotion can be directed by external gradients of diffusible substances leading to chemotaxis. Recently, the mechanisms of gradient sensing, the cell guidance system, came under scrutiny both in experimental analysis and computational modeling. Here, we review several recent computational models of gradient sensing in eukaryotic cells, demonstrating why some of them predict little sensitivity to changes in the gradient and response "locking," whereas others predict high gradient sensitivity at the expense of signal gain. We also propose a way to view chemotaxis regulation as a highly coupled combination of semi-independent control modules, leading to simplifying modeling of this complex cellular behavior.
Enumeration, Phenotyping, and Identification of Activation Events in Conjugates Between T cells and Antigen-Presenting Cells by Flow Cytometry
Authors: Grebe Kristie M. , Potter Terry A.
Published: May 2003

Microscopic analysis of T cell-antigen-presenting cell (T cell:APC) interactions at the single cell level has been a powerful, but tedious and subjective, technique. In this paper, we describe a rapid and quantitative method to identify T cell:APC conjugates using succinimidyl ester dyes, which irreversibly label free amine groups on the cell surface. The labeled cell conjugates and subsequent activation events are detected by flow cytometry.
Nuclear Lipid Signaling
Authors: Irvine Robin F.
Published: May 2003

Abundant evidence now supports the existence of phospholipids in the nucleus that resist washing of nuclei with detergents. These lipids are apparently not in the nuclear envelope as part of a bilayer membrane, but are actually within the nucleus in the form of proteolipid complexes with unidentified proteins. This review discusses the experimental evidence that attempts to explain their existence. Among these nuclear lipids are the polyphosphoinositol lipids which, together with the enzymes that synthesize them, form an intranuclear phospholipase C (PI-PLC) signaling system that generates diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)
P
3
]. The isoforms of PI-PLC that are involved in this signaling system, and how they are regulated, are not yet entirely clear. Generation of DAG within the nucleus is believed to recruit protein kinase C (PKC) to the nucleus to phosphorylate intranuclear proteins. Generation of Ins(1,4,5)
P
3
may mobilize Ca
2+
from the space between the nuclear membranes and thus increase nucleoplasmic Ca
2+
. Less well understood are the increasing number of variations and complications on the "simple" idea of a PI-PLC system. These include, all apparently within the nucleus, (i) two routes of synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)
P
2
]; (ii) two sources of DAG, one from the PI-PLC pathway and the other probably from phosphatidylcholine; (iii) several isoforms of PKC translocating to nuclei; (iv) increases in activity of the PI-PLC pathway at two points in the cell cycle; (v) a pathway of phosphorylation of Ins(1,4,5)
P
3
, which may have several functions, including a role in the transfer of mRNA out of the nucleus; and (vi) the possible existence of other lipid signaling pathways that may include sphingolipids, phospholipase A
2
, and, in particular, 3-phosphorylated inositol lipids, which are now emerging as possible major players in nuclear signaling.
At the Crossroads of Necrosis and Apoptosis: Signaling to Multiple Cellular Targets by HMGB1
Authors: Bustin Michael
Published: May 2003

Cells can die through two mechanisms: necrosis and apoptosis. Necrosis occurs in response to stimuli that cause cellular damage and result in the release of intracellular proteins, which stimulate the inflammatory response. Necrosis is associated with tissue damage and organ deterioration. Apoptosis is a programmed cell death during which the cellular membrane remains intact, but activated enzymes destroy the cell from within. Bustin discusses how the mobility of the nuclear protein high mobility group B1 (HMGB1) may be a key signal for determining whether certain cells undergo necrotic or apoptotic death. Necrosis may result when HMGB1 is released from the cells and stimulates the inflammatory response. HMGB1 appears to have extracellular signaling functions, such as stimulation of cell motility and tumor metastasis and activation of the cytokine signaling pathways. Through its action on the cytokine signaling targets, HMGB1 may be an important therapeutic target for diseases of cytokine excess, including septic shock.
Id: A Target of BMP Signaling
Authors: Miyazono Kohei , Miyazawa Keiji
Published: May 2003

Cytokines of the transforming growth factor-β (TGF-β) superfamily transduce their signals by activating receptor-regulated Smads (R-Smads). Distinct R-Smads or combinations of R-Smads are activated by TGF-β, activin, or bone morphogenetic proteins (BMPs). R-Smads activated by BMPs induce expression of Id proteins, which act as inhibitors of differentiation and stimulators of cell growth by inhibiting the function of basic helix-loop-helix transcription factors. In endothelial cells, TGF-β binds to two distinct type I receptor serine-threonine kinases, ALK-5 and ALK-1; the latter activates the same R-Smads that are activated by BMP and induces synthesis of Id (inhibitor of differentation or inhibitor of DNA binding) proteins. Growing evidence suggests that Id proteins may play crucial roles in angiogenesis, neurogenesis, and osteogenesis and act as key molecules in regulating biological responses induced by BMPs and TGF-β.
Insider Information: How Palmitoylation of Ras Makes It a Signaling Double Agent
Authors: Berthiaume Luc G.
Published: May 2003

Ras small guanosine triphosphatases (GTPases) are involved in the regulation of cell growth, differentiation, and survival and are mutated in as many as 30% of human cancers. These proto-oncogenic GTPases are mostly involved in the activation of signaling cascades downstream from growth factor receptors and lead to transcriptional activation of specific genes. Because of a complex series of posttranslational COOH-terminal modifications, Ras proteins are found on various intracellular membranes, in addition to the plasma membrane. Using a novel fluorescent probe monitoring GTP-bound Ras in live cells (GFP-Raf-1-RBS), Golgi-associated H-Ras was shown to be activated in situ after growth factor stimulation, with kinetics distinct from that of H-Ras activation at the plasma membrane. Furthermore and also noteworthy, an oncogenic H-Ras chimera that was tethered to the endoplasmic reticulum activated the extracellular signal-regulated kinase (ERK) and Akt pathways preferentially, whereas a Golgi-tethered oncogenic H-Ras chimera activated predominantly the Jun-NH
2
-terminal kinase (JNK) pathway. Thus, the subcellular localization of Ras influenced which downstream effector pathways were engaged. The activation of Golgi-H-Ras may be mediated by second messengers through the action of a Golgi-localized guanine nucleotide exchange factor, Ras-GRP.
Phosphorylation-Independent Desensitization of G Protein-Coupled Receptors?
Authors: Pao Christina S. , Benovic Jeffrey L.
Published: May 2003

G protein-coupled receptors (GPCRs) are involved in a multitude of signaling processes and respond to a wide range of ligands. The activity of GPCRs is subject to three principal modes of regulation: desensitization, trafficking, and down-regulation. Desensitization is defined as a loss in the responsiveness of a signaling system. The generally established paradigm for GPCR desensitization involves receptor phosphorylation by GPCR kinases (GRKs), initiated by agonist-induced conformational changes in the receptor or by kinases activated by specific signaling pathways. GRKs have several interaction domains and may be able to contribute to receptor desensitization through mechanisms that do not involve the kinase activity of GRK. Pao and Benovic discuss some of these interactions and their relevance for the regulation of GPCR signaling.
In-Gel Kinase Assay as a Method to Identify Kinase Substrates
Authors: Wooten Marie W.
Published: May 2003

Phosphorylation of proteins by kinases is central to many cellular processes, including signal transduction. Thus, assays to identify or characterize kinases are a key tool for research in this area. Kinase substrates can be incorporated into polyacrylamide gels and used to characterize kinase activity in mixed samples. This methodology can be adapted for the identification of novel kinase-substrates or kinase-kinases that participate in the regulation of cell signaling. Here, I review the rationale and principles of an in-gel kinase assay. This strategy relies on co-polymerization of a substrate within the gel matrix, followed by detection of enzymatic activity in situ. The following Protocol provides a detailed method for performing the in-gel kinase assay and discusses the uses of the assay to evaluate kinase activity in the context of proliferation, differentiation, and survival pathways.
Analysis of Tyrosine Phosphorylation Sites in Signaling Molecules by a Phosphotyrosine-Specific Immonium Ion Scanning Method
Authors: Steen Hanno , Pandey Akhilesh , Andersen Jens S. , Mann Matthias
Published: May 2003

Signal transduction pathways involve cascades of events, such as formation of second messengers and protein complexes that alter the activities of proteins. This can ultimately lead to changes in gene expression in response to the stimuli. Reversible phosphorylation of proteins is an important mechanism for activating or inhibiting enzymes and for the assembly of multiprotein complexes. Here, we describe a mass spectrometry-based phosphotyrosine-specific immonium ion scanning (PSI scanning) method for selective detection of tyrosine-phosphorylated peptides. Once the tyrosine-phosphorylated peptides are identified, they can be directly sequenced in the same experiment to localize the phosphorylation site. We provide protocols for isolation and preparation of samples for analysis, and detailed instructions for operation of a quadrupole time-of-flight (TOF) mass spectrometer for this method. Because of its simplicity and specificity, PSI scanning is likely to become an important tool in proteomic studies of pathways involving tyrosine phosphorylation.
Will the Real LHC II Kinase Please Step Forward?
Authors: Allen John F. , Race Helen L.
Published: May 2003

Many laboratories have searched for the protein kinase responsible for phosphorylation of the chloroplast light-harvesting complex of photosynthesis, LHC II. The LHC II kinase provides a vital link in a redox signaling pathway of ecological, developmental, and evolutionary significance. Various candidates for the LHC II kinase, some stronger than others, have come and gone. Recently, a family of three thylakoid-associated kinases (TAKs) has been identified and purified; they too catalyze in vitro phosphorylation of LHC II. The LHC II kinase is part of an integrated network of signal transduction to which input is provided by a number of environmental factors. The implications of understanding these processes stretch beyond the important, central question of how plants adapt their photosynthetic machinery to changing wavelengths of light.
Semaphorins: Green Light for Redox Signaling?
Authors: Ventura Andrea , Pelicci Pier Giuseppe
Published: May 2003

How semaphorins stimulate plexins to regulate axon steering is an actively investigated question. Ventura and Pelicci discuss new evidence indicating that MICAL, a putative monoxygenase, transmits the signal from the receptor plexin to the actin cytoskeleton through a redox mechanism. MICAL could act either indirectly, causing a local increase in the concentration of reactive oxygen species (ROS), or directly, inducing redox changes in downstream effectors, such as actin and members of the Rho and Rac family of guanosine triphosphatases. Because semaphorin-plexin signaling plays a role in a number of clinically relevant settings, including recovery from spinal cord trauma, regulation of the immune system, and cancer metastasis, these findings could have important therapeutic implications.
AMPA Receptors Do the Electric Slide
Authors: deSouza Sunita , Ziff Edward B.
Published: May 2003

How the synapse is organized and how its organization changes during events that result in long-term changes in synaptic efficacy is the subject of intense study. Various anchoring proteins work in concert to organize the postsynaptic side of the membrane, and the interactions of these proteins can be altered by synaptic activity. DeSouza and Ziff discuss the evidence that the reversible palmitoylation of the postsynaptic density protein PSD-95 may result in the movement of AMPA-type glutamate receptors into and out of lipid raft domains, ultimately controlling AMPA receptor accumulation at the postsynaptic membrane.
Glutamate Receptor Trafficking in Synaptic Plasticity
Authors: Contractor Anis , Heinemann Stephen F.
Published: May 2003

Ionotropic glutamate receptors mediate excitatory synaptic transmission at most central mammalian synapses. In addition to converting the chemical signal released from the presynaptic terminal to an electrical response in the postsynaptic neuron, these receptors are critically involved in activity-dependent, long-term changes in synaptic strength and, therefore, are central to processes thought to underlie learning and memory. Several mechanisms have been proposed to play roles in altering synaptic strength, and it is clear that there are several different forms of long-term synaptic plasticity in the mammalian brain. Here, we review recent evidence that some forms of synaptic strengthening rely on the modification of the glutamate receptor complement at synapses in response to activity-dependent processes.
OxyR: A Molecular Code for Redox Sensing?
Authors: Helmann John D.
Published: May 2003

Helmann discusses the controversy surrounding the activation of the bacterial redox-regulated transcription factor OxyR. Evidence from different sources, including crystallographic data, has led to opposing models for the chemical changes that activate OxyR. Is it an intramolecular disulfide-linkage? Is it oxidation of a single cysteine residue to a sulfenic acid? Are there different active forms depending on the type of cysteine modification: intramolecular disulfide bond, sulfenic acid,
S
-nitrosothiol, or mixed disulfide with glutathione? These issues are discussed in the broader context of transcriptional regulation and how particular regulators may activate distinct genetic programs depending on the precise state of the regulator produced in response to environmental cues.
Cripto, a Multifunctional Partner in Signaling: Molecular Forms and Activities
Authors: Rosa Frédéric M.
Published: May 2003

Within a multicellular organism, communication between cells is essential during development to ensure proper execution of cell migration, cell fate decisions, and differentiation events. It is also essential in the adult for the coordination of many physiological functions. Cell-to-cell communications often rely on the interaction of cell surface receptors with soluble or membrane-bound ligands. Receptors or ligands may interact with additional partners to trigger specific signaling cascades inside the cell. In most cases, partners act in a specific configuration, either as a diffusible "co-ligand" or a membrane-bound co-receptor. Here, we examine the case of Cripto, a signaling molecule that has prominent functions during vertebrate development. Conflicting results have suggested that Cripto has the unusual capacity to act both as a secreted ligand and as a cell surface component to control a single signaling pathway. Here, we review the recent experiments that attempt to reconcile those results. Furthermore, three reports have described the fact that Cripto is modified by the addition of sugar residues, including a rare case of fucosylation. These modifications are essential for Cripto function, suggesting that, as is the case for other key developmental or physiological regulators such as Notch or selectins, the activity of Cripto may be controlled by the extent of its glycosylation or fucosylation (or both).
Regulation of Growth Factor Receptors by Gangliosides
Authors: Miljan Erik A. , Bremer Eric G.
Published: May 2003

Since their discovery in the 1940s, gangliosides have been associated with a number of biological processes, such as growth, differentiation, and toxin uptake. Hypotheses about regulation of these processes by gangliosides are based on indirect observations and lack a clear definition of their mechanisms within the cell. The first insights were provided when a reduction in cell proliferation in the presence of gangliosides was attributed to inhibition of the epidermal growth factor receptor (EGFR). Since that initial finding, most, if not all, growth factor receptors have been described as regulated by gangliosides. In this review, we describe the effects of gangliosides on growth factor receptors, beginning with a list of known effects of gangliosides on growth factor receptors; we then present three models based on fibroblast growth factor (FGFR), platelet-derived growth factor receptor (PDGFR), and EGFR. We focus first on ganglioside modulation of ligand binding; second, we discuss ganglioside regulation of receptor dimerization; and third, we describe a model that implicates gangliosides with receptor activation state and subcellular localization. The methodology used to develop the three models may be extended to all growth factor receptors, bearing in mind that the three models may not be mutually exclusive. We believe that gangliosides do not act independently of many well-established mechanisms of receptor regulation, such as clathrin-coated pit internalization and ubiquitination, but that gangliosides contribute to these functions and to signal transduction pathways. We hypothesize a role for the diverse structures of gangliosides in biology through the organization of the plasma membrane into lipid raft microdomains of unique ganglioside composition, which directly affect the signal duration and membrane localization of the growth factor receptor.
Internalization of Inactive EGF Receptor into Endosomes and the Subsequent Activation of Endosome-Associated EGF Receptors
Authors: Wang Yi , Pennock Steven , Chen Xinmei , Wang Zhixiang
Published: May 2003

Despite intensive efforts to understand cell signaling from endosomes, there is no direct evidence demonstrating that endosomal signaling is sufficient to activate signal transduction pathways or that endosomal signaling can produce biological responses. The lack of breakthrough is due in part to the inability to generate endosomal signals in isolation from plasma membrane signals. In this Protocol, we describe a system in which epidermal growth factor (EGF) receptor (EGFR) is specifically activated when it is endocytosed into endosomes. We treated cells with EGF in the presence of AG1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks recycling of EGFR. This treatment led to the internalization of nonactivated EGF-EGFR complex into endosomes. The endosome-associated EGFR was then activated by removing AG1478 and monensin. During this procedure, we did not observe any detectable surface EGFR phosphorylation. We also achieved specific activation of endosome-associated EGFR without using monensin. Specific activation of endosome-associated EGFR provides a unique tool to study endosomal signaling of EGFR. This method may also be applied to other receptor tyrosine kinases to study whether they, too, can signal from endosomes.
Aurora-A in Cell Fate Control
Authors: Gonzalez Cayetano
Published: May 2003

Key cells divide asymmetrically during the development of multicellular organisms to give rise to offspring with different fates. In the
Drosophila
external sensory organ, asymmetrical division depends on polarization of the precursor cells during interphase and the consequent unequal distribution during mitosis of the protein Numb, which determines cell fate. Gonzalez discusses recent research implicating the mitotic kinase Aurora-A in the asymmetric localization of Numb in sensory organ pI precursor cells, a new function that appears to be independent of Aurora-A's known roles in regulating centrosomal maturation and the organization of mitotic spindle microtubules.
Hitting the Target: Emerging Technologies in the Search for Kinase Substrates
Authors: Manning Brendan D. , Cantley Lewis C.
Published: May 2003

Through phosphorylation, protein kinases can alter the activity, localization, protein association, and stability of their targets. Despite the importance to our understanding of all aspects of cell biology, progress toward identifying bona fide substrates of specific protein kinases has been slow. Traditionally used techniques to identify true kinase substrates, such as genetics, yeast two-hybrid screens, and biochemical purification, are often laborious and unreliable. However, several new approaches have recently been developed and used successfully to identify genuine in vivo substrates of certain protein kinases. These methods include screening for phosphorylation of proteins from phage expression libraries, peptide library screens to determine optimal motifs favored by specific kinases, the use of phospho-motif antibodies, and an approach that uses structurally altered kinases and allele-specific adenosine triphosphate analogs and kinase inhibitors. We describe these approaches and discuss their utility and inherent caveats.
Plasticity of the Kinomes in Monkey and Rat Tissues
Authors: Pelech Steven , Zhang Hong
Published: May 2003

The Kinetworks KPKS 1.0 immunoblotting screen was used to investigate the differential expression of 58 protein serine-threonine kinases, five protein threonine-tyrosine kinases, and 15 other nonreceptor protein tyrosine kinases in three types of human blood cells, 14 monkey organs, and six rat organs. The results demonstrate striking differences in the kineome profiles of diverse organs and even between the same organs in different species.
Pulsating Ion Fluxes and Growth at the Pollen Tube Tip
Authors: Robinson Kenneth R. , Messerli Mark A.
Published: May 2003

Reproduction in higher plants requires the directed growth of pollen tubes in order to transmit sperm cells to the ovules at the base of the style. Many signaling processes have been implicated in polarized pollen tube growth. Here, changes in the concentration of calcium, potassium, hydrogen (pH), and chloride are discussed, as they may all contribute to the process of oscillatory growth and guidance observed in pollen tubes.
Creating Asymmetric Cell Divisions by Skewing Endocytosis
Authors: Shen Qin , Temple Sally
Published: May 2003

How can one cell divide to form two cells, which are genetically identical to the mother cell, with different cell fates? That is the question discussed by Shen and Temple. Specifically, they address how Notch signaling is attenuated in one daughter cell through the asymmetric inheritance of the protein Numb, which may be responsible for mediating the endocytosis of the receptor Notch in the Numb-enriched cell.
Design and Use of a Mammalian Protein-Protein Interaction Trap (MAPPIT)
Authors: Eyckerman Sven , Lemmens Irma , Lievens Sam , Van der Heyden Jose , Verhee Annick , Vandekerckhove Joël , Tavernier Jan
Published: May 2003

Identifying the interaction partners of a protein is a straightforward way to gain insight into the protein's function and to position it in an interaction network such as a signal transduction pathway. Various techniques have been developed to serve this purpose, and some are specifically designed to study posttranslational modifications in mammalian proteins and to clarify their normal physiological context. However, several intrinsic constraints limit the use of these technologies, and most are not suitable for screening for new interacting partners. In the Mammalian Protein-Protein Interaction Trap (MAPPIT) Protocol described here, knowledge of cytokine receptor signaling has been used to design a versatile genetic tool that can be used analytically and for detection of new protein-protein interactions in mammalian cells.
Mono-ADP-Ribosylation: A Tool for Modulating Immune Response and Cell Signaling
Authors: Corda Daniela , Di Girolamo Maria
Published: May 2003

Mono-ADP-ribosylation is a posttranslational modification of cellular proteins that has the potential to regulate various cell functions. This reaction consists of the enzymatic transfer of ADP-ribose to specific acceptor amino acid residues (predominantly arginine and cysteine). The best-known cellular ADP-ribosyltransferases (the enzymes that catalyze this reaction) are the seven ectoenzymes, members of the ART family.

Recently, ADP-ribosylated human neutrophil-derived peptide (HNP-1, an antimicrobial peptide secreted by immune cells) has been identified in the bronchoalveolar lavage fluid from individuals who smoke cigarettes. This demonstrates that ADP-ribosylation of HNP-1 occurs in vivo. In vitro experiments have indicated that ART-1, an enzyme also present in the airway epithelium, specifically modifies Arg
14
of the HNP-1, causing the loss of the peptide's antimicrobial and cytotoxic activity, while preserving its chemotactic activity. From a functional point of view, these data support a role of ADP-ribosylation in the innate immune response.

Additional functions proposed for the ADP-ribosylation reaction involve the intracellular ADP-ribosyltransferases, which are molecularly unrelated to the ARTs and intervene in cell signaling and metabolism cascades. The growing understanding of the biological roles of protein and peptide ADP-ribosylation represents a powerful tool for novel pharmacological interventions.
GAF Domains: Cyclic Nucleotides Come Full Circle
Authors: Hurley James H.
Published: May 2003

Cyclic nucleotides are ubiquitous signaling molecules that are present in life forms ranging from bacteria to yeast to humans. In higher eukaryotes, conserved molecular machinery processes signals through both cyclic adenosine monophosphate (AMP) and cyclic guanosine monophosphate (GMP). Hurley describes how one widespread cyclic nucleotide recognition module, the GAF (cyclic GMP, adenylyl cyclase, FhlA) domain, formerly thought to bind only cyclic GMP, has now been found to bind cyclic AMP as well. Recent structural analysis of a cyclic GMP-binding GAF domain shows how conserved elements among the cyclic nucleotide-binding subgroup of GAF domains recognize the common chemical moieties in the two compounds.
DARPP Chocolate: A Caffeinated Morsel of Striatal Signaling
Authors: Bastia Elena , Schwarzschild Michael A.
Published: May 2003

The psychomotor stimulant effects of caffeine, the most widely consumed psychoactive substance, are mediated through its antagonism of extracellular adenosine receptors in the basal ganglia. In the absence of caffeine, adenosine stimulates inhibitory striatopallidal neurons that suppress motor activity by binding to A
2A
receptors, thereby activating a cyclic adenosine 3′,5′-monophosphate (cAMP) and protein kinase A signaling pathway. Bastia and Schwarzschild discuss recent research implicating DARRP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kilodaltons) as an attractive mediator of the sustained psychomotor stimulant effect seen with low doses of caffeine. They highlight the role of postsynaptic A
2A
receptor blockade, but leave open the possibility that antagonism of presynaptic or postsynaptic A
1
receptors also contributes to DARPP-32-dependent psychomotor stimulation by caffeine.
Shedding Light on Cell Signaling: Interpretation of FRET Biosensors
Authors: Gaits Frédérique , Hahn Klaus
Published: May 2003

This Perspective compares recently developed approaches for studying live-cell signaling dynamics. It is now possible not only to study the changing localizations of proteins within living cells but to correlate these dynamics with quantitation of protein activities, including ligand interactions and phosphorylation. Initial applications of an increasingly varied toolchest of techniques have revealed strengths and weaknesses in each approach, clarifying which tool is best for which job.
Excitation-Transcription Coupling: Signaling by Ion Channels to the Nucleus
Authors: Dolmetsch Ricardo
Published: May 2003

Changes in the concentration of intracellular Ca
2+
([Ca
2+
]
i
) in response to various stimuli play a role in regulating numerous cellular processes, including the activation of gene expression. In neurons, the extraordinary diversity of the response to Ca
2+
signaling depends on the location, intensity, and duration of the Ca
2+
transient. Interestingly, Ca
2+
-dependent gene transcription appears to be sensitive both to increases in nuclear Ca
2+
, which occur after relatively intense stimuli, and to highly localized increases in Ca
2+
near the sites of Ca
2+
influx. Activation of intracellular signaling pathways by specific types of Ca
2+
channels depends on localization of specific Ca
2+
receptors close to the channel mouth. The dual regulation of signaling pathways by Ca
2+
near channels and in the nucleus may permit neurons to precisely tailor transcriptional activation to specific types of electrical or chemical stimuli and at the same time ensure that only robust stimuli that generate nuclear Ca
2+
elevations are converted into long-term changes in gene expression.
FXYD Proteins: New Tissue-Specific Regulators of the Ubiquitous Na,K-ATPase
Authors: Crambert Gilles , Geering Käthi
Published: May 2003

Maintenance of the Na
+
and K
+
gradients between the intracellular and extracellular milieus of animal cells is a prerequisite for basic cellular homeostasis and for functions of specialized tissues. The Na,K-ATPase, an oligomeric P-type adenosine triphosphatase (ATPase), is composed of a catalytic α subunit and a regulatory β subunit and is the main player that fulfils these tasks. A variety of regulatory mechanisms are necessary to guarantee appropriate Na,K-ATPase expression and activity adapted to changing physiological demands. Recently, a regulatory mechanism was defined that is mediated by interaction of Na,K-ATPase with small proteins of the FXYD family, which possess a single transmembrane domain and so far have been considered as channels or regulators of ion channels. The mammalian FXYD proteins FXYD1 through FXYD7 exhibit tissue-specific distribution. Phospholemman (FXYD1) in heart and skeletal muscle, the γ subunit of Na,K-ATPase (FXYD2) and corticosteroid hormone-induced factor (FXYD4, also known as CHIF) in the kidney, and FXYD7 in the brain associate preferentially with the widely expressed Na,K-ATPase α1-β1 isozyme and modulate its transport activity in a way that conforms to tissue-specific requirements. Thus, tissue- and isozyme-specific interaction of Na,K-ATPase with FXYD proteins contributes to proper handling of Na
+
and K
+
by the Na,K-ATPase, and ensures correct function in such processes as renal Na
+
-reabsorption, muscle contraction, and neuronal excitability.
Activation of Procaspases by FK506 Binding Protein-Mediated Oligomerization
Authors: Chang David W. , Yang Xiaolu
Published: May 2003

Oligomerization is an important biological mechanism for regulating signal transduction. Activation of caspases during apoptosis is triggered by adaptor protein-mediated oligomerization of initiator procaspases. To facilitate the study of initiator caspase activation, a system that allows inducible activation of various caspases both in vitro and in vivo is highly desired. Here we describe such a caspase activation system that is based on FK506 binding protein (FKBP)-mediated oligomerization. The NH
2
-terminal prodomains of initiator procaspases that facilitate the interaction between procaspases and their adaptor proteins are replaced by a derivative of FKBP called Fv. The Fv-caspase fusions can then be dimerized by a synthetic divalent Fv ligand, AP20187, which binds strongly to Fv but weakly to the endogenous FKBPs. This FKBP-based system may be widely applicable to the study of the regulation and functions of caspases.
Mitogen-Activated Protein Kinase Activation in UV-Induced Signal Transduction
Authors: Bode Ann M. , Dong Zigang
Published: May 2003

Experimental evidence supported by epidemiological findings suggests that solar ultraviolet (UV) irradiation is the most important environmental carcinogen leading to the development of skin cancers. Because the ozone layer blocks UVC (wavelength, 180 to 280 nm) exposure, UVA (UVA I, 340 to 400 nm; UVA II, 320 to 340 nm) and UVB (280 to 320 nm) are probably the chief carcinogenic components of sunlight with relevance for human skin cancer. Substantial contributions to the elucidation of the specific signal transduction pathways involved in UV-induced skin carcinogenesis have been made over the past few years, and most evidence suggests that the cellular signaling response is UV wavelength-dependent. The mitogen-activated protein kinase (MAPK) signaling cascades are targets for UV and are important in the regulation of the multitude of UV-induced cellular responses. Experimental studies have used a range of UVA, UVB, UVC, and various combinations in multiple doses, and the observed effects on activation and phosphorylation of MAPKs are varied. This review focuses on the mechanistic data supporting a role for MAPKs in UV-induced skin carcinogenesis. Progress in understanding the mechanisms of UV-induced signal transduction could lead to the use of these protein kinases as specific targets for the prevention and control of skin cancer.
Alkaline Phosphatase Fusion Proteins as Affinity Probes for Protein Localization Studies
Authors: Brennan Caroline , Fabes Jez
Published: May 2003

The receptor affinity probe or receptor alkaline phosphatase (RAP) staining method uses soluble protein ectodomains fused to secreted placental alkaline phosphatase to locate ectodomain binding sites within cells or tissues. We have used this approach to identify expressing cells in tissue culture, in tissue sections, or in whole-mount embryos. The RAP method is especially useful in situations where a reliable monoclonal antibody is not available or if an orphan receptor is the focus of the study. The technique permits localization of both receptors and ligands and is readily quantifiable for cell-surface binding assays. Soluble ectodomain placental alkaline phosphatase fusion proteins are therefore highly sensitive reagents that permit the direct localization of available binding sites through simple chromogenic assays without purification, radioactive labeling, or secondary reagents.
Determination of Cellular H
2
O
2
Production
Authors: Werner Erica
Published: May 2003

The detection of radical oxygen species (ROS) is central to the understanding of their role in signal transduction. ROS detection is based on the radical-dependent reduction of a compound with a measurable change in a chemical property. However, in vitro several of the compounds are reduced by several oxidants, which results in a lack of specificity when used to detect intracellular ROS. In contrast, by using methods that detect extracellular ROS, specificity can be corroborated by adding the appropriate competitor; for example, superoxide dismutase competes for superoxide, and catalase competes for H
2
O
2
. In addition, because of the efficient activity of superoxide dismutases on superoxide and H
2
O
2
membrane permeability, determination of extracellular H
2
O
2
can detect all potential cellular sources of these ROS. In this protocol, extracellular H
2
O
2
is measured as the limiting factor of peroxidase-mediated oxidation of homovanillic acid into a fluorescent dimer. The specificity of this reaction for H
2
O
2
is demonstrated by the addition of catalase as an H
2
O
2
scavenger. Because the assay detects small changes in fluorescence, it is highly sensitive. The high sensitivity and the specificity of this assay make it well suited to measure ROS in nonphagocytic cells where the ROS levels are in the low micromolar range. To further increase sensitivity, H
2
O
2
measurements are performed over time to ensure the detection of maximum response and to minimize the variability in response arising from cellular heterogeneity, an attribute of primary cultures.
A Long Search for Glut4 Activation
Authors: Kandror Konstantin V.
Published: May 2003

Insulin stimulates glucose transport in its target cells by translocation of the glucose transporter isoform 4 (Glut4) from an intracellular storage pool to the plasma membrane. A large body of evidence indicates that activity of Glut4 at the plasma membrane may vary. Recent findings suggest that p38 MAPK may be involved in regulation of the intrinsic activity of the transporter.
A New Role for SOCS in Insulin Action
Authors: Krebs Danielle L. , Hilton Douglas J.
Published: May 2003

Suppressor of cytokine signaling (SOCS) family proteins were initially identified as inhibiting cytokine signaling pathways through a negative feedback loop involving the inhibition of Janus kinase activity. More recent data suggest that SOCS proteins may also modulate signaling mediated through receptor tyrosine kinases. Krebs and Hilton discuss research implicating SOCS-1 and SOCS-3 as inhibitors of insulin receptor-mediated and insulin-like growth factor-1 receptor-mediated signaling pathways, as well as the increasing evidence that SOCS proteins may act in part by participating in a ubiquitin ligase complex to promote the degradation of target proteins.
Leptin--a critical body weight signal and a "master" hormone?
Authors: Trayhurn Paul
Published: May 2003

Leptin, initially identified as a hormone produced by white fat that acted as a satiety signal, has since been found to be synthesized in various tissues and to subserve diverse functions, including regulation of blood glucose concentrations and blood vessel growth, and signaling to the reproductive and immune systems. Recent research suggests that leptin, acting through the sympathetic nervous system, may also regulate bone density. Trayhurn discusses this research in the context of the established bidirectional interaction between leptin and the sympathetic nervous system, and the need to elucidate a unifying theme with which to make sense of leptin's myriad, seemingly quite disparate, functions.
Single Cell Imaging of PI3K Activity and Glucose Transporter Insertion Into the Plasma Membrane by Dual Color Evanescent Wave Microscopy
Authors: Tengholm Anders , Teruel Mary N. , Meyer Tobias
Published: May 2003

Many signaling events involve the translocation of signaling molecules to or from the plasma membrane; however, suitable techniques to quantify the temporal relationships between such signaling events are lacking. Here, we describe an evanescent wave microscopy technique that allows parallel measurement of the recruitment and dissociation of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) labeled proteins to and from the plasma membrane in individual living cells. The selective excitation of fluorescence in a zone less than 100 nm above a cover glass enables selective imaging within the plasma membrane of adherent cells, with markedly improved resolution, lower background, and minimal phototoxicity compared to confocal microscopy and other microscopy-based assays. In the microscope design we have developed, the beams from helium-cadmium (442 nm) and argon (514 nm) lasers are merged and focused through a dove prism at an angle that yields total internal reflection. In this configuration, evanescent wave-excited fluorescence at the glass-water interface can be detected with either high or low magnification, to allow for high-resolution imaging or the study of many cells in parallel. We applied this technique to make parallel measurements of the time-course of insulin-triggered activation of phosphatidylinositol 3-kinase (PI3K) and GLUT4 glucose transporter insertion into the plasma membrane of individual differentiated 3T3L1 adipocytes using a phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P
3
]-binding pleckstrin homology domain fused to CFP, and GLUT4 conjugated to YFP. The technique should have wide applicability to various cell types and diverse signaling processes.
Meeting Report: In Silico Cell Signaling UndergroundA report of the meeting "International Conference on Systems Biology," Karolinska Institute, Stockholm, Sweden, 13 to 15 December 2002.
Authors: Werner Eric
Published: May 2003

Cell signaling is becoming a darling of systems biology. Because cell signaling is a relatively well understood, complex, but not overwhelming area of biology, it has become an attractive focal point for computational modeling and simulation efforts by engineers, computer scientists, mathematicians, and systems biologists. The flow of information within and between cells points to a possible conceptual and theoretical kinship with similar phenomena in computer networks, communication theory, information theory, and information flow in engineered systems. Although still in its infancy, the field is experiencing a growth in interest, as evidenced by the poster sessions at the recent International Conference on Systems Biology (ICSB 2002). This report focuses on some of those efforts to mathematically model and simulate cell signaling and signal transduction networks.