The balance between the novel protein target of wingless and the Drosophila Rho-associated kinase pathway regulates planar cell polarity in the Drosophila wing

Planar cell polarity (PCP) signaling is mediated by the serpentine receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling utilizes Drosophila Frizzled 2 (DFz2) as a receptor and also requires Dsh for transducing signals to regulate cell proliferation and differentiation in many developmental contexts. Distinct pathways are activated downstream of Dsh in Wg- and Fz-signaling pathways. Recently, a number of genes, which have essential roles as downstream components of PCP signaling, have been identified in Drosophila. They include the small GTPase RhoA/Rho1, its downstream effector Drosophila rho-associated kinase (Drok), and a number of genes such as inturned (in) and fuzzy (fy), whose biochemical functions are unclear. RhoA and Drok provide a link from Fz/Dsh signaling to the modulation of actin cytoskeleton. Here we report the identification of the novel gene target of wingless (tow) by enhancer trap screening. tow expression is negatively regulated by Wg signaling in wing imaginal discs, and the balance between tow and the Drok pathway regulates wing-hair morphogenesis. A loss-of-function mutation in tow does not result in a distinct phenotype. Genetic interaction and gain-of-function studies provide evidence that Tow acts downstream of Fz/Dsh and plays a role in restricting the number of hairs that wing cells form.     

Co-localization of neural cell adhesion molecule and fibroblast growth factor receptor 2 in early embryo development

During development there is a multitude of signaling events governing the assembly of the developing organism. Receptors for signaling molecules such as fibroblast growth factor receptor 2 (FGFR2) enable the embryo to communicate with the surrounding environment and activate downstream pathways. The neural cell adhesion molecule (NCAM) was first characterized as a cell adhesion molecule highly expressed in the nervous system, but recent studies have shown that it is also a signaling receptor. Using a novel single oocyte adaptation of the proximity ligation assay, we here show a close association between NCAM and FGFR2 in mouse oocytes and 2-cell embryos. Real-time PCR analyses revealed the presence of messenger RNA encoding key proteins in downstream signaling pathways in oocytes and early mouse embryos. In summary these findings show a co-localization of NCAM and FGFR2 in early vertebrate development with intracellular signaling pathways present to enable a cellular response.     

TNFR1 promotes tumor necrosis factor-mediated mouse colon epithelial cell survival through RAF activation of NF-kappaB

Tumor necrosis factor (TNF) is a therapeutic target in the treatment of inflammatory bowel disease; however, the exact role of TNF signaling in the colon epithelium remains unclear. We demonstrate that TNF activation of TNF receptor (R)1 stimulates both pro- and anti-apoptotic signaling pathways in the colon epithelium; however, TNFR1 protects against colon epithelial cell apoptosis following TNF exposure. To investigate anti-apoptotic signaling pathways downstream of TNFR1, we generated an intestinal epithelium-specific Raf knock-out mouse and identified Raf kinase as a key regulator of colon epithelial cell survival in response to TNF. Surprisingly, Raf promotes NF-kappaB p65 phosphorylation, independent of MEK signaling, to support cell survival. Taken together, these data demonstrate a novel pathway in which Raf promotes colon epithelial cell survival through NF-kappaB downstream of TNFR1 activation. Thus, further understanding of colon epithelial cell-specific TNFR signaling may result in the identification of new targets for inflammatory bowel disease treatment and define novel mediators of colitis-associated cancer.     

The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation

B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.     

Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions

Chondrocyte differentiation is a multi-step process characterized by successive changes in cell morphology and gene expression. In addition to tight regulation by numerous soluble factors, these processes are controlled by adhesive events. During the early phase of the chondrocyte life cycle, cell-cell adhesion through molecules such as N-cadherin and neural cell adhesion molecule (N-CAM) is required for differentiation of mesenchymal precursor cells to chondrocytes. At later stages, for example in growth plate chondrocytes, adhesion signaling from extracellular matrix (ECM) proteins through integrins and other ECM receptors such as the discoidin domain receptor (DDR) 2 (a collagen receptor) and Annexin V is necessary for normal chondrocyte proliferation and hypertrophy. Cell-matrix interactions are also important for chondrogenesis, for example through the activity of CD44, a receptor for Hyaluronan and collagens. The roles of several signaling molecules involved in adhesive signaling, such as integrin-linked kinase (ILK) and Rho GTPases, during chondrocyte differentiation are beginning to be understood, and the actin cytoskeleton has been identified as a common target of these adhesive pathways. Complete elucidation of the pathways connecting adhesion receptors to downstream effectors and the mechanisms integrating adhesion signaling with growth factor- and hormone-induced pathways is required for a better understanding of physiological and pathological skeletal development.     

MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling.

The receptor tyrosine kinases Sevenless (SEV) and the Epidermal growth factor receptor (EGFR) are required for the proper development of the Drosophila eye. The protein tyrosine phosphatase Corkscrew (CSW) is a common component of many RTK signaling pathways, and is required for signaling downstream of SEV and EGFR. In order to identify additional components of these signaling pathways, mutations that enhanced the phenotype of a dominant negative form of Corkscrew were isolated. This genetic screen identified the novel signaling molecule MASK, a large protein that contains two blocks of ankyrin repeats as well as a KH domain. MASK genetically interacts with known components of these RTK signaling pathways. In the developing eye imaginal disc, loss of MASK function generates phenotypes similar to those generated by loss of other components of the SEV and EGFR pathways. These phenotypes include compromised photoreceptor differentiation, cell survival and proliferation. Although MASK is localized predominantly in the cellular cytoplasm, it is not absolutely required for MAPK activation or nuclear translocation. Based on our results, we propose that MASK is a novel mediator of RTK signaling, and may act either downstream of MAPK or transduce signaling through a parallel branch of the RTK pathway.     

The functional role of the second NPXY motif of the LRP1 beta-chain in tissue-type plasminogen activator-mediated activation of N-methyl-D-aspartate receptors

The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-D-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors.     

Identification of a specific inhibitor of the dishevelled PDZ domain

The Wnt signaling pathways are involved in embryo development as well as in tumorigenesis. Dishevelled (Dvl) transduces Wnt signals from the receptor Frizzled (Fz) to downstream components in canonical and noncanonical Wnt signaling pathways. The Dvl PDZ domain is thought to play an essential role in both pathways, and we recently demonstrated that the Dvl PDZ domain binds directly to Fz receptors. In this study, using structure-based virtual ligand screening, we identified an organic molecule (NSC668036) from the National Cancer Institute small-molecule library that can bind to the Dvl PDZ domain. We then used molecular dynamics simulation to analyze the binding between the PDZ domain and NSC668036 in detail. In addition, we showed that, in Xenopus, as expected, NSC668036 inhibited the signaling induced by Wnt3A. This compound provides a basis for rational design of high-affinity inhibitors of the PDZ domain, which can block Wnt signaling by interrupting the Fz-Dvl interaction.     

Coordinating TLR-activated signaling pathways in cells of the immune system

Toll-like receptor (TLR) signaling leads to the activation of mitogen-activated protein kinase and nuclear factor-kappaB signaling pathways. While the upstream signaling events initiated at the level of adaptors and the activation of the downstream signaling pathways have received a lot of attention, our understanding of how these signaling pathways are coordinated to regulate gene expression is poorly understood. This review gives a selective overview on our current understanding of signaling downstream of TLRs, with an emphasis on how the upstream kinases like the mitogen-activated protein kinase kinase kinases (TAK1 and Tpl2) and inhibitor of kappa-B kinase (IKK) coordinate the signaling events that steer the course of an immune response.     

Optical biosensor provides insights for bradykinin B(2) receptor signaling in A431 cells

The spatial and temporal targeting of proteins or protein assemblies to appropriate sites is crucial to regulate the specificity and efficiency of protein-protein interactions, thus dictating the timing and intensity of cell signaling and responses. The resultant dynamic mass redistribution could be manifested by label free optical biosensor, and lead to a novel and functional optical signature for studying cell signaling. Here we applied this technology, termed as mass redistribution cell assay technology (MRCAT), to study the signaling networks of bradykinin B(2) receptor in A431 cells. Using MRCAT, the spatial and temporal relocation of proteins and protein assemblies mediated by bradykinin was quantitatively monitored in microplate format and in live cells. The saturability to bradykinin, together with the specific and dose-dependent inhibition by a B(2) specific antagonist HOE140, suggested that the optical signature is a direct result of B(2) receptor activation. The sensitivity of the optical signature to cholesterol depletion by methyl-beta-cyclodextrin argued that B(2) receptor signaling is dependent on the integrity of lipid rafts; disruption of these microdomains hinders the B(2) signaling. Modulations of several important intracellular targets with specific inhibitors suggested that B(2) receptor activation results in signaling via at least dual pathways - G(s)- and G(q)-mediated signaling. Remarkably, the two signaling pathways counter-regulate each other. Several critical downstream targets including protein kinase C, protein kinase A, and epidermal growth factor receptor had been identified to involve in B(2) signaling. The roles of endocytosis and cytoskeleton modulation in B(2) signaling were also demonstrated.     

Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells

Notch and bone morphogenetic protein signaling pathways are important for cellular differentiation, and both have been implicated in vascular development. In many cases the two pathways act similarly, but antagonistic effects have also been reported. The underlying mechanisms and whether this is caused by an interplay between Notch and BMP signaling is unknown. Here we report that expression of the Notch target gene, Herp2, is synergistically induced upon activation of Notch and BMP receptor signaling pathways in endothelial cells. The synergy is mediated via RBP-Jkappa/CBF-1 and GC-rich palindromic sites in the Herp2 promoter, as well as via interactions between the Notch intracellular domain and Smad that are stabilized by p/CAF. Activated Notch and its downstream effector Herp2 were found to inhibit endothelial cell (EC) migration. In contrast, BMP via upregulation of Id1 expression has been reported to promote EC migration. Interestingly, Herp2 was found to antagonize BMP receptor/Id1-induced migration by inhibiting Id1 expression. Our results support the notion that Herp2 functions as a critical switch downstream of Notch and BMP receptor signaling pathways in ECs.     

Additive effects of PDGF receptor beta signaling pathways in vascular smooth muscle cell development

The platelet-derived growth factor beta receptor (PDGFRbeta) is known to activate many molecules involved in signal transduction and has been a paradigm for receptor tyrosine kinase signaling for many years. We have sought to determine the role of individual signaling components downstream of this receptor in vivo by analyzing an allelic series of tyrosine-phenylalanine mutations that prevent binding of specific signal transduction components. Here we show that the incidence of vascular smooth muscle cells/pericytes (v/p), a PDGFRbeta-dependent cell type, can be correlated to the amount of receptor expressed and the number of activated signal transduction pathways. A decrease in either receptor expression levels or disruption of multiple downstream signaling pathways lead to a significant reduction in v/p. Conversely, loss of RasGAP binding leads to an increase in this same cell population, implicating a potential role for this effector in attenuating the PDGFRbeta signal. The combined in vivo and biochemical data suggest that the summation of pathways associated with the PDGFRbeta signal transduction determines the expansion of developing v/p cells.     

TRAF molecules in cell signaling and in human diseases

The tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of intracellular proteins were originally identified as signaling adaptors that bind directly to the cytoplasmic regions of receptors of the TNF-R superfamily. The past decade has witnessed rapid expansion of receptor families identified to employ TRAFs for signaling. These include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), T cell receptor, IL-1 receptor family, IL-17 receptors, IFN receptors and TGFβ receptors. In addition to their role as adaptor proteins, most TRAFs also act as E3 ubiquitin ligases to activate downstream signaling events. TRAF-dependent signaling pathways typically lead to the activation of nuclear factor-κBs (NF-κBs), mitogen-activated protein kinases (MAPKs), or interferon-regulatory factors (IRFs). Compelling evidence obtained from germ-line and cell-specific TRAF-deficient mice demonstrates that each TRAF plays indispensable and non-redundant physiological roles, regulating innate and adaptive immunity, embryonic development, tissue homeostasis, stress response, and bone metabolism. Notably, mounting evidence implicates TRAFs in the pathogenesis of human diseases such as cancers and autoimmune diseases, which has sparked new appreciation and interest in TRAF research. This review presents an overview of the current knowledge of TRAFs, with an emphasis on recent findings concerning TRAF molecules in signaling and in human diseases.     

c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells

Key participants in G protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. A prototypic GPCR that has been widely used to study these signaling mechanisms is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via G alpha i, EGF receptor without the involvement of Hb-EGF, and c-Src, but independently of PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and phosphatidylinositol 3-kinase (PI3K). ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Besides the main pathway, the dissociated G beta gamma and beta-arrestin may initiate additional, albeit minor, pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other cell lines bearing GnRHR, indicating that GnRH can utilize various signaling mechanisms for the activation of MAPK cascades. The unique pathway elucidated here in which c-Src and PI3K are sequentially activated downstream of the EGF receptor may serve as a prototype of signaling mechanisms by GnRHR and by additional GPCRs in various cell types.     

The lysosomal transmembrane protein 9B regulates the activity of inflammatory signaling pathways

The intracellular signaling pathway by which tumor necrosis factor (TNF) induces its pleiotropic actions is well characterized and includes unique components as well as modules shared with other signaling pathways. In addition to the currently known key effectors, further molecules may however modulate the biological response to TNF. In our attempt to characterize novel regulators of the TNF signaling cascade, we have identified transmembrane protein 9B (TMEM9B, c11orf15) as an important component of TNF signaling and a module shared with the interleukin 1beta (IL-1beta) and Toll-like receptor (TLR) pathways. TMEM9B is a glycosylated protein localized in membranes of the lysosome and partially in early endosomes. The expression of TMEM9B is required for the production of proinflammatory cytokines induced by TNF, IL-1beta, and TLR ligands but not for apoptotic cell death triggered by TNF or Fas ligand. TMEM9B is essential in TNF activation of both the NF-kappaB and MAPK pathways. It acts downstream of RIP1 and upstream of the MAPK and IkappaB kinases at the level of the TAK1 complex. These findings indicate that TMEM9B is a key component of inflammatory signaling pathways and suggest that endosomal or lysosomal compartments regulate these pathways.     

CB(1) cannabinoid receptor-G protein association: a possible mechanism for differential signaling

Effects of cannabinoid compounds on neurons are predominantly mediated by the CB(1) cannabinoid receptor. Onset of signaling cascades in response to cannabimimetic drugs is triggered by the interaction of the cannabinoid receptor with G(i/o) proteins. Much work has been done to delineate the cannabinoid agonist-induced downstream signaling events; however, it remains to define the molecular basis of cannabinoid receptor-G protein interactions that stimulate these signaling pathways. In this review, we discuss several signal transduction pathways, focusing on studies that demonstrate the efficacy of CB(1) receptor agonists through G protein mediated pathways.     

The neurite outgrowth multiadaptor RhoGAP, NOMA-GAP, regulates neurite extension through SHP2 and Cdc42

Neuronal differentiation involves the formation and extension of neuronal processes. We have identified a novel regulator of neurite formation and extension, the neurite outgrowth multiadaptor, NOMA-GAP, which belongs to a new family of multiadaptor proteins with RhoGAP activity. We show that NOMA-GAP is essential for NGF-stimulated neuronal differentiation and for the regulation of the ERK5 MAP kinase and the Cdc42 signaling pathways downstream of NGF. NOMA-GAP binds directly to the NGF receptor, TrkA, and becomes tyrosine phosphorylated upon receptor activation, thus enabling recruitment and activation of the tyrosine phosphatase SHP2. Recruitment of SHP2 is required for the stimulation of neuronal process extension and for sustained activation of ERK5 downstream of NOMA-GAP. In addition, we show that NOMA-GAP promotes neurite outgrowth by tempering activation of the Cdc42/PAK signaling pathway in response to NGF. NOMA-GAP, through its dual function as a multiadaptor and RhoGAP protein, thus plays an essential role downstream of NGF in promoting neurite outgrowth and extension.