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β 2 -Adrenergic receptors (β 2 -ARs) transduce the effects of (nor)epinephrine on a variety of cell types and act as key mediators of the body’s reaction to stress. β 2 -ARs are also expressed on immune cells and there is ample evidence for their role in immunomodulation. A key regulator of the immune response and a target for regulation by stress-induced signals is the transcription factor Nuclear Factor-kappaB (NF-κB). NF-κB shapes the course of both innate and adaptive immune responses and plays an important role in susceptibility to disease. In this review, we summarise the literature that has been accumulated in the past 20 years on adrenergic modulation of NF-κB function. We here focus on the molecular basis of the reported interactions and show that both physiological and pharmacological triggers of β 2 -ARs intersect with the NF-κB signalling cascade at different levels. Importantly, the action of β 2 -AR-derived signals on NF-κB activity appears to be highly cell type specific and gene selective, providing opportunities for the development of selective NF-κB modulators. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptor (TLR) signalling is initiated by the recruitment of one or more adaptor proteins to the activated receptor complex. At present, four of these proteins are identified, namely MyD88, Mal, Trif and Tram and their selective usage by different TLRs in part accounts for TLR-specific transcriptional responses. Recent findings described unique biochemical properties for each of these TIR-domain containing adaptors and revealed that these adapters are subjected to post-translational modification. We used mammalian protein–protein interaction trap (MAPPIT), a two-hybrid technique that functions in a mammalian cell context, to study the molecular interactions downstream of TLR activation. We demonstrate pathway walking from TLR4 to IRAK-1 and identified Mal as a bridging adaptor, linking MyD88 to the activated TLR4. [Copyright &y& Elsevier]

Abstract: Besides the transcription-promoting role of histone acetyltransferases (HATs) and the transcription-delimiting function of histone deacetylases (HDACs) through histone acetylation and deacetylation respectively, HATs and HDACs also regulate the activity of several non-histone proteins. This includes signal transducers and activators of transcription (STATs), key proteins in cytokine signaling. Unlike Tyr phosphorylation/dephosphorylation, which mainly acts as an on/off switch of STAT activity, the control exerted by HATs and HDACs appears multifaceted and far more complex than initially imagined. Our review focuses on the latest trends and novel hypotheses to explain differential context-dependent STAT regulation by complex posttranslational modification patterns. We chart the knowledge on how STATs interact with HATs and HDACs, and additionally bring a transcriptional regulatory and gene-set specific role for HDACs in the picture. Indeed, a growing amount of evidence demonstrates, paradoxically, that not only HAT but also HDAC activity can be required for STAT-dependent transcription, in a STAT subtype- and cell type-dependent manner. Referring to recent reports, we review and discuss the various molecular mechanisms that have recently been proposed to account for this peculiar regulation, in an attempt to shed more light on the difficult yet important question on how STAT specificity is being generated. [Copyright &y& Elsevier]

Summary Members of the Toll-like receptor and interleukin-1 (IL-1) receptor families all signal via Toll/IL-1R (TIR) domain-driven assemblies with adaptors such as MyD88. We here combine the mammalian two-hybrid system MAPPIT and saturation mutagenesis to complement and extend crystallographic and nuclear magnetic resonance data, and reveal how TIR domains interact. We fully delineate the interaction sites on the MyD88 TIR domain for homo-oligomerization and for interaction with Mal and TLR4. Interactions between three sites drive MyD88 homo-oligomerization. The BB-loop interacts with the αE-helix, explaining how BB-loop mimetics inhibit MyD88 signaling. The αC′-helix interacts symmetrically. The MyD88 TIR domains thus assemble into a left-handed helix, compatible with the Myddosome death domain crystal structure. This assembly explains activation of MyD88 by Mal and by an oncogenic mutation, and regulation by phosphorylation. These findings provide a paradigm for the interaction of mammalian TIR domains. [ABSTRACT FROM AUTHOR]

The regulation of intracellular Ca 2+ signaling is an important aspect of how anti-apoptotic B-cell lymphoma 2 (Bcl-2) proteins regulate cell death and cell survival. At the endoplasmic reticulum (ER) the Bcl-2 homology (BH) 4 domain of Bcl-2 is known to bind to and inhibit both inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and ryanodine receptors (RyRs). Besides this, drugs that target the hydrophobic cleft of Bcl-2 have been reported to deplete ER Ca 2+ stores in an IP 3 R- and RyR-dependent way. This suggests that the hydrophobic cleft of Bcl-2 may also be involved in regulating these ER-located Ca 2+ -release channels. However, the contribution of the hydrophobic cleft on the binding and regulatory properties of Bcl-2 to either IP 3 Rs or RyRs has until now not been studied. Here, the importance of the hydrophobic cleft of Bcl-2 in binding to and inhibiting the RyR was assessed by using a genetic approach based on site-directed mutagenesis of Bcl-2's hydrophobic cleft and a pharmacological approach based on the selective Bcl-2 hydrophobic cleft inhibitor, ABT-199. Both binding assays and single-cell Ca 2+ measurements indicated that RyR binding and the inhibition of RyR-mediated Ca 2+ release by Bcl-2 is independent of its hydrophobic cleft. [ABSTRACT FROM AUTHOR]

The timely orchestration of multiple signalling pathways is crucial for the integrity of an organism and therefore tightly controlled. Gab family proteins coordinate signal transduction at the plasma membrane (PM) by acting as docking platforms for signalling components involved in MAP kinase (MAPK), PI3 kinase (PI3K), phospholipase C (PLC) and Rho family GTPase signalling. The interaction with these components as well as the targeting of the docking platform to the PM underlies complex spatial and temporal regulatory mechanisms. Deregulated Gab1 activation and membrane binding have been observed in some haematopoietic malignancies and solid tumours, thereby contributing, for example, to the development of Philadelphia chromosome-negative myeloproliferative neoplasms and certain lung cancers. Previously, we could demonstrate that the presence of PIP3 in the PM, which is increased in many cancer cells, is not sufficient for constitutive Gab1 membrane recruitment. In addition, MAPK-dependent phosphorylation of Gab1 at serine 552 (Ser552) is vital for Gab1 membrane binding. Here, we confirm our hypothesis that in the absence of MAPK activity an intrinsic part of Gab1 prevents binding to PIP3 at the PM. This epitope of Gab1, which encompasses Ser552, interacts directly with the Gab1 PH domain. Two arginines located in positions + 4 and + 8 of Ser552 are essential for the interaction with the PH domain, as well as for the inhibition of membrane recruitment of unphosphorylated Gab1. Ser552 phosphorylation is dispensable in respective arginine to alanine mutants of Gab1. Gab1 recruitment to the PM is highly dynamic and continuous PI3K and MAPK activities are both essential for sustained Gab1 membrane localisation. Our data document the existence of a sophisticated and robust control mechanism that prevents Gab1 translocation and signalling complex assembly after the activation of either MAPK or PI3K alone. [ABSTRACT FROM AUTHOR]

Abstract: MAPPIT (mammalian protein–protein interaction trap) is a two-hybrid interaction mapping technique based on functional complementation of a type I cytokine receptor signaling pathway. Over the last decade, the technology has been extended into a platform of complementary assays for the detection of interactions among proteins and between chemical compounds and proteins, and for the identification of small molecules that interfere with protein–protein interactions. Additionally, several screening approaches have been developed to broaden the utility of the platform. In this review we provide an overview of the different components of the MAPPIT toolbox and highlight a number of applications in interactomics, drug screening and compound target profiling. [Copyright &y& Elsevier]

Abstract: The suppressors of cytokine signalling (SOCS) box is a structural domain found at the C-terminus of over 70 human proteins. It is usually coupled to a protein interaction module such as an SH2 domain in case of SOCS proteins, a family of modulators of cytokine signaling. The SOCS box participates in the formation of E3 ligase complexes, marking activated cytokine receptor complexes for proteasomal degradation. A similar mechanism was recently uncovered for controlling SOCS activity itself, since SOCS2 was found to enhance the turnover of other SOCS proteins. The SOCS box can also add unique features to individual SOCS proteins: it can function as an adaptor domain as was demonstrated for SOCS3, or as a modulator of substrate binding in case of CIS. In this review we discuss these multiple roles of the SOCS box, which emerges as a versatile module controlling cytokine signaling via multiple mechanisms. [Copyright &y& Elsevier]

Abstract: Toll-like receptors (TLRs) are crucial components of the innate immune system, coupling pathogen recognition to a cellular response. We used the MAPPIT mammalian two-hybrid technique to investigate protein–protein interactions in the early steps in TLR signalling. A partial TLR-adaptor interaction map was constructed confirming several known but also documenting novel interactions. We show that the TLR adaptor Mal is critical for linking Myeloid Differentiation primary response protein 88 (MyD88) to TLR2 and TLR4. Analysis of the contributions of the different sub-domains of MyD88-adaptor-like protein (Mal) and MyD88 in adaptor homo- and hetero-dimerisation provides an initial mechanistic insight in this bridging function of Mal. [Copyright &y& Elsevier]

Ligand-induced clustering of type I cytokine receptor subunits leads to trans-phosphorylation and activation of associated cytosolic janus kinases (JAKs). In turn, JAKs phosphorylate tyrosine residues in the receptor tails, leading to recruitment and activation of signalling molecules. Among these, signal transducers and activators of transcription (STATs) are important in the direct transmission of signals to the nucleus. Here, we show that incorporation of an interaction trap in a signalling-deficient receptor allows the identification of protein?protein interactions, using a STAT-dependent complementation assay. Mammalian protein?protein interaction trap (MAPPIT) adds to existing yeast two-hybrid procedures, as originally explored by Fields and Song, and permits the detection of both modification-independent and of phosphorylation-dependent interactions in intact human cells. We also demonstrate that MAPPIT can be used to screen complex complementary DNA libraries, and using this approach, we identify cytokine-inducible SH2-containing protein (CIS) and suppressor of cytokine signalling-2 (SOCS-2) as interaction partners of the phosphotyrosine 402 (Tyr 402)-binding motif in the erythropoietin receptor (EpoR). Importantly, this approach places protein?protein interactions in their normal physiological context, and is especially applicable to the in situ analysis of signal transduction pathways. [ABSTRACT FROM AUTHOR]

Summary Loss of ER Ca 2+ homeostasis triggers endoplasmic reticulum (ER) stress and drives ER-PM contact sites formation in order to refill ER-luminal Ca 2+ . Recent studies suggest that the ER stress sensor and mediator of the unfolded protein response (UPR) PERK regulates intracellular Ca 2+ fluxes, but the mechanisms remain elusive. Here, using proximity-dependent biotin identification (BioID), we identified the actin-binding protein Filamin A (FLNA) as a key PERK interactor. Cells lacking PERK accumulate F-actin at the cell edges and display reduced ER-PM contacts. Following ER-Ca 2+ store depletion, the PERK-FLNA interaction drives the expansion of ER-PM juxtapositions by regulating F-actin-assisted relocation of the ER-associated tethering proteins Stromal Interaction Molecule 1 (STIM1) and Extended Synaptotagmin-1 (E-Syt1) to the PM. Cytosolic Ca 2+ elevation elicits rapid and UPR-independent PERK dimerization, which enforces PERK-FLNA-mediated ER-PM juxtapositions. Collectively, our data unravel an unprecedented role of PERK in the regulation of ER-PM appositions through the modulation of the actin cytoskeleton. [ABSTRACT FROM AUTHOR]

Background: Stromal fibroblasts participate in the development of a permissive environment for tumor growth, yet molecular pathways to therapeutically target fibroblasts are poorly defined. CD248, also known as endosialin or tumor endothelial marker 1 (TEM1), is a transmembrane glycoprotein expressed on activated fibroblasts. We recently showed that the cytoplasmic domain of CD248 is important in facilitating an inflammatory response in a mouse model of arthritis. Others have reported that CD248 gene inactivation in mice results in dampened tumor growth. We hypothesized that the conserved cytoplasmic domain of CD248 is important in regulating tumor growth.Methods: Mice lacking the cytoplasmic domain of CD248 (CD248CyD/CyD) were generated and evaluated in tumor models, comparing the findings with wild-type mice (CD248WT/WT).Results: As compared to the response in CD248WT/WT mice, growth of T241 fibrosarcomas and Lewis lung carcinomas was significantly reduced in CD248CyD/CyD mice. Tumor size was similar to that seen with CD248-deficient mice. Conditioned media from CD248CyD/CyD fibroblasts were less effective at supporting T241 fibrosarcoma cell survival. In addition to our previous observation of reduced release of activated matrix metalloproteinase (MMP)-9, CD248CyD/CyD fibroblasts also had impaired PDGF-BB-induced migration and expressed higher transcripts of tumor suppressor factors, transgelin (SM22α), Hes and Hey1.Conclusions: The multiple pathways regulated by the cytoplasmic domain of CD248 highlight its potential as a therapeutic target to treat cancer. [ABSTRACT FROM AUTHOR]