A network analysis of changes in molecular interactions in cellular signaling

Multiprotein complexes play an essential role in the propagation and integration of cellular signals. However, systems level analyses of signaling-dependent changes in the pattern of molecular interactions are still missing. Signaling in T-lymphocytes is one prominent example in which multiprotein complexes orchestrate signal transduction. We implemented peptide microarrays comprising a set of interaction motifs of signaling proteins for network-based analyses of signaling-dependent changes in molecular interactions. Lysates of resting or stimulated cells were incubated on these arrays, and the binding of signaling proteins was detected by immunofluorescence. Signaling-dependent complex formation led to changes of signals on the microarrays in two ways. 1) Masking of a binding site of a signaling protein for a peptide on the array resulted in a signal decrease. 2) Interaction of a protein with a second protein, which in turn binds to a peptide on the array, resulted in a signal increase for the first protein. Dissipation of complexes led to the reverse changes. Competition with peptides corresponding to interaction motifs provided detailed information on the architecture of complexes; lack of individual signaling proteins revealed the functional interdependence of interactions in the network. We show that complex formation through phosphorylation of the scaffolding protein LAT (linker for activation of T-cells) acted as a signal amplifier. PLCgamma1 deficiency increased the resting state levels of LAT-dependent complexes and augmented the recruitment of the phosphatase SHPTP2 into complexes. For the analysis of signaling networks, the parallel detection of changes in interactions enabled the identification of functional interdependencies with minimum a priori knowledge.     

Emerging role for ERM proteins in cell adhesion and migration

The highly related ERM (Ezrin, Radixin, Moesin) proteins provide a regulated linkage between the membrane and the underlying actin cytoskeleton. They also provide a platform for the transmission of signals in responses to extracellular cues. Studies in different model organisms and in cultured cells have highlighted the importance of ERM proteins in the generation and maintenance of specific domains of the plasma membrane. A central question is how do ERM proteins coordinate actin filament organization and membrane protein transport/stability with signal transduction pathways to build up complex structures? Through their interaction with numerous partners including membrane proteins, actin cytoskeleton and signaling molecules, ERM proteins have the ability to organize multiprotein complexes in specific cellular compartments. Likewise, ERM proteins participate in diverse functions including cell morphogenesis, endocytosis/exocytosis, adhesion and migration. This review focuses on aspects still poorly understood related to the function of ERM proteins in epithelial cell adhesion and migration.Key words: epithelial cells, membrane-cytoskeleton interface, morphogenesis, ERM proteins, cell adhesion     

Coordination of DNA damage responses via the Smc5/Smc6 complex

The detection of DNA damage activates DNA repair pathways and checkpoints to allow time for repair. Ultimately, these responses must be coordinated to ensure that cell cycle progression is halted until repair is completed. Several multiprotein complexes containing members of the structural maintenance of chromosomes family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization. Here we show that the Smc5/Smc6 (Smc5/6) complex is required for a coordinated response to DNA damage and normal chromosome integrity. Fission yeast cells lacking functional Smc6 initiate a normal checkpoint response to DNA damage, culminating in the phosphorylation and activation of the Chk1 protein kinase. Despite this, cells enter a lethal mitosis, presumably without completion of DNA repair. Another subunit of the complex, Nse1, is a conserved member of this complex and is also required for this response. We propose that the failure to maintain a checkpoint response stems from the lack of ongoing DNA repair or from defective chromosomal organization, which is the signal to maintain a checkpoint arrest. The Smc5/6 complex is fundamental to genome integrity and may function with the condensin and cohesin complexes in a coordinated manner.     

Binding specificity of multiprotein signaling complexes is determined by both cooperative interactions and affinity preferences

The generation of multiprotein complexes at receptors and adapter proteins is crucial for the activation of intracellular signaling pathways. In this study, we used multiple biochemical and biophysical methods to examine the binding properties of several SH2 and SH3 domain-containing signaling proteins as they interact with the adapter protein linker for activation of T-cells (LAT) to form multiprotein complexes. We observed that the binding specificity of these proteins for various LAT tyrosines appears to be constrained both by the affinity of binding and by cooperative protein-protein interactions. These studies provide quantitative information on how different binding parameters can determine in vivo binding site specificity observed for multiprotein signaling complexes.     

Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex

The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface.     

The membrane proximal proline-rich region and correct order of C-terminal tyrosines on the adaptor protein LAT are required for TCR-mediated signaling and downstream functions

The primary activating receptor for T cells is the T cell receptor (TCR), which is stimulated upon binding to an antigen/MHC complex. TCR activation results in the induction of regulated signaling pathways vital for T cell differentiation, cellular adhesion and cytokine release. A critical TCR-induced signaling protein is the adaptor protein LAT. Upon TCR stimulation, LAT is phosphorylated on conserved tyrosines, which facilitates the formation of multiprotein complexes needed for propagation of signaling pathways. Although the role of the conserved tyrosines in LAT-mediated signaling has been investigated, few studies have examined the role of larger regions of LAT in TCR-induced pathways. In this study, a sequence alignment of 97 mammalian LAT proteins was used to identify several “functional” domains on LAT. Using LAT mutants expressed in Jurkat E6.1 cells, we observed that the membrane proximal, proline-rich region of LAT and the correct order of domains containing conserved tyrosines are necessary for optimal TCR-mediated early signaling, cytokine production, and cellular adhesion. Together, these data show that LAT contains distinct regions whose presence and correct order are required for the propagation of TCR-mediated signaling pathways.     

GPCR-Kir channel signaling complexes: defining rules of engagement

Ion channels and G protein-coupled receptors (GPCRs) are integral transmembrane proteins vital to a multitude of cell signaling and physiological functions. Members of these large protein families are known to interact directly with various intracellular protein partners in a dynamic and isoform-dependent manner, ultimately shaping their life cycle and signal output. The family of G protein-gated inwardly rectifying potassium channels (Kir3 or GIRK) expressed in brain, heart, and endocrine tissues were recently shown to stably associate with several different GPCRs, forming the basis of a macromolecular ion channel-GPCR signaling complex. The molecular determinants that mediate and maintain GPCR-Kir3 channel complexes are currently not well understood. Recent findings and emerging hypotheses on the assembly and stability of multiprotein GPCR-Kir channel signaling complexes are discussed, highlighting distinct mechanisms used by different Kir channel families. These protein-protein interaction processes are crucial in determining both the synaptic response times and the extent of GPCR "cross-talk" in Kir3-mediated inhibitory synaptic transmission.     

The LIM protein Ajuba influences interleukin-1-induced NF-kappaB activation by affecting the assembly and activity of the protein kinase Czeta/p62/TRAF6 signaling complex

The Zyxin/Ajuba family of cytosolic LIM domain-containing proteins has the potential to shuttle from sites of cell adhesion into the nucleus and thus can be candidate transducers of environmental signals. To understand Ajuba's role in signal transduction pathways, we performed a yeast two-hybrid screen with the LIM domain region of Ajuba. We identified the atypical protein kinase C (aPKC) scaffold protein p62 as an Ajuba binding partner. A prominent function of p62 is the regulation of NF-kappaB activation in response to interleukin-1 (IL-1) and tumor necrosis factor signaling through the formation of an aPKC/p62/TRAF6 multiprotein signaling complex. In addition to p62, we found that Ajuba also interacted with tumor necrosis factor receptor-associated factor 6 (TRAF6) and PKCzeta. Ajuba recruits TRAF6 to p62 and in vitro activates PKCzeta activity and is a substrate of PKCzeta. Ajuba null mouse embryonic fibroblasts (MEFs) and lungs were defective in NF-kappaB activation following IL-1 stimulation, and in lung IKK activity was inhibited. Overexpression of Ajuba in primary MEFs enhances NF-kappaB activity following IL-1 stimulation. We propose that Ajuba is a new cytosolic component of the IL-1 signaling pathway modulating IL-1-induced NF-kappaB activation by influencing the assembly and activity of the aPKC/p62/TRAF6 multiprotein signaling complex.     

Cancer cell motility--on the road from c-erbB-2 receptor steered signaling to actin reorganization.

Cell migration depends mainly on actin polymerization and intracellular organization, which are influenced by a vast variety of actin binding proteins (ABPs). Regulation of ABP activity is mediated by second messengers such as phosphoinositides and calcium. Signaling via these second messengers is initiated and regulated by membrane receptors, e.g., receptor tyrosine kinases (RTKs), and by adhesion molecule interactions (e.g., integrins and selectins) and focal adhesion kinases. A major role in steering second-messenger signaling and thus in actin cytoskeleton reorganization and motility of cancer cells is played by the RTK c-erbB-2. This occurs through a number of signaling pathways which involve mainly enzymes, e.g., phospholipase Cgamma1 and GTPases, which modify signaling molecules. Furthermore large multiprotein complexes including actin-related protein 2/3, Wiskott-Aldrich syndrome protein, profilin, and capping protein among others play an important role in regulating actin reorganization. The complex picture of the mode of actin reorganization, which is involved in tumor cell migration, is slowly emerging from the mists of cellular signaling pathways, but this is still by no means a clear view.     

Examining multiprotein signaling complexes from all angles

Dynamic protein-protein interactions are involved in most physiological processes and, in particular, for the formation of multiprotein signaling complexes at transmembrane receptors, adapter proteins and effector molecules. Because the unregulated induction of signaling complexes has substantial clinical relevance, the investigation of these complexes is an active area of research. These studies strive to answer questions about the composition and function of multiprotein signaling complexes, along with the molecular mechanisms of their formation. In this review, the adapter protein, linker for activation of T cells (LAT), will be employed as a model to exemplify how signaling complexes are characterized using a range of techniques. The intensive investigation of LAT highlights how the systematic use of complementary techniques leads to an integrated understanding of the formation, composition and function of multiprotein signaling complexes that occur at receptors, adapter proteins and effector molecules.     

The functions of mTOR in ischemic diseases

Mammalian Target of Rapamycin (mTOR) is a serine/threonine kinase and that forms two multiprotein complexes known as the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTOR regulates cell growth, proliferation and survival. mTORC1 is composed of the mTOR catalytic subunit and three associated proteins: raptor, mLST8/GβL and PRAS40. mTORC2 contains mTOR, rictor, mLST8/GβL, mSin1, and protor. Here, we discuss mTOR as a promising anti-ischemic agent. It is believed that mTORC2 lies down-stream of Akt and acts as a direct activator of Akt. The different functions of mTOR can be explained by the existence of two distinct mTOR complexes containing unique interacting proteins. The loss of TSC2, which is upstream of mTOR, activates S6K1, promotes cell growth and survival, activates mTOR kinase activities, inhibits mTORC1 and mTORC2 via mTOR inhibitors, and suppresses S6K1 and Akt. Although mTOR signaling pathways are often activated in human diseases, such as cancer, mTOR signaling pathways are deactivated in ischemic diseases. From Drosophila to humans, mTOR is necessary for Ser473 phosphorylation of Akt, and the regulation of Akt-mTOR signaling pathways may have a potential role in ischemic disease. This review evaluates the potential functions of mTOR in ischemic diseases. A novel mTOR-interacting protein deregulates over-expression in ischemic disease, representing a new mechanism for controlling mTOR signaling pathways and potential therapeutic strategies for ischemic diseases.     

Caspase-12 modulates NOD signaling and regulates antimicrobial peptide production and mucosal immunity

Bacterial sensing by intracellular Nod proteins and other Nod-like receptors (NLRs) activates signaling pathways that mediate inflammation and pathogen clearance. Nod1 and Nod2 associate with the kinase Rip2 to stimulate NF-kappaB signaling. Other cytosolic NLRs assemble caspase-1-activating multiprotein complexes termed inflammasomes. Caspase-12 modulates the caspase-1 inflammasome, but unlike other NLRs, Nod1 and Nod2 have not been linked to caspases, and mechanisms regulating the Nod-Rip2 complex are less clear. We report that caspase-12 dampens mucosal immunity to bacterial infection independent of its effects on caspase-1. Caspase-12 deficiency enhances production of antimicrobial peptides, cytokines, and chemokines to entric pathogens, an effect dependent on bacterial type III secretion and the Nod pathway. Mechanistically, caspase-12 binds to Rip2, displacing Traf6 from the signaling complex, inhibiting its ubiquitin ligase activity, and blunting NF-kappaB activation. Nod activation and resulting antimicrobial peptide production constitute an early innate defense mechanism, and caspase-12 inhibits this mucosal antimicrobial response.     

Secretion without Golgi

A growing number of proteins devoid of signal peptides have been demonstrated to be released through the non-classical pathways independent of endoplasmic reticulum and Golgi. Among them are two potent proangiogenic cytokines FGF1 and IL1alpha. Stress-induced transmembrane translocation of these proteins requires the assembly of copper-dependent multiprotein release complexes. It involves the interaction of exported proteins with the acidic phospholipids of the inner leaflet of the cell membrane and membrane destabilization. Not only stress, but also thrombin treatment and inhibition of Notch signaling stimulate the export of FGF1. Non-classical release of FGF1 and IL1alpha presents a promising target for treatment of cardiovascular, oncologic, and inflammatory disorders.     

Comprehensive proteomic analysis of human Par protein complexes reveals an interconnected protein network

The polarization of eukaryotic cells is controlled by the concerted activities of asymmetrically localized proteins. The PAR proteins, first identified in Caenorhabditis elegans, are common regulators of cell polarity conserved from nematode and flies to man. However, little is known about the molecular mechanisms by which these proteins and protein complexes establish cell polarity in mammals. We have mapped multiprotein complexes formed around the putative human Par orthologs MARK4 (microtubule-associated protein/microtubule affinity-regulating kinase 4) (Par-1), Par-3, LKB1 (Par-4), 14-3-3zeta and eta (Par-5), Par-6a, -b, -c, and PKClambda (PKC3). We employed a proteomic approach comprising tandem affinity purification (TAP) of protein complexes from cultured cells and protein sequencing by tandem mass spectrometry. From these data we constructed a highly interconnected protein network consisting of three core complex "modules" formed around MARK4 (Par-1), Par-3.Par-6, and LKB1 (Par-4). The network confirms most previously reported interactions. In addition we identified more than 50 novel interactors, some of which, like the 14-3-3 phospho-protein scaffolds, occur in more than one distinct complex. We demonstrate that the complex formation between LKB1.Par-4, PAPK, and Mo25 results in the translocation of LKB1 from the nucleus to the cytoplasm and to tight junctions and show that the LKB1 complex may activate MARKs, which are known to introduce 14-3-3 binding sites into several substrates. Our findings suggest co-regulation and/or signaling events between the distinct Par complexes and provide a basis for further elucidation of the molecular mechanisms that govern cell polarity.     

EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains

Transmembrane ephrinB proteins have important functions during embryonic patterning as ligands for Eph receptor tyrosine kinases and presumably as signal-transducing receptor-like molecules. Consistent with "reverse" signaling, ephrinB1 is localized in sphingo-lipid/cholesterol-enriched raft microdomains, platforms for the localized concentration and activation of signaling molecules. Glutamate receptor-interacting protein (GRIP) and a highly related protein, which we have termed GRIP2, are recruited into these rafts through association with the C-terminal PDZ target site of ephrinB1. Stimulation of ephrinB1 with soluble EphB2 receptor ectodomain causes the formation of large raft patches that also contain GRIP proteins. Moreover, a GRIP-associated serine/threonine kinase activity is recruited into ephrinB1-GRIP complexes. Our findings suggest that GRIP proteins provide a scaffold for the assembly of a multiprotein signaling complex downstream of ephrinB ligands.     

The SUMO system: a master organizer of nuclear protein assemblies

Cellular signaling pathways largely depend on the plasticity of multiprotein complexes. A central mechanism that assures the coordinated assembly and disassembly of protein complexes is the reversible post-translational modification of the individual components for example by phosphorylation, acetylation, or ubiquitylation. Accumulating evidence indicates that the small ubiquitin-related modifier (SUMO) system is another master organizer of protein complexes. Here, we will focus on the role of SUMO in the regulation of nuclear protein complexes that are involved in chromatin remodeling, double-strand break repair, and ribosome biogenesis. On the basis of these selected pathways, we will summarize current ideas of SUMO signaling, including the concept of group modification and the intersection of the ubiquitin and SUMO pathways.