Differential association of cytoplasmic signalling molecules SHP-1, SHP-2, SHIP and phospholipase C-gamma1 with PECAM-1/CD31.

Recent studies have shown that, in addition to its role as an adhesion receptor, platelet endothelial cell adhesion molecule 1/CD31 becomes phosphorylated on tyrosine residues Y663 and Y686 and associates with protein tyrosine phosphatases SHP-1 and SHP-2. In this study, we screened for additional proteins which associate with phosphorylated platelet endothelial cell adhesion molecule 1, using surface plasmon resonance. We found that, besides SHP-1 and SHP-2, platelet endothelial cell adhesion molecule 1 binds the cytoplasmic signalling proteins SHIP and PLC-gamma1 via their Src homology 2 domains. Using two phosphopeptides, NSDVQpY663TEVQV and DTETVpY686SEVRK, we demonstrate differential binding of SHP-1, SHP-2, SHIP and PLC-gamma1. All four cytoplasmic signalling proteins directly associate with cellular platelet endothelial cell adhesion molecule 1, immunoprecipitated from pervanadate-stimulated THP-1 cells. These results suggest that overlapping immunoreceptor tyrosine-based inhibition motif/immunoreceptor tyrosine-based activation motif-like motifs within platelet endothelial cell adhesion molecule 1 mediate differential interactions between the Src homology 2 containing signalling proteins SHP-1, SHP-2, SHIP and PLC-gamma1.     

Protein Disorder and Short Conserved Motifs in Disordered Regions Are Enriched near the Cytoplasmic Side of Single-Pass Transmembrane Proteins

Intracellular juxtamembrane regions of transmembrane proteins play pivotal roles in cell signalling, mediated by protein-protein interactions. Disordered protein regions, and short conserved motifs within them, are emerging as key determinants of many such interactions. Here, we investigated whether disorder and conserved motifs are enriched in the juxtamembrane area of human single-pass transmembrane proteins. Conserved motifs were defined as short disordered regions that were much more conserved than the adjacent disordered residues. Human single-pass proteins had higher mean disorder in their cytoplasmic segments than their extracellular parts. Some, but not all, of this effect reflected the shorter length of the cytoplasmic tail. A peak of cytoplasmic disorder was seen at around 30 residues from the membrane. We noted a significant increase in the incidence of conserved motifs within the disordered regions at the same location, even after correcting for the extent of disorder. We conclude that elevated disorder within the cytoplasmic tail of many transmembrane proteins is likely to be associated with enrichment for signalling interactions mediated by conserved short motifs.     

Genetic isolation of transport signals directing cell surface expression

Membrane proteins represent approximately 30% of the proteome in both prokaryotes and eukaryotes. The spatial localization of membrane-bound proteins is often determined by specific sequence motifs that may be regulated in response to physiological changes, such as protein interactions and receptor signalling. Identification of signalling motifs is therefore important for understanding membrane protein expression, function and transport mechanisms. We report a genetic isolation of novel motifs that confer surface expression. Further characterization showed that SWTY, one class of these isolated motifs with homology to previously reported forward transport motifs, has the ability to both override the RKR endoplasmic reticulum localization signal and potentiate steady-state surface expression. The genetically isolated SWTY motif is functionally interchangeable with a known motif in cardiac potassium channels and an identified motif in an HIV coreceptor, and operates by recruiting 14-3-3 proteins. This study expands the repertoire of and enables a screening method for membrane trafficking signals.     

Analysis of leptin signalling in hematopoietic cells using an adapted MAPPIT strategy

The adipocyte-secreted hormone leptin participates in the regulation of hematopoiesis and enhances proliferation of hematopoietic cells. We used an adaptation of the MAPPIT mammalian two-hybrid method to study leptin signalling in a hematopoietic setting. We confirmed the known interactions of suppressor of cytokine signalling 3 (SOCS3) and STAT5 with the Y985 and Y1077 motifs of the leptin receptor, respectively. We also provide evidence for novel interactions at the Y1077 motif, including phospholipase C gamma and several members of the SOCS protein family, further underscoring the important role of the Y1077 motif in leptin signalling.     

The unfolding tale of PECAM-1

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a member of the immunoglobulin (Ig) superfamily that has distinctive features of an immunoreceptor based upon its genomic structure and the presence of intrinsic immunoreceptor tyrosine inhibitory motifs (ITIMs) in its ligand binding polypeptide. This has lead to its subclassification into the Ig-ITIM superfamily. Its amino-terminal Ig-like domain of PECAM-1 is necessary for its homophilic binding, which plays an important role in cell–cell interactions. Its intracellular ITIMs serve as scaffolds for recruitment of signalling molecules including protein-tyrosine phosphatases to mediate its inhibitory co-receptor activity. Increasing evidence has implicated PECAM-1 in a plethora of biological phenomena, including modulation of integrin-mediated cell adhesion, transendothelial migration, angiogenesis, apoptosis, cell migration, negative regulation of immune cell signalling, autoimmunity, macrophage phagocytosis, IgE-mediated anaphylaxis and thrombosis. In this review, we discuss some of the new developments attributed to this molecule and its unique roles in biology.     

Immunoglobulin superfamily cell adhesion molecules: zippers and signals

The latest structural studies of immunoglobulin superfamily cell adhesion molecules are driving a shift in perspective; increasingly the view is not focused solely on the individual molecule but rather is on the molecular assembly. Two common themes are emerging, revealing mechanisms for ectodomain-dependent regulation of cell surface receptors' signalling abilities. The first is the propensity of many such molecules to arrange in zipper-type or array-type assemblies driven by a network of highly specific cis and trans interactions. The second is the use of the extracellular dimensions of a molecule or adhesion complex as properties which, in combination with characteristic intercellular spacings, can determine the co-localisation or exclusion of particular protein populations at cell interfaces and junctions.     

Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides

Signalling by fibroblast growth factors (FGFs) through FGF receptors (FGFRs) depends on the cell-surface polysaccharide heparan sulphate (HS) [1] [2]. HS has an ordered domain structure of highly diverse saccharide motifs that present unique displays of sulphate, carboxyl and hydroxyl groups [3]. These motifs interact with many proteins, particularly growth factors. HS binds both to FGFs [4] [5] [6] and FGFRs [7], and probably activates signalling by facilitating ligand-induced receptor dimerisation [8] [9]. Nevertheless, the extent to which specific HS saccharide sequences play a regulatory role has not been established. By screening a library of structurally diverse HS decasaccharides in bioassays of FGF signalling mediated by three different FGFR isoforms, we found that saccharides showed specificity for both ligands and receptors; some saccharides selectively activated FGF signalling through different FGFR isoforms, others acted as negative regulators. We conclude that HS saccharides play critical roles in dictating the specificity of ligand-receptor interactions in FGFR signalling. Controlled alterations in HS structures [10] would provide a mechanism for regulation of cellular responsiveness to growth factors that bind HS.     

SAP couples Fyn to SLAM immune receptors

SAP (SLAM-associated protein) is a small lymphocyte-specific signalling molecule that is defective or absent in patients with X-linked lymphoproliferative syndrome (XLP). Consistent with its single src homology 2 (SH2) domain architecture and unusually high affinity for SLAM (also called CD150), SAP has been suggested to function by blocking binding of SHP-2 or other SH2-containing signalling proteins to SLAM receptors. Additionally, SAP has recently been shown to be required for recruitment and activation of the Src-family kinase FynT after SLAM ligation. This signalling 'adaptor' function has been difficult to conceptualize, because unlike typical SH2-adaptor proteins, SAP contains only a single SH2 domain and lacks other recognized protein interaction domains or motifs. Here, we show that the SAP SH2 domain binds to the SH3 domain of FynT and directly couples FynT to SLAM. The crystal structure of a ternary SLAM-SAP-Fyn-SH3 complex reveals that SAP binds the FynT SH3 domain through a surface-surface interaction that does not involve canonical SH3 or SH2 binding interactions. The observed mode of binding to the Fyn-SH3 domain is expected to preclude the auto-inhibited conformation of Fyn, thereby promoting activation of the kinase after recruitment. These findings broaden our understanding of the functional repertoire of SH3 and SH2 domains.     

Molecular interactions between neurotrophin receptors

Neurotrophins signal via a dual-receptor system comprising receptor tyrosine kinases, the Trks, and a tumor necrosis factor (TNF) receptor like molecule, p75. Interest in these receptors was spurred on by the finding that they are employed by their neurotrophin ligands to activate opposing cellular mechanisms. Signalling via Trk receptors promotes the survival of embryonic neurons, whereas activation of p75 can trigger apoptosis. However, this antagonistic view is an oversimplification. It is more accurate to refer to this system as a signalling network in which ligands, receptors and their intracellular target proteins are linked by balanced biochemical interactions. This article reviews recent advances in our understanding of these molecular mechanisms which critically determine many cell-type-specific responses to neurotrophins. Emphasis is given to the formation of receptor complexes, the generation of receptor diversity by alternative splicing and the influence exerted by the local membrane environment on neurotrophin signalling.     

An investigation of spatial signal transduction in cellular networks

ABSTRACT: BACKGROUND: Spatial signal transduction plays a vital role in many intracellular processes such as eukaryotic chemotaxis, polarity generation, cell division. Furthermore it is being increasingly realized that the spatial dimension to signalling may play an important role in other apparently purely temporal signal transduction processes. It is being recognized that a conceptual basis for studying spatial signal transduction in signalling networks is necessary. RESULTS: In this work we examine spatial signal transduction in a series of standard motifs/networks. These networks include coherent and incoherent feedforward, positive and negative feedback, cyclic motifs, monostable switches, bistable switches and negative feedback oscillators. In all these cases, the driving signal has spatial variation. For each network we consider two cases, one where all elements are essentially non diffusible, and the other where one of the network elements may be highly diffusible. A careful analysis of steady state signal transduction provides many insights into the behaviour of all these modules. While in the non-diffusible case for the most part, spatial signalling reflects the temporal signalling behaviour, in the diffusible cases, we see significant differences between spatial and temporal signalling characteristics. Our results demonstrate that the presence of diffusible elements in the networks provides important constraints and capabilities for signalling. CONCLUSIONS: Our results provide a systematic basis for understanding spatial signalling in networks and the role of diffusible elements therein. This provides many insights into the signal transduction capabilities and constraints in such networks and suggests ways in which cellular signalling and information processing is organized to conform to or bypass those constraints. It also provides a framework for starting to understand the organization and regulation of spatial signal transduction in individual processes.     

Single molecule studies of nucleocytoplasmic transport

Molecular traffic between the cytoplasm and the nucleoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). Hundreds, if not thousands, of molecules interact with and transit through each NPC every second. The pore is blocked by a permeability barrier, which consists of a network of intrinsically unfolded polypeptides containing thousands of phenylalanine-glycine (FG) repeat motifs. This FG-network rejects larger molecules and admits smaller molecules or cargos bound to nuclear transport receptors (NTRs). For a cargo transport complex, minimally consisting of a cargo molecule plus an NTR, access to the permeability barrier is provided by interactions between the NTR and the FG repeat motifs. Numerous models have been postulated to explain the controlled accessibility and the transport characteristics of the FG-network, but the amorphous, flexible nature of this structure has hindered characterization. A relatively recent development is the ability to monitor the real-time movement of single molecules through individual NPCs via single molecule fluorescence (SMF) microscopy. A major advantage of this approach is that it can be used to continuously monitor a series of specific molecular interactions in an active pore with millisecond time resolution, which therefore allows one to distinguish between kinetic and thermodynamic control. Novel insights and prospects for the future are outlined in this review. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

Role of Abl and Src family kinases in actin-cytoskeletal rearrangements induced by the Helicobacter pylori CagA protein

The clinical outcome of infections with Helicobacter pylori is determined by a complex interplay of host-pathogen interactions, and persistent infection with this pathogen is the major cause of developing chronic gastritis, peptic ulcers and gastric cancer. Highly virulent strains encode a so-called type IV secretion system which translocates the CagA effector protein into gastric epithelial target cells. Injected CagA becomes tyrosine-phosphorylated on EPIYA sequence motifs by Src and Abl family kinase members. CagA then binds to and activates/inactivates various signalling proteins in a phosphorylation-dependent and phosphorylation-independent manner. In this way injected CagA can act as a master key that evolved during evolution the ability to highjack multiple downstream signalling cascades. Here we review our knowledge on the tyrosine phosphorylation motifs in CagA, the recent advances in the interaction of CagA with Src and Abl tyrosine kinases and their role in signalling events leading to changes of the phosphorylation status of actin-binding proteins cortactin, ezrin and vinculin followed by actin-cytoskeletal rearrangements, cell scattering and elongation. Detailed investigation of these pathways will help to yield novel insights and to elucidate the mechanisms of H. pylori-induced pathogenesis.     

细胞黏附分子互作网络的构建与分析

借助网络分析可对基因调控、蛋白质互作和信号转导等细胞活动进行全局和局部性质分析.以细胞黏附的蛋白质相互作用为对象,通过数据挖掘和可视化软件构建了整合蛋白介导的黏附分子互作网络,该分子互作网络由156种蛋白质通过690种相互作用相连,其平均节点度为8.66、平均聚集系数为0.24,平均路径长度为2.6.黏附分子互作网络中包含数个功能模块,这些模块涉及网络内部多种分子相互作用的启动与停止,并进一步影响细胞的黏附、迁移和骨架组织.对黏附分子网络进行模体筛选和比较,发现一些数量相对较少、以三元复合物为主要结构的关键模体,同时对各网络模块和模体对细胞黏附的调控作用进行了探讨.     

Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis

Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). Here, we identify two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels.     

Simulation and sensitivity analysis of phosphorylation of EGFR signal transduction pathway in PC12 cell model

The epidermal growth factor receptor (EGFR) signalling pathway is a complex signalling process with a wide network of interactions. The activation of the mitogen-activated protein kinases (MAPKs) cascade by this activated EGFR has been well studied. MAPKs form a highly integrated network, which is essential for certain specialised cell functions. This paper presents a kinetic model for the MAPK pathway downstream of the EGFR using a biochemical simulator. The model includes 30 signalling events and 29 signalling molecules. The time course data were examined for the activation of each signalling component. The simulation provides a large volume of data, by monitoring the kinetics of the signalling components, which were compared experimentally using the PC12 cell line. The kinetic model corresponded well with the experimental results observed in the EGFR induced activation of proteins. An examination of the kinetic analysis of the multiple signalling events provides a quantitative framework for representing the EGFR signalling network.     

The role of peptide motifs in the evolution of a protein network

Naturally occurring proteins in cellular networks often share peptide motifs. These motifs have been known to play a pivotal role in protein interactions among the components of a network. However, it remains unknown how these motifs have contributed to the evolution of the protein network. Here we addressed this issue by a synthetic biology approach. Through the motif programming method, we have constructed an artificial protein library by mixing four peptide motifs shared among the Bcl-2 family proteins that positively or negatively regulate the apoptosis networks. We found one strong pro-apoptotic protein, d29, and two proteins having moderate, but unambiguous anti-apoptotic functions, a10 and d16, from the 28 tested clones. Thus both the pro- and anti-apoptotic modulators were present in the library, demonstrating that functional proteins with opposing effects can emerge from a single pool prepared from common motifs. Motif programming studies have exhibited that the annotated function of the motifs were significantly influenced by the context that the motifs embedded. The results further revealed that reshuffling of a set of motifs realized the promiscuous state of protein, from which disparate functions could emerge. Our finding suggests that motifs contributed to the plastic evolvability of the protein network.     

Spatially distributed cell signalling

Emerging evidence indicates that complex spatial gradients and (micro)domains of signalling activities arise from distinct cellular localization of opposing enzymes, such as a kinase and phosphatase, in signal transduction cascades. Often, an interacting, active form of a target protein has a lower diffusivity than an inactive form, and this leads to spatial gradients of the protein abundance in the cytoplasm. A spatially distributed signalling cascade can create step-like activation profiles, which decay at successive distances from the cell surface, assigning digital positional information to different regions in the cell. Feedback and feedforward network motifs control activity patterns, allowing signalling networks to serve as cellular devices for spatial computations.